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Fancy meeting you here! A fresh look at 'prokaryotic' protein phosphorylation
Journal of Bacteriology
Abstract
Bacteria play host to a wide range of protein phosphorylation-dephosphorylation systems (Fig. 1). As little as five years ago the known systems were thought to be late-emerging and absolutely prokaryote specific. Today we know that most protein kinases and protein phosphatases are descended from a set of common, and possibly quite ancient, prototypes. Prokaryote- and eukaryote-specific protein kinases and protein phosphatases are rare and represent exceptions, not the rule as previously thought. Commonality suggests that a dynamic and versatile regulatory mechanism was first adapted to the modulation of protein function as early if not earlier than more "basic" mechanisms such as allosterism, etc. The existence of common molecular themes confirms that the microbial world offers a unique, largely untapped library and a powerful set of tools for the understanding of a regulatory mechanism which is crucial to all organisms, tools whose diversity and experimental malleability will provide new avenues for exploring and understanding key modes of cellular regulation.
... El descubrimiento de otro importante grupo de proteínas quinasas, las tirosina quinasas, ocurrió a principios de 1980, cuando se determinó que la proteína v -Src, inicialmente reportada como una quinasa por Collett y Erikson (1978), era capaz de fosforilar residuos de tirosina (Hunter y Sefton, 1980 En contraste con la intensa investigación realizada en los organismos e ucariotas desde 1950, la fosforilación de proteínas en los organismos procariotas es de fecha reciente. Los primeros estudios datan de finales de 1970 y todas las descripciones mostraban, al sistema de dos componentes consistente en sensores de histidina quinasa y sus reguladores de respuesta asociados, como responsables de la fosforilación de proteínas (Stock, 1989;Kennelly y Potts, 1996). El sistema de dos componentes es un mecanismo acoplado de estímulo-respuesta que permite a las bacterias sensar y responder a diferentes condiciones ambientales y actúan en una variedad de mecanismos celulares como la quimiotaxis, regulación de nitrógeno, fósforo y oxígeno, osmoregulación, esporulación, síntesis de exoproteínas, virulencia, entre otros (Stock y col., 1 989; Cozzone, 1993 (Kennelly y Potts, 1996;Zhang y col., 1996). ...
... Los primeros estudios datan de finales de 1970 y todas las descripciones mostraban, al sistema de dos componentes consistente en sensores de histidina quinasa y sus reguladores de respuesta asociados, como responsables de la fosforilación de proteínas (Stock, 1989;Kennelly y Potts, 1996). El sistema de dos componentes es un mecanismo acoplado de estímulo-respuesta que permite a las bacterias sensar y responder a diferentes condiciones ambientales y actúan en una variedad de mecanismos celulares como la quimiotaxis, regulación de nitrógeno, fósforo y oxígeno, osmoregulación, esporulación, síntesis de exoproteínas, virulencia, entre otros (Stock y col., 1 989; Cozzone, 1993 (Kennelly y Potts, 1996;Zhang y col., 1996). Por otra parte, también se han encontrado sistemas de dos componentes en organismos eucariotas (Kennelly y Potts, 1996;Stock, y col., 2000). ...
... El sistema de dos componentes es un mecanismo acoplado de estímulo-respuesta que permite a las bacterias sensar y responder a diferentes condiciones ambientales y actúan en una variedad de mecanismos celulares como la quimiotaxis, regulación de nitrógeno, fósforo y oxígeno, osmoregulación, esporulación, síntesis de exoproteínas, virulencia, entre otros (Stock y col., 1 989; Cozzone, 1993 (Kennelly y Potts, 1996;Zhang y col., 1996). Por otra parte, también se han encontrado sistemas de dos componentes en organismos eucariotas (Kennelly y Potts, 1996;Stock, y col., 2000). ...
... El descubrimiento de otro importante grupo de proteínas quinasas, las tirosina quinasas, ocurrió a principios de 1980, cuando se determinó que la proteína v -Src, inicialmente reportada como una quinasa por Collett y Erikson (1978), era capaz de fosforilar residuos de tirosina (Hunter y Sefton, 1980 En contraste con la intensa investigación realizada en los organismos e ucariotas desde 1950, la fosforilación de proteínas en los organismos procariotas es de fecha reciente. Los primeros estudios datan de finales de 1970 y todas las descripciones mostraban, al sistema de dos componentes consistente en sensores de histidina quinasa y sus reguladores de respuesta asociados, como responsables de la fosforilación de proteínas (Stock, 1989;Kennelly y Potts, 1996). El sistema de dos componentes es un mecanismo acoplado de estímulo-respuesta que permite a las bacterias sensar y responder a diferentes condiciones ambientales y actúan en una variedad de mecanismos celulares como la quimiotaxis, regulación de nitrógeno, fósforo y oxígeno, osmoregulación, esporulación, síntesis de exoproteínas, virulencia, entre otros (Stock y col., 1 989; Cozzone, 1993 (Kennelly y Potts, 1996;Zhang y col., 1996). ...
... Los primeros estudios datan de finales de 1970 y todas las descripciones mostraban, al sistema de dos componentes consistente en sensores de histidina quinasa y sus reguladores de respuesta asociados, como responsables de la fosforilación de proteínas (Stock, 1989;Kennelly y Potts, 1996). El sistema de dos componentes es un mecanismo acoplado de estímulo-respuesta que permite a las bacterias sensar y responder a diferentes condiciones ambientales y actúan en una variedad de mecanismos celulares como la quimiotaxis, regulación de nitrógeno, fósforo y oxígeno, osmoregulación, esporulación, síntesis de exoproteínas, virulencia, entre otros (Stock y col., 1 989; Cozzone, 1993 (Kennelly y Potts, 1996;Zhang y col., 1996). Por otra parte, también se han encontrado sistemas de dos componentes en organismos eucariotas (Kennelly y Potts, 1996;Stock, y col., 2000). ...
... El sistema de dos componentes es un mecanismo acoplado de estímulo-respuesta que permite a las bacterias sensar y responder a diferentes condiciones ambientales y actúan en una variedad de mecanismos celulares como la quimiotaxis, regulación de nitrógeno, fósforo y oxígeno, osmoregulación, esporulación, síntesis de exoproteínas, virulencia, entre otros (Stock y col., 1 989; Cozzone, 1993 (Kennelly y Potts, 1996;Zhang y col., 1996). Por otra parte, también se han encontrado sistemas de dos componentes en organismos eucariotas (Kennelly y Potts, 1996;Stock, y col., 2000). ...
This is my Doctoral Thesis from which the paper: "Mycobacterium tuberculosis transporter MmpL7 is a potential substrate for kinase PknD" (Biochemical and Biophysical Research Communications 348 (2006) 6–12,) was published. As a researcher, I know that not always the manuscript of a Doctoral Thesis is easily available. But I think that its content is very important because not all the results are published as "papers". Most of the times, unexplained results or "not beautiful" results are unpublished and are forgotten. Nevertheless, these results could be very useful for other researchers working in similar areas. So, here is my work and I hope to be useful. The language is Spanish but if someone needs a translation of a particular text , I could do it in English. Thank you very much.
... Phosphorylation is a widely studied post-translational protein modification that can be observed in both prokaryotic and eukaryotic systems. This process is controlled by two types of enzymes, kinases and phosphatases, that enable the transfer of phosphate from a nucleotide triphosphates such as adenosine triphosphate (ATP) to a protein substrate or the removal of phosphate from a protein substrate as a conduit of information to enable different cellular functions to occur (49,117). It was initially thought that histidine and aspartate kinases were exclusive to bacteria, while serine/threonine and tyrosine kinases were found only in eukaryotes. ...
... This has been disproved since serine/threonine and tyrosine kinases have been found in bacteria, while histidine and aspartate kinases have been observed in eukaryotes (5,49,94). Though these kinases have been found in two different domains of life, studies have shown and suggested that their functions are not always similar (61). ...
... Historically, histidine kinases were initially discovered and studied in bacteria, while tyrosine and ser/thr kinases were exclusively found in eukaryotic cells (5). Since the 1990s, tyrosine kinases and ser/thr kinases were identified in prokaryotes as well (5,49,94). Studies comparing the protein sequences of known eukaryotic kinases with predicted prokaryotic and archaeal kinases showed that although their amino acid sequences greatly differ, there were conserved motifs that suggested a common ancestral protein kinase (61). ...
... Early attempts during the 1960s and 1970s to detect protein phosphorylation in microbial organisms, by using the techniques that had been successful with mammals, proved negative. Many scientists concluded that protein phosphorylation-dephosphorylation was the exclusive province of higher organisms, a relatively late evolutionary invention devised to meet the special demands of organisms composed of multiple, differentiated cells -one for which simple organisms had no need and hence may do without 43,45 . ...
... IphP appears to be a secreted protein that displayed phosphomonoesterase activity toward a broad range of exogenous peptides, proteins, as well as pyrophosphatase activity toward ADP and ATP 341 . Thus, it might function either as dedicated protein phosphatases or as nonspecific phosphate in the mold of the acid and alkaline phosphatases 45 . The physiological roles of IphP remain uncertain 35 . ...
Genome sequencing data has revealed genes encoding uncharacterized protein family UPF0079 which are exclusively found in bacteria; broadly distributed in this kingdom and possess an ATP-binding motif in their sequences. Biochemical characterization and physiological role elucidation of UPF0079 will undoubtedly increase our fundamental biology knowledge, and also remain a prerequisite towards the development of new antimicrobial compounds. Our investigation on YdiB, an archetype of this family in Bacillus subtilis revealed both autophosphorylating and protein phosphotransferase activities. The dual-specificity Ser/Thr and Tyr kinase activity of YdiB seems to require oligomerization is upregulated by basic molecule activators such as natural polyamines or poly-L-lysine. The 10 most conserved residues were studied to gain insights into molecular mechanism of the kinase YdiB. To characterize the function of phosphorylation events linked to YdiB, starting with the B. subtilis ydiA-B-C-D-E operon we showed that YdiB and YdiC function as cognate protein kinase/phosphatase towards two ribosome-related protein substrates YdiD and YdiE. Some co-localization between YdiB and ribosomes were observed. Furthermore, YdiB is capable of phosphorylating both ribosomal 50S and 30S subunits as well as two ribosome-binding GTPases EngA and EngB. We also demonstrated that phosphorylated EngA by YdiB is an in vitro substrate of the phosphatase YdiC. Finally, based on the phosphoproteome pf Bacillus subtilis, peptides mimicking the in vivo phosphorylation sites were used. Some of them were found to be phosphorylated in vitro by YdiB, including two peptides which belongs to the superoxide dismutase SodA. The activity of purified SodA was then shown to be upregulated via phosphorylation by YdiB. We furthermore found that B. subtilis cells lacking ydiB become more sensitive to oxidative stress-causing agents such as paraquat or norfloxacin. We propose that in vivo, YdiB functions as a protein kinase involved in ribosome function in normal condition; and in protecting cells from oxidative stress damage.
... Until the later 1970s, protein phosphorylation was considered unique to eukaryotic organisms, ever since the first discovery of this phenomenon in human neuroendocrine system (39). The discovery of protein phosphorylation and the identification of protein kinases in prokaryotic organisms proved protein phosphorylation to be a cellular process that is common to both types of organisms (3,80). As we learned more about protein phosphorylation, however, a new doctrine was formed stating that eukaryotic cells exclusively target the hydroxyl amino acids (serine, threonine, and tyrosine) for phosphorylation, while bacteria specifically use histidine and carboxyl amino acids (e.g., aspartic acid) in their so-called two-component regulatory systems. ...
... Rapid progress in bacterial genome-sequencing projects revealed a group of novel protein kinases in bacterial systems (80,96,179). These kinases are highly homologous to the eukaryotic Ser/Thr protein kinases; however, most of their roles remain a mystery. ...
... For several decades, the phosphorylation of proteins on serine and threonine residues was generally regarded as exclusively eukaryotic in origin and distribution, an adaptation to the coordination and communication requirements of a more complex compartmentalized cell form (1). In this scenario, tyrosine phosphorylation emerged to meet the expanded signal transduction needs of "higher" eukaryotes composed of multiple differentiated cells. ...
... As the 1990s dawned, occasional reports surfaced indicating the presence of eukaryotic protein kinases and protein phosphatases in bacteria and viruses (1,2). However, their close association with pathogenic organisms, tendency to target proteins endogenous to their eukaryotic hosts, and frequent coding by mobile elements such as plasmids were consistent with acquisition from the Eukarya by lateral gene transfer. ...
The third domain of life, the Archaea (formerly Archaebacteria), is populated by a physiologically diverse set of microorganisms,
many of which reside at the ecological extremes of our global environment. Although ostensibly prokaryotic in morphology,
the Archaea share much closer evolutionary ties with the Eukarya than with the superficially more similar Bacteria. Initial
genomic, proteomic, and biochemical analyses have revealed the presence of “eukaryotic” protein kinases and phosphatases and
an intriguing set of serine-, threonine-, and tyrosine-phosphorylated proteins in the Archaea that may offer new insights
into this important regulatory mechanism.
... Many cellular processes such as transcription, metabolism and cell movement are controlled by phosphorylated proteins (Raggiaschi et al., 2005). For many years however, protein phosphorylation was considered limited to complex organisms, those composed of numerous differentiated cells (Johnson and Lewis, 2001;Kennelly and Potts, 1996). The existence of bacterial kinases was not accepted until the late 1970s. ...
... A decade later, the discovery of the two-component system proved that protein kinases were ubiquitous amongst prokaryotes. Even so, kinases targeting hydroxyl amino acids like serine, threonine and tyrosine were considered unique to eukaryotic organisms while prokaryotes favoured phosphorylation of histidine or carboxyl amino acids like aspartate (Johnson and Lewis, 2001;Kennelly and Potts, 1996). As the cartoon below illustrates (Figure 1), researchers have been challenged to prove that when it comes to protein phosphorylation, what eukaryotic organisms do, the prokaryotes are capable of as well. ...
... Besides two-component sensory systems, cyanobacteria possess an another possibility to respond to environmental changes even faster. This involves specific post-translational modification of existing proteins by phosphorylation (for reviews see Kennelly and Potts 1996; Mann 1994). Generally, the phosphorylation of polypeptides is catalyzed by serine–threonine (ser–thr) protein kinases that exhibit autophosphorylating activities at the serine and threonine residues, and these are the same residues at which other polypeptides are specifically phosphorylated. ...
... However, the presence of tyrosine-phosphorylated proteins has been reported for cyanobacteria like Nostoc commune Burger-Wiersina, Prochlorothrix hollandica UTEX 584, and Synechococcus sp. strain PCC7942 (summarized by Kennelly and Potts 1996), but it is unclear at the moment whether tyrosine phosphorylation is the result of a rather unspecific ser–thr protein kinase or a specific tyrosine kinase. A number of phosphorylated proteins were shown to occur in cyanobacteria depending on the environmental stimuli (Table 1 ). ...
Cyanobacteria as well as eukaryotic algae possess a CO2 concentrating mechanism that enables the cells to use low CO2 concentrations very efficiently for photosynthesis. The efficiency of the CO2 concentrating mechanism changes in response to environmental changes, especially the availability of inorganic carbon, but the underlying mechanisms that are involved in the regulation of the induction are unknown. This review deals with the occurrence of protein phosphorylation in cyanobacteria and highlights the possible involvement of post-translational modifications of existing proteins in the induction process, which leads to a high-affinity state of the CO2 concentrating mechanism.Key words: cyanobacteria, CO2 concentrating mechanism, protein kinase, protein phosphorylation, post-translational regulation.
... But, as there is still induction of the σ E signal transduction system in ΔvanJ, there are likely to be additional mechanisms that lead to the activation of this regulatory system in the presence of teicoplanin.Cluster 5 was enriched for genes predicted to encode 'STK activity' (GO:0004674, P-value = 4.95 x 10 -2 ) which are also involved in signal transduction within bacteria. They resemble the eukaryotic STKs (or Hanks-type kinases) that phosphorylate serine or threonine residues when activated157,159,160 . All genes within this ontology were significantly upregulated in both strains(Figure 4.1). ...
The glycopeptide antibiotics vancomycin and teicoplanin are clinically important as a secondline therapy to treat nosocomial infections caused by Gram-positive pathogens. Glycopeptideantibiotics universally target the terminal residues, D-Alanyl-D-Alanine on the cell wallpeptidoglycan intermediate lipid II, interfering with peptidoglycan biosynthesis and weakeningthe cell wall. A general resistance mechanism to these antibiotics requires a core set of genes,vanRSHAX, that detect a glycopeptide (VanS) and upregulate genes (VanR) which orchestratethe remodelling of D-Ala-D-Ala on lipid II to D-Ala-D-Lactate (VanHAX), reducingglyopeptide affinity by 1000-fold. Our previous study demonstrated that altering the termini oflipid II by VanHAX action is insufficient for providing resistance to teicoplanin in S.coelicolor, which is instead mediated mainly by the elusive membrane protein, VanJ.
This study further characterised VanJ by comparing the transcriptomes of a wt S. coelicolor A3(2) M600 strain and an isogenic ΔvanJ knock-out mutant after exposing cells to teicoplanin, identifying that ΔvanJ exhibited increased signs of cellular stress that were attributed to a delayed induction of genes involved in the osmotic, redox, and cell envelope stress responses. This dataset led to the functional characterisation of a group of genes with phosphatidic acid phosphatase activity which affected the sensitivity of S. coelicolor to a broad range of cell wall targeting antibiotics. One of these genes, SCO6355, significantly counteracted the intrinsic vanRSHAX resistance system of S. coelicolor, lowering its high-level vancomycin resistance (80 µg/mL) by four-fold, to intermediate levels (20 µg/mL). This work demonstrates a novel mechanism which can antagonise the function of intrinsic van resistance clusters that will be important in the development of strategies that can circumvent glycopeptide resistance in clinical pathogens.
... This shows broad phosphomonoesterase activity towards a range of molecules, including proteins, peptides and low molecular weight organic phosphates. It also has measurable pyrophosphatase activity, and may have a further role in sensing the environment (Kennelly and Potts, 1996;Potts, 2000). In view of the range of organic phosphates in the environment and the need to maximize uptake where phosphorus is low or limiting, it seems probable that the ability to detect diverse molecules in the environment will prove an important feature of periplasmic proteins in cyanobacteria (Mann, 2000). ...
This book contains state-of-the-art reviews of organic phosphorus characterization and transformations in the environment. It includes 17 chapters organized into 3 main sections. Section 1 (3 chapters) describes analytical techniques used to characterize organic phosphorus compounds in environmental samples, with chapters dedicated to state-of-the-art procedures involving chromatographic separation, nuclear magnetic resonance spectroscopy and mass spectrometry. Section 2 (7 chapters) addresses the processes that control organic phosphorus behaviour in terrestrial and aquatic environments, including the origins, stabilization, hydrolysis (biotic and abiotic) and biological utilization of organic phosphorus. Section 3 (7 chapters) integrates these processes at the ecosystem level, with chapters synthesizing information on organic phosphorus in soils and aquatic systems, its interaction with other nutrient cycles and its transfer between terrestrial and aquatic environments. Two chapters (Chapters 15 and 16) in this section describe state-of-the-art models used to investigate the behaviour of organic phosphorus in both terrestrial and aquatic environments. The final chapter in this section summarizes the importance of organic phosphorus in the environment and identifies key areas towards which future research effort should be directed.
... The reversible regulatory phosphorylation events operate on a much faster time scale than changes in protein expression [14]. The earliest studies on protein phosphorylation and dephosphorylation as regulatory events in bacteria focused on phosphorylation of histidine and aspartate residues in relation to two-component systems [15]. However, phosphorylation events on serine, threonine, and tyrosine (Ser/Thr/Tyr) have also been found to play important roles in prokaryotic intracellular signalling. ...
Background:
To respond and adapt to environmental challenges, prokaryotes regulate cellular processes rapidly and reversibly through protein phosphorylation and dephosphorylation. This study investigates the intracellular proteome and Ser/Thr/Tyr phosphoproteome of the oral commensal Streptococcus gordonii. Intracellular proteins from planktonic cells of S. gordonii DL1 were extracted and subjected to 2D-gel electrophoresis. Proteins in general were visualized using Coomassie Brilliant Blue and T-Rex staining. Phosphorylated proteins were visualized with Pro-Q Diamond Phosphoprotein Gel Stain. Proteins were identified by LC-MS/MS and sequence analysis.
Results:
In total, sixty-one intracellular proteins were identified in S. gordonii DL1, many of which occurred at multiple isoelectric points. Nineteen of these proteins were present as one or more Ser/Thr/Tyr phosphorylated form. The identified phosphoproteins turned out to be involved in a variety of cellular processes.
Conclusion:
Nineteen phosphoproteins involved in various cellular functions were identified in S. gordonii. This is the first time the global intracellular Ser/Thr/Tyr phosphorylation profile has been analysed in an oral streptococcus. Comparison with phosphoproteomes of other species from previous studies showed many similarities. Proteins that are consistently found in a phosphorylated state across several species and growth conditions may represent a core phosphoproteome profile shared by many bacteria.
... Protein kinase C phosphorylation site phosphorylation then this must also be possible in bacteria. Relatively little has been published on bacterial serine and threonine phosphorylation [206][207][208][209] however, recent reports suggest there may be many families of bacterial enzymes with high similarity to protein serine/threonine kinases which have yet to be described in any detail [210,211]. Hence, phosphorylation at sites on eukaryote proteins expressed recombinantly in bacteria may be possible and cannot be ruled out in this case. ...
This thesis describes the elucidation of the genetic basis of primary hyperoxaluria type 2 (PH2), an autosomal recessive disease characterised by recurrent calcium oxalate kidney stone formation leading to renal failure and, in some cases to an early death. The enzyme deficient in this condition, glyoxylate / hydroxypyruvate reductase (GRHPR), has been partially purified and characterised from human liver and is distinct from lactate dehydrogenase which also has hydroxypyruvate reductase activity. GRHPR utilises hydroxypyruvate and glyoxylate as substrates with a preference for hydroxypyruvate with NADPH as preferred cofactor. Chromatofocused partially purified human liver proteins show no OR activity associated with LDH, using NADPH as cofactor. Although sharing low protein sequence similarity to the GR/HPR enzymes of plants and bacteria, a human cDNA encoding GRHPR has been identified from an EST database. Purified recombinant GRHPR has similar kinetic properties to the partially purified liver protein, with a preference for hydroxypyruvate as substrate and NADPH as cofactor at pH7.0. Antibodies raised against this recombinant enzyme identify a single protein in human liver of approximately 38kDa which is absent in patients with PH2. The protein demonstrates charge heterogeneity on chromatofocusing and isoelectric focusing, although at a different pi range unless subunits are chemically crosslinked as a dimer (the preferred enzyme conformation). Northern blot analysis of GRHPR mRNA shows its primary location to be the liver but with RNA present in many other tissues. This ubiquitous expression is not reflected by distribution of immunoreactive protein which was essentially restricted to the liver. DNA from patients with PH2 has been screened for mutations in the GRHPR gene by direct sequencing of cDNA where available, or SSCP analysis and sequencing of genomic DNA. 6 mutations and a single polymorphism have been identified including a 1 bp deletion in codon 35, found in 33% of all PH2 alleles.
... immune reactions as well as other physiological processes including cell growth, differentiation, mobility, metabolism, and survival [36]. In addition, in some bacteria, protein phosphorylation plays an important role in sensing extracellular signals and coordinating intracellular events [37]. Thus, it is not surprising that in pathogenic bacteria, such as ...
A genome project has been launched and aims to sequence total genome of Cotesia plutellae bracovirus (CpBV). On this process, several putative open reading frames have been proposed, among which there was a large gene family coding for protein tyrosine phosphatases (PTPs). This study analyzed the deduced amino acid sequences of 14 CpBV-PTPs in terms of conserved domains with other known polydnaviral PTPs and determined their expression patterns in diamondback moth, Plutella xylostella, parasitized by C. plutellae. The analyzed CpBV-PTPs share the common 10 motifs with classical type of PTPs. However, there are variations among CpBV-PTPs in active site sequence and phosphorylation sites. Quantitative real-time polymerase chain reaction (PCR) indicated that most PTPs in the parasitized P. xylostella were expressed from the first day of parasitization and increased the expression levels during parasitization. All 14 PTPs were expressed in both immune-associated tissues of fat body and hemocytes in the parasitized host. During last instar, the PTP enzyme activity of the parasitized P. xylostella was significantly lower than that of the nonparasitized. The reduction of the PTP activity was observed in cytosolic fraction, but not in membrane fraction. The hemocytes of parasitized P. xylostella markedly lost their spreading ability in response to a cytokine (PSP1: plasmatocyte-spreading peptide 1). The functional link between the reduced PTP activity and the suppressed hemocytic behavior was evidenced by the inhibitory effect of sodium orthovanadate (a specific PTP inhibitor) on hemocyte-spreading behavior of nonparasitized P. xylostella. These results suggest that CpBV-PTPs are expressed in the parasitized P. xylostella and affect cellular PTP activity, which may be associated with host immunosuppression. r
... In prokaryotes and lower eukaryotes (yeast, fungi, and plants), protein-histidine kinase activity of metabolic enzymes was identified in two-component and multicomponent signaling systems, and this activity plays an important role in sugar phosphorylation [47][48][49]. In the PEP/ sugar phosphotransferase system, enzyme I, which converts phosphoenolpyruvate to pyruvate, is autophosphorylated at His189, with PEP being used as the phosphate donor. ...
Protein kinases regulate every aspect of cellular activity, whereas metabolic enzymes are responsible for energy production and catabolic and anabolic processes. Emerging evidence demonstrates that some metabolic enzymes, such as pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase (KHK) isoform A (KHK-A), hexokinase (HK), and nucleoside diphosphate kinase 1 and 2 (NME1/2), that phosphorylate soluble metabolites can also function as protein kinases and phosphorylate a variety of protein substrates to regulate the Warburg effect, gene expression, cell cycle progression and proliferation, apoptosis, autophagy, exosome secretion, T cell activation, iron transport, ion channel opening, and many other fundamental cellular functions. The elevated protein kinase functions of these moonlighting metabolic enzymes in tumor development make them promising therapeutic targets for cancer.
... Under osmotic stress conditions, bacteria have developed signal transduction systems to sense changes in the environment and to control the coordinated expression of genes involved in cellular defense mechanisms (Kennelly and Potts, 1996). When the osmotic pressure in the surrounding environment increases, cells activate osmoregulation systems in order to balance the intracellular and environmental pres- sures (Wood, 1999). ...
Bacterial cells respond to changes in the environment by adjusting their physiological reactions. In cascades of cellular responses to stresses of various origins, rapid modulation of RNA function is known to be an effective biochemical adaptation. Among many factors affecting RNA function, RNase III, a member of the phylogenetically highly conserved endoribonuclease III family, plays a key role in posttranscriptional regulatory pathways in Escherichia coli. In this review, we provide an overview of the factors affecting RNase III activity in E. coli.
... 1,2 On the other hand, in prokaryotes phosphorylation mainly occurs on His and Asp residues via His kinases and response regulators as the two-component signal transduction system. However, it is known that the eukaryotic-type protein kinases are also present in some bacteria and archea, [3][4][5] while the elements of the two-component signal transduction system have been reported in archaea and eukaryotes. 6,7 The first putative gene for a eukaryotic-type protein kinase in the bacterial world was identified in Myxococcus xanthus, a Gram-negative soil bacterium. ...
The complete genome of the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for a subfamily Pkn2 of the eukaryotic-type (or “Hanks-type”) protein kinase. Previously,
SpkA and SpkB were shown to have protein kinase activity and to be required for cell motility. Here, the other five genes
were examined. These genes, except for spkG (slr0152), were successfully expressed in Escherichia coli. Eukaryotic-type protein kinase activity of the expressed SpkC (Slr0599), SpkD (Sll0776) and SpkF (Slr1225) was demonstrated
as autophosphorylation and phosphorylation of the general substrate proteins. SpkE (Slr1443) did not show any activity, a
finding consistent with its lack of several key amino acid residues in its kinase motif. Gene-disrupted mutants showed no
discernible defect in phenotype except that spkD was apparently essential for survival.
... Although the phosphorylation of histidine residues (phosphoHis) was already observed in bovine liver in the early 1960s (Boyer et al. 1962), only sporadic reports during the following decades have been addressing this issue in mammals. In contrast, in prokaryotes (Hess et al. 1988) as well as in lower eukaryotes, like yeast, fungi, and plants (Swanson et al. 1994;Kennelly and Potts 1996), two-or multi-component signaling systems were discovered in which protein histidine kinases and histidine phosphatases are important mediators in cellular responses. Homology screens did not reveal similar systems in vertebrates and mammals. ...
Like phosphorylation of serine, threonine, and tyrosine
residues in many organisms, reversible histidine phosphorylation
is a well-known regulatory signal in prokaryotes
and lower eukaryotes. In vertebrates, phosphohistidine has
been mainly described as a phosphorylated intermediate in
enzymatic reactions, and it was believed that regulatory histidine
phosphorylation is of minor importance. During the last
decade, it became evident however, that nucleoside diphosphate
kinase (NDPK), an ubiquitously expressed enzyme required
for nucleotide homeostasis, can additionally act as a
protein histidine kinase. Especially for the isoformNDPK B, at
least three defined substrates, the β subunit of heterotrimeric G
proteins (Gβ), the intermediate conductance potassium channel
KCa3.1, and the Ca2+-conducting TRP channel family
member, TRPV5, have been identified. In all three proteins,
the phosphorylation of a specific histidine residue is of regulatory
importance for protein function, and these
phosphohistidines are cleaved by a counteracting 14 kDa
phosphohistidine phosphatase (PHP). This article will therefore
give an overview of our current knowledge on protein
histidine phosphorylation in prokaryotes and lower eukaryotes
and compare it with the regulatory phosphorylation and dephosphorylation
of histidine residues in vertebrates by NDPK
and PHP, respectively.
... This shows broad phosphomonoesterase activity towards a range of molecules, including proteins, peptides and low molecular weight organic phosphates. It also has measurable pyrophosphatase activity, and may have a further role in sensing the environment (Kennelly and Potts, 1996;Potts, 2000). In view of the range of organic phosphates in the environment and the need to maximize uptake where phosphorus is low or limiting, it seems probable that the ability to detect diverse molecules in the environment will prove an important feature of periplasmic proteins in cyanobacteria (Mann, 2000). ...
... Results reported earlier in case of cytosolic aconitase (Pitula et al., 2004) and Human Pregnane X Receptor (Pondugula et al., 2009) also showed the differential effects of phosphor-ablative and phosphor-mimetic mutant derivatives. Protein phosphorylation/dephosphorylation are wellcharacterized mechanism that living cells use for communicating the surrounding changes to cellular and genetic levels (Kennelly and Potts, 1996). PprA has been shown to exist with other DNA repair proteins, protein kinases and phosphoproteins in a multiprotein DNA processing complex identified from D. radiodurans (Kota and Misra, 2008). ...
... Nevertheless, the phosphorylation of histidine residues (P-His) was already observed in bovine liver in the early 1960's (Boyer et al., 1962), but only sporadic reports during the following decades have addressed this issue in vertebrate systems. In contrast, in prokaryotes (Hess et al., 1988) as well as in lower eukaryotes, like yeast, fungi, and plants (Kennelly and Potts, 1996;Swanson et al., 1994) two or multicomponent signaling systems were discovered in which protein histidine kinases and histidine phosphatases are important mediators of cellular responses such as bacterial chemotaxis. Homology screens did not uncover similar systems and thus a role of P-His, besides its existence as phosphorylated intermediate in enzymatic reactions, was for quite sometime believed to be of minor importance for vertebrates. ...
Regulation of protein phosphorylation by kinases and phosphatases is involved in many signaling pathways in mammalian cells. In contrast to prokaryotes and lower eukaryotes a role for the reversible phosphorylation of histidine residues is just emerging. The β subunit of heterotrimeric G proteins, the metabolic enzyme adenosine 5′-triphosphate-citrate lyase (ACL), and the Ca2+-activated K+ channel KCa3.1 have been identified as targets for nucleoside diphosphate kinase (NDPK) acting as protein histidine kinase and the so far only identified mammalian protein histidine phosphatase (PHPT-1). Herein, we describe the analysis of the phosphorylation and dephosphorylation of histidine residues by NDPK and PHPT-1. In addition, experimental protocols for studying the consequences of heterotrimeric G protein activation via NDPK/Gβγ mediated phosphorelay, the regulation of ACL activity and of KCa3.1 conductivity by histidine phosphorylation will be presented.
... The involvement of eukaryotic-like serine/threonine kinase (STPKs) during cell division and cell wall biosynthesis is well documented [1,2]. STPK and their associated phosphatases (STPP) play major regulatory roles in eukaryotes [3] and in prokaryotes [4,5], particularly in Gram-positive bacteria. Extensive studies have shown that STPKs play essential roles in cell competence, biofilm production [6], cell shape/division [7], cell envelope biosynthesis [7,8], sporulation [9,10] and stress response [11]. ...
Recent genetic, biochemical and structural studies have established that eukaryotic-like Ser/Thr protein-kinases are critical mediators of developmental changes and host pathogen interactions in bacteria. Although with lower abundance compared to their homologues from eukaryotes, Ser/Thr protein-kinases are widespread in gram-positive bacteria. These data underline a key role of reversible Ser/Thr phosphorylation in bacterial physiology and virulence. Numerous studies have revealed how phosphorylation/dephosphorylation of Ser/Thr protein-kinases governs cell division and cell wall biosynthesis and that Ser/Thr protein kinases are responsible for distinct phenotypes, dependent on different environmental signals. In this review we discuss the current understandings of Ser/Thr protein-kinases functional processes based on structural data.
... Over the past decade it has become clear that 'eukaryote-like' tyrosine and serine/threonine protein kinases and phosphatases can be found in many bacteria [1,2]. The tubercle bacillus genome project revealed the presence of a large number of candidate protein kinases and phosphatases [3]. ...
Probing protein extracts from exponentially growing and stationary phase cultures of Mycobacterium bovis BCG with anti-phospho amino acid antibodies revealed a 31-kDa anti-phospho threonine antibody-reactive protein specific to growing culture. The corresponding protein was purified via two-dimensional gel electrophoresis and identified via mass spectrometry to be malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a component of the fatty acid biosynthetic pathway. MCAT tagged with histidine reacted with anti-phospho threonine antibody and was positive in an in-gel chemical assay for phospho proteins. Analysis of the growth phase dependence of MCAT-His phosphorylation and protein levels showed that phosphorylated MCAT-His can be detected only in growing culture. In contrast, MCAT-His protein level was growth phase-independent. These results suggest that MCAT may be a substrate of a protein kinase and phosphatase, and that aspects of fatty acid synthesis in tubercle bacilli are regulated by protein phosphorylation.
... This reveals that Lys336 exhibits a much lower reactivity and/or a lack of accessibility towards FSBA than members of the eukaryotic kinase family, and this possibly reflects a different topography of their respective ATP-binding site. To strengthen this assumption, further comparison between IDHK/P and eukaryotic protein kinases failed to detect any other homologous regions in their sequence [44]. Particularly, the 'catalytic triad' of eukaryotic protein kinases, corresponding for instance to residues Asp166, Asn171, and Asp184 of the cAMP-dependent protein kinase [12]), has not been identified in IDHK/P. ...
The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5′-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.
... Under stress conditions, microorganisms have developed signal transduction systems (movement of signals from outside the cell to inside) to sense environmental stresses and to control the coordinated expression of genes involved in cellular defense mechanisms (Kennelly and Potts 1996). These evolved protective or adaptive networks assist microorganisms to modify their environments and/or to survive the stress condition. ...
Escherichia coli encounter numerous different stresses during their growth, survival, and infection. These stresses are relevant to survival in foods and food processing environments. E. coli and other bacteria respond to stress conditions by activating small or large groups of genes under the control of common regulator proteins. Stress conditions result in the accumulation of these regulator proteins and subsequent transcription of many genes allows cells to cope with specific stress situations, conferring stress tolerance and survival. In addition, induced stress tolerance of cells is attributed to enhanced virulence and enhanced tolerance to other stresses (cross-protection). In this review, regulation of stress and the stress tolerance response of E. coli to heat, acid, starvation, and cold stresses that are commonly used in food preservation and food production will be addressed. The effect of different stress on survival, adaptation, and cross-protection of E. coli studied using laboratory media, and real foods will be briefly summarized. Finally, the relationship of stress response and subsequent virulence and cross-protection will also be discussed.
Microorganisms that adhere to surfaces in order to protect themselves from many adverse environmental conditions form a layer called biofilm. Biofilms protect bacteria from changing environmental conditions such as starvation, antibiotics, disinfectants, pH and temperature fluctuation, dryness and UV rays. In this study, biofilms were formed on surfaces of glass coupons in a cooling tower model system over a period of 180 days. The biofilms were treated with various stress factors monthly. These stress factors were: exposure to temperatures of 4°C and 60°C, pH of 3, 5, and 11, 3 M aqueous NaCl and distilled water, as well as, monochloramine at 2, 500, and 1 000 mg/L (ppm). Following the treatment with stress factors, both the numbers of actively respiring bacteria and the total bacteria in the biofilms were determined by CTC-DAPI staining. The aerobic heterotrophic plate counts (HPC) in the biofilms were determined by the conventional culture method of spread plating on R2A agar. The aim of this study was to determine the impact of these stressors on the model cooling-tower biofilms. Of the stressors tested, those that had the greatest impact were a temperature of 60°C, pH of 3, 3 M NaCl, and monochloramine at both 500 and 1 000 mg/L. However, when using a non-culture-based viability assay (CTC-DAPI staining), an extremely high number of live bacteria were detected even after applying the most effective stress factors (with the exception of pH 3) of 60°C, 3 M NaCl, monochloramine at 500 and 1 000 mg/L. Results showed that biofilm protects the bacteria from extreme physical and chemical stress conditions. Additionally, the conventional culture technique cannot detect the presence of bacteria that have entered the viable but non-culturable (VBNC) phase; the use of different techniques, such as microscopy and cytometry (flow and solid-phase), is therefore important to obtain more accurate results.
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An important element of communications is the machinery utilized to formulate and interpret messages. This process can occur in a hardwired reflexive manner or under the aegis of an integrated information processing network composed of linked biomolecular bits. The most versatile and adaptable of these bits is the phosphoryl group, a potent general perturber of protein conformation and function. This chapter explores our current knowledge of the protein phosphorylation-dephosphorylation events that take place in the members of the domain Archaea, and asks whether the members of this domain have achieved “microprocessor” status.
Single-cell organisms can respond to stimuli from the environment, including chemical and tactile stimuli, to survive and propagate. Thus, single-cell organisms appear to show neuron-like functions. In this review, I investigate neuron-like functions in bacteria (Escherichia coli) and ciliates (Paramecium and Tetrahymena). In E. coli, six chemotaxis-specific (che) genes have been identified as critical in the ability of organisms to react to stimuli from the environment. The che genes encode signaling molecules to transmit information from receptors to motor proteins that regulate some E. coli behavior. Thus, the Che proteins are thought to form a “central processing unit (CPU)”-like complex in E. coli. The eukaryotic single-cell organisms Paramecium and Tetrahymena have also been employed for understanding the molecular mechanisms underlying ciliate behaviors. Paramecia uptake calcium ions and show membrane excitation when they receive a repulsive stimulus, similar to neurons. In addition, the calcium ions function as the second messengers through calmodulin activity and regulate the concentration of cAMP in cilia. Increment changes in cAMP concentration in the cilia result in changes in their beating pattern, which alters the behaviors of paramecia. Moreover, our recent results indicate that the neurotransmitter serotonin is involved in physical functions in Tetrahymena thermophila. These results indicate clearly that bacteria and ciliates are equipped with neuron-like functions. The discussion addresses whether single-cell organisms have intelligence, emotion, and mind.
Protein post-translational modifications (PTMs) are crucial in regulating cellular biology by playing key roles in processes such as the rapid on and off switching of signaling network and the regulation of enzymatic activities without affecting gene expressions. PTMs lead to conformational changes in the tertiary structure of protein and resultant regulation of protein function such as activation, inhibition, or signaling roles. PTMs such as phosphorylation, acetylation, and S-nitrosylation of specific sites in proteins have key roles in regulation of mitochondrial functions, thereby contributing to the progression to heart failure. Despite the extensive study of PTMs in mitochondrial proteins much remains unclear. Further research is yet to be undertaken to elucidate how changes in the proteins may lead to cardiovascular and metabolic disease progression in particular. We aimed to summarize the various types of PTMs that occur in mitochondrial proteins, which might be associated with heart failure. This study will increase the understanding of cardiovascular diseases through PTM.
Survival in a dynamic external environment demands the ability to monitor and respond to a wide range of internal and external variables, or signals. The binding of a signal to its receptor initiates a sequence or “cascade” of molecular events inside the cell that modulate relevant metabolic, nuclear, motile, or other processes. The transmission of an often extracellular receptor binding event into the interior of the cell and its translation into a catalytic or other response is called signal transduction. For an organism to be consistently successful in nature’s continual competition for scarce resources, such responses must be rapid and efficient. Efficiency demands that the response be comprehensive in scope to ensure against wastage of either materials or energy. For example, the activation of the contractile machinery in a muscle cell is accompanied by several secondary and tertiary effects. To meet the requirements of initiating and sustaining contractile activity, a concomitant reconfiguration of the metabolic processes that generate cellular energy and mobilize stored energy sources takes place. The diversion of cellular resources to meet the overriding demands of the contractile process, in turn, requires compensatory fine-tuning of other pathways.
The classic pseudomonads are characterized by polar flagella, ranging from a single polar flagellum, Pseudomonas aeruginosa, to several polar flagella in Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas syringae. The only exception is Burkholderia mallei, which is permanently immotile and interestingly requires a living host for survival. It is the causal agent of the disease, glanders, in horses. Several of the multipolar flagellar types have been removed from the Pseudomonas genus based on r-RNA probe comparisons (Holloway, 1996), most recently to the Burkholderia spp. Some of these groups are discussed separately for comparison. The bulk of this chapter, however, focuses on P. aeruginosa because most studies of flagella have centered on this organism, primarily because of its clinical importance, but also because of its own metabolic uniqueness and versatility.
In the Proceedings of Endocytobiology III, I had the opportunity to outline some ideas about how the evolution of cellular clocks may have occurred concomitantly with the evolution of eukaryotic cells (Kippert 1987). I speculated whether internal timekeeping might have been the initial selective advantage that cellular clocks provided to an endocytobiotic consortium developing into the early eukaryotic cell-a hypothesis which constituted a link between the fields of chronobiology and endocytobiology. Since then, there have been exciting developments in both fields. Much of the progress made in chronobiological research is of significance in evolutionary terms and is more ore less related to the field of endocytobiology. Thus, for Endocytobiology VI it may be timely to review this progress. The aim of the present review is to present briefly those recent findings that may be of relevance to our understanding of the evolution of cellular clocks, and discuss what this may tell us about today’s oscillator mechanisms.
Photosynthetic cells use the energy from absorbed light to synthesize partially or fully reduced organic compounds such as carbohydrates, fatty acids and amino acids from oxidized substrates. A series of elaborate redox reactions links the fundamental events of light absorption to the ultimate supply of reduced assimilates within the cell and also for export to sink tissues. When one looks from a modeling point of view, three sets of parameters appear to be of interest and — if possible — should be controlled in order to optimize the process of photosynthesis, to minimize the waste of energy and to prevent the development of damage (Fig. 1). These three sets of parameters are (1) the input parameters, mainly the incident photon flux density, other environmental parameters and gene activity, (2) the output parameters, particularly the accumulated amount of assimilate products and the energy status, and (3) process parameters, the redox state of intermediate reactions which are of key importance for the whole process.
The aminoglycoside-aminocyclitol antibiotics (hereafter termed aminoglycosides) are a large family of water soluble, cationic molecules which exhibit broad antimicrobial spectra. While the moniker aminoglycoside refers to a vast array of structurally diverse compounds, they all share the incorporation of a six-membered aminocyclitol ring (Fig. 1). The aminoglycosides find use in the treatment of many bacterial infections caused by both Gram-positive and Gram-negative organisms, and are generally administered by injection or intravenously as a result of their relatively poor oral absorption (Edson and Terrell, 1991). Despite some problems of toxicity and bacterial resistance (described in detail below), these antibiotics continue to be a critically important component of our modern antimicrobial arsenal.
Eukaryotic-like protein kinases have been identified recently in several prokaryotes by comparative studies of DNA sequences and Western blotting techniques using antiphosphoprotein antibodies. Examination of the Mycobacterium tuberculosis genome by means of PCR amplification with consensus primers, Southern hybridization, and comparative analysis of DNA sequences with genomic databases revealed the existence of at least seven eukaryotic-like protein kinases in this pathogen. In addition, we report the biochemical identification of phosphorylated proteins in M. tuberculosis. Taken together, these findings show that M. tuberculosis possesses elements of cell signaling similar to those observed in eukaryotic organisms. We suggest that some of these processes may play roles in the pathogenesis of M. tuberculosis.
This review presents the data on the role of eukaryotic-like serine/threonine protein kinases in the members of various groups of cyanobacteria. Information is provided for the two most studied model species (Anabaena and Synechocystis), differing in their morphology and ecophysiological features, and covers the entire period of study of this group of enzymes in cyanobacteria.
Protein phosphorylation-dephosphorylation is the principal mechanism for translation of external signals into cellular responses. Eukaryotic-like serine/threonine kinases have been reported to play important roles in bacterial development and/or virulence. The PknI protein is one of the 11 eukaryotic-like serine/threonine kinases in Mycobacterium tuberculosis H37Rv. From the bioinformatic studies, PknI protein has been shown to have an N-terminal cytoplasmic domain followed by a transmembrane region and an extracellular C-terminus suggestive of a sensor molecule. In this study, we have cloned, overexpressed, and characterized the entire coding region and the cytoplasmic domain of PknI as a fusion protein with an N-terminal histidine tag, and used immobilized metal affinity chromatography for purification of recombinant proteins. The purified recombinant proteins were found to be functionally active through in vitro phosphorylation assay and phosphoamino acid analysis. In vitro kinase assay of both proteins revealed that PknI is capable of autophosphorylation and showed manganese-dependent activity. Phosphoamino acid analysis indicated phosphorylation at serine and threonine residues. Southern blot analysis with genomic DNA highlighted the conserved nature of pknI among the various mycobacterial species. In silico analysis revealed a close homology of PknI to Stk1 from Streptococcus agalactiae, shown to have a role in virulence and cell segregation of the organism.
Protein phosphorylation is one of the major post-translational modifications to allow for signal transmission and fine tuning of metabolism on the cellular proteomic level. As such it is “one of the last instances” to modulate the activity of enzymes and hence to impact the cellular life irrespective of the basic conditions provided by the genome – and epigenome– controlled gene expression. The evolutionary increase in cellular complexity is reflected by highly sophisticated regulatory networks in multicellular eukaryotes based on the transfer of phosphate mostly onto the side chains of serine, threonine and tyrosine residues. Nature has chosen phosphate for inter- and intracellular communication, which is also an integral component of nucleic acids and can be regarded as the molecule of choice for life.
Currently, life science is interested to unravel the network of reversible protein phosphorylation that is catalyzed by two antagonistic enzyme classes: the protein kinases and protein phosphatases. We are currently in the era of proteomics and enormously benefit from the progress of mass-spectrometry methods. This is documented by a huge number of “proteomic studies” that mostly provide a simple inventory of the existence of proteins – and/or their phosphorylated forms – under more or less defined conditions.
So far, the physiological correlations could be established only in a few cases, e.g. by comparing two physiological conditions. Another strategy, which was addressed in this work, is the systematic screening of mutants defective in genes encoding either protein kinases or protein phosphatases. This approach benefits from the ease to predict these enzymes due to the presence of characteristic protein motifs. In combination with the major goal of this work – to shed light on the impact of protein phosphorylation in the mitochondrial (mt) compartment – the yeast Saccharomyces cerevisiae was chosen as a model system because of its respiro-fermentative metabolism, that allows for the maintenance of respiratory defective mutants.
Indeed, this reverse genetic approach successfully revealed two kinases (Pkp1p, Pkp2p) and two phosphatases (Ppp1p, Ppp2p) as the key components regulating the pyruvate dehydrogenase complex by phosphorylation of serine 313 of its α- subunit Pda1p. In addition, evidence is provided that Pkp1p has an additional role in the assembly process of the PDH complex.
Also, the effect of the deletion of the COQ8 gene (gene engaged in coenzyme Q synthesis; originally named ABC1) leading to respiratory deficiency, could be correlated with the phosphorylation of subunit Coq3p of the mitochondrial ubiquinone biosynthesis complex.
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Finally, in the case of the kinase Sat4p (protein involved in salt tolerance), overexpression of the enzyme was used as an alternative approach to unravel the molecular basis of the originally observed salt sensitivity of sat4 mutants. The data suggest that Sat4p has a direct or indirect role in the late steps of iron-sulfur (Fe/S) cluster assembly of the so-called “aconitase-type” enzymes in mitochondria, accompanied by a strongly reduced steady state concentration of the Fe/S-cluster protein aconitase. Interestingly, a secondary phenotype became apparent upon overexpression of Sat4p: the abundance of the lipoic acid containing mitochondrial proteome was markedly reduced. Most likely this phenotype is due to the fact that the synthesis and/or attachment of lipoic acid depend on a Fe/S-cluster bearing enzyme.
In the course of the work it became clear that the regulatory (mt) protein phosphorylation network of yeast evolved to meet the criteria of a life adapted to the ecological niche on temporarily available sugar rich sources. Clearly, the transfer of the respective data to higher eukaryotes is limited. However, it shows that yeast is primarily an excellent model system for the principal molecular reactions shared with higher eukaryotes.
Repression of alkaline phosphatase (APase) activity in four taxonomically different cyanobacteria Anabaena flosaquae, Calothrix brevissima, Scytonema javanicum and Hapalosiphon intricatus by rifampicin and chloramphenicol led to the conclusion that enzyme synthesis is under transcriptional control. A mutant isolated in Nostoc muscorum after mutagenesis with NTG was found to be defective in APase activity (APase
def). The mutant showed nearly 90% growth compared to wild type strain in the presence of inorganic phosphate (Pi). The wild type Nostoc muscorum showed some growth (33% compared to Pi) in the presence of organic phosphate but the growth of the APase
def mutant was negligible under these conditions since it lacks APase activity. APase activity and phosphate uptake functions were phosphate repressible in the wild type strain. Phosphate uptake in the mutant was comparable to the wild type cyanobacterium. The mutant was also able to accumulate polyphosphate granules like the wild type.
Ser/Thr protein kinases, members of signal transduction systems, play important roles in responses to the environmental changes and intra-cellular signals. In comparison with extensive literature describing the function, comprehensive evolutionary analyses of STK gene family are rare. In this study, we employed a maximum likelihood method to detect evidence of positive selection on STKs. Several site-models in the PAML package were used and identified 17 positive selected sites. Sites mostly likely undergoing positive selection were found in the additional domain suggesting these sites may play a major role in their functional divergence. We postulate that STK domain and additional domain have different evolutionary patterns. The sites identified to be under positive selection would provide targets for further structural and functional analyses.
This chapter focuses on the chromatography of amino acids (AAs) and peptide analysis. The approach to AA analysis depends on the origin of a specimen. Specimens may be divided into three classes: (1) mixtures of AAs derived from peptide hydrolysis, (2) AAs derived from peptide sequencing, and (3) AAs present in some biological fluid. The major task in AA analysis is the determination of free AAs in biological fluids. A specimen may contain many unknown substances that can affect the reliability of the analysis. Conventional AA chromatography is currently performed mainly on octadecyl silica by reversed-phase, high-performance liquid chromatography (RP-HPLC) with water/organic solvent gradients. The most common method for peptide analysis utilizes a HPLC procedure with a column of 25-cm length and 4.1-mm ID, packed with a 5-μm, wide-pore octadecylor octyl-bonded stationary phase, eluted with a water/acetonitrile gradient and 0.1–0.2% of trifluoroacetic acid (TFA) as ion-pairing and solubilizing agents.
The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin). These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center. The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled. To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 Angstrom resolution. The overall fold resembles that of PPI and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif. Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center. The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes. In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule. Two additional solvent molecules are coordinated to Mn1. In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2. The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands. In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162. The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues.
Cell type-specific transcription during Bacillus sporulation is established by σF. SpoIIAB is an anti-σ that binds and negatively regulates σF, as well as a serine kinase that phosphorylates and inactivates the anti-anti-σ SpoIIAA. The crystal structure of σF bound to the SpoIIAB dimer in the low-affinity, ADP form has been determined at 2.9 Å resolution. SpoIIAB adopts the GHKL superfamily fold of ATPases and histidine kinases. A domain of σF contacts both SpoIIAB monomers, while 80% of the σ factor is disordered. The interaction occludes an RNA polymerase binding surface of σF, explaining the SpoIIAB anti-σ activity. The structure also explains the specificity of SpoIIAB for its target σ factors and, in combination with genetic and biochemical data, provides insight into the mechanism of SpoIIAA anti-anti-σ activity.
In heme-deficient reticulocytes, protein synthesis is inhibited due to the activation of heme-regulated eIF2α kinase (HRI). Activation of HRI is accompanied by its phosphorylation. We have investigated the role of autophosphorylation in the formation of active and stable HRI. Two autophosphorylated species of recombinant HRI expressed in Escherichia coli were resolved by SDS−PAGE. Both species of HRI were multiply autophosphorylated on serine, threonine, and to a lesser degree also tyrosine residues. Species II HRI exhibited a much higher extent of autophosphorylation and thus migrates slower in SDS−PAGE than species I HRI. Similarly, HRI naturally present in reticulocytes also exhibited these species with different degrees of phosphorylation. Importantly, in heme-deficient intact reticulocytes, inactive species I HRI was converted completely into species II. We further separated and characterized these two species biochemically. We found that species I was inactive and had a tendency to aggregate while the more extensively autophosphorylated species II was an active heme-regulated eIF2α kinase and stable homodimer. Our results strongly suggest that autophosphorylation regulates HRI in a two-stage mechanism. In the first stage, autophosphorylation of newly synthesized HRI stabilizes species I HRI against aggregation. Although species I is an active autokinase, it is still without eIF2α kinase activity. Additional multiple autophosphorylation in the second stage is required for the formation of stable dimeric HRI (species II) with eIF2α kinase activity that is regulated by heme.
Signal transduction via protein phosphorylationMetallophosphatases: classificationMetallophosphatase active site architectureMetal ion requirements and regulation by redoxCalcineurin redox: implications for catalysisSite-directed mutagenesis: implications for catalysisCalcineurin redox: implications for in vivo regulation
Cytoplasmic proteins from the antarctic psychrotrophic bacterium Pseudomonas syringae showed two phosphorylated proteins of molecular mass 66 kDa and 62 kDa. The phosphorylation of 66 kDa protein was enhanced in the presence of Triton X-100 solubilised membrane proteins at a higher temperature (30°C) only. Western blot analysis and phosphoamino acid analysis indicated that the 66 kDa protein is phosphorylated at a tyrosine residue. Surprisingly, sodium orthovanadate, which is a known phosphotyrosine phosphatase (PTPase) inhibitor, inhibited the phosphorylation of the protein. The possible importance of this tyrosine phosphorylated protein to growth at low temperature is suggested.
Evidence for phosphorylation of proteins by protein kinases has been found in Salmonella typhimurium despite previous indications that protein kinase action is absent in prokaryotes. At least four proteins have been found to be phosphorylated. Serine and threonine phosphates have been isolated from acid hydrolysates of these proteins after in vivo and in vitro labeling. The kinases do not phosphorylate histones, casein, or phosvitin. It would appear that phosphorylation as a regulatory control exists in prokaryotes.
A cDNA for branched-chain alpha-ketoacid dehydrogenase kinase was cloned from a rat heart cDNA library. The cDNA had an open reading frame encoding a protein of 382 amino acid residues with a calculated molecular weight of 43,280. The clone codes for the branched-chain alpha-ketoacid dehydrogenase kinase based on the following: 1) the deduced amino acid sequence contained the partial sequence of the kinase determined by direct sequencing; 2) expression of the cDNA in Escherichia coli resulted in synthesis of a 43,000-Da protein that was recognized specifically by kinase antibodies; and 3) enzyme activity that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex was found in extracts of E. coli expressing the protein. Northern blot analysis indicated the mRNA for the branched-chain alpha-ketoacid dehydrogenase kinase was more abundant in rat heart than in rat liver, as expected from the relative amounts of kinase activity expressed in these tissues. The deduced sequence of the kinase aligned with a high degree of similarity within subdomains characteristic of procaryotic histidine protein kinases. This first mitochondrial protein kinase to be cloned appears more closely related in sequence to procaryotic histidine protein kinases than to eucaryotic serine/threonine protein kinases.
A methylated membrane protein of 97 kDa was suggested on the basis of mutant analysis to transduce signals from the phototaxis receptor sensory rhodopsin I to the flagellar motor in Halobacterium halobium. Here we report isolation of the proposed transducer protein, cloning of its gene based on partial protein sequences, the complete gene sequence, and analysis of the encoded primary structure. The 1611-base-pair gene termination codon overlaps the initiator ATG of the sopI gene, which encodes the sensory rhodopsin I apoprotein. The predicted size of 57 kDa for the methylated protein indicates an aberrant electrophoretic migration on SDS/polyacrylamide gels, as occurs with other acidic halophilic proteins. Putative promotor elements are located in an A+T-rich region upstream of the gene. Comparison of the translated nucleotide sequence with N-terminal sequence of the purified protein shows the protein is synthesized without a processed leader peptide and the N-terminal methionine is removed in the mature protein. The deduced protein sequence predicts two transmembrane helices near the N terminal that would anchor the protein to the membrane. Beyond this hydrophobic region of 46 residues, the remainder of the protein (536-amino acid residues total) is hydrophilic. The C-terminal 270 residues contain a region homologous to the signaling domains of eubacterial transducers (e.g., Escherichia coli Tsr protein), flanked by two regions homologous to the methylation domains of the transducer family. The protein differs from E. coli Tsr in that it does not have an extramembranous-receptor binding domain but instead has a more extended cytoplasmic region. Coexpression of the methyl-accepting protein gene (designated htrI) and sopI restores sensory rhodopsin I phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI region. These results extend the eubacterial transducer family to the archaebacteria and substantiate the proposal that the methylated membrane protein functions as a signal-transducing relay between sensory rhodopsin I and cytoplasmic sensory-pathway components.
We have investigated a tyrosine kinase activity from Pseudomonas solanacearum, an economically important plant pathogen. In vitro incubation of membrane fractions with [gamma-32P]ATP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85-kDa phosphoprotein. Phosphorylation of this protein on tyrosine residues was demonstrated by phosphoamino acid analysis of base hydrolysis products and by immunoanalysis of Western blots (immunoblots) with antiphosphotyrosine monoclonal antibody. In vitro incubation of membranes with ATP was not required for recognition by the antibody, indicating that the 85-kDa protein is phosphorylated in vivo. These results demonstrate that membranes from P. solanacearum exhibit a tyrosine kinase activity toward an endogenous membrane protein. This bacterium provides an opportunity to study the structure and function of a prokaryotic tyrosine kinase.
A recombinant protein-tyrosine-phosphatase has been expressed in Escherichia coli and purified to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using affinity chromatography. When the phosphatase was allowed to react with 32P-labeled substrates and then rapidly denaturated, a 32P-labeled phosphoprotein could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Transient formation of a 32P-labeled phosphoprotein was observed, and the 32P-labeled protein disappeared as substrate was consumed. In the presence of 32P-labeled p-nitrophenyl phosphate, 0.27 mol of phosphate was incorporated per mol of protein-tyrosine-phosphatase. Site-directed mutagenesis of a catalytically essential cystine residue (position 215) in the recombinant protein resulted in an inactive enzyme, and no phosphoprotein was formed. The 32P-labeled phosphoprotein showed a maximum lability between pH 2.5 and 3.5 and was rapidly decomposed in the presence of iodine. These properties, along with additional site-directed mutations, suggest that the protein-tyrosine-phosphatase forms a covalent thiol phosphate linkage between Cys215 and phosphate.
Protein phosphorylation was investigated in the bacterium Acinetobacter calcoaceticus both in vivo and in vitro. In cells grown with [32P]orthophosphate, several radioactive phosphoproteins were detected by gel electrophoresis and autoradiography. These proteins were shown to contain phosphoserine, phosphothreonine, and a relatively large proportion of phosphotyrosine residues. Incubation of cellular extracts with [gamma-32P] ATP also resulted in the phosphorylation of several proteins. At least four of them, namely an 81-kDa protein, were modified at tyrosine. No protein labeling occurred when extracts were incubated with [gamma-32P] ATP or [14C]ATP. Moreover, phosphoproteins were insensitive to snake venom phosphodiesterase. All together these results indicate that A. calcoaceticus harbors different protein kinases including a protein-tyrosine kinase activity. Further analysis of this activity showed that it has little, if any, functional similarity with eukaryotic protein-tyrosine kinases.
In Escherichia coli, the phosphorylation and dephosphorylation of isocitrate dehydrogenase (IDH) are catalyzed by a bifunctional protein kinase/phosphatase. We have determined the nucleotide sequence of aceK, the gene encoding IDH kinase/phosphatase. This gene consists of a single open reading frame of 1,734 base pairs preceded by a Shine-Dalgarno ribosome-binding site. Examination of the deduced amino acid sequence of IDH kinase/phosphatase revealed sequences which are similar to the consensus sequence for ATP-binding sites. This protein did not, however, exhibit the extensive sequence homologies which are typical of other protein kinases. Multiple copies of the REP family of repetitive extragenic elements were found within the intergenic region between aceA (encoding isocitrate lyase) and aceK. These elements have the potential for combining to form an exceptionally stable stem-loop structure (delta G = -54 kcal/mol [ca. -226 kJ/mol]) in the mRNA. This structure, which masks the ribosome-binding site and start codon for aceK, may contribute to the downshift in expression observed between aceA and aceK. Another potential stem-loop structure (delta G = -29 kcal/mol [ca. 121 kJ/mol]), unrelated to the REP sequences, was found within aceK.
Protein kinases play essential roles in the development of eukaryotic cells. These enzymes display various degrees of sequence similarity in their catalytic domains. This conservation has allowed the identification of protein kinases in a variety of organisms, including the Gram-negative bacterium Myxococcus xanthus. In this study, sequences related to those encoding eukaryotic protein kinases were amplified by PCR from DNA of Anabaena PCC 7120, a filamentous cyanobacterium that differentiates cells specifically for nitrogen fixation, called heterocysts, under conditions of combined nitrogen limitation. Results from Southern hybridization and sequencing of PCR products suggest the presence of a family of similar protein kinases in this strain. One of the corresponding genes (pknA) was isolated from a gene library. The N-terminal region of its amino acid sequence shows significant similarity to the catalytic domains of eukaryotic-type protein kinases. Expression of this gene was found to be developmentally regulated. Inactivation of pknA led to colonies that appeared light green and rough in the absence of combined nitrogen. Mutant filaments produce fewer heterocysts than wild-type ones. These results suggest that pknA is required for both normal cellular growth and differentiation of Anabaena PCC 7120.
A divalent metal ion-stimulated protein-serine/threonine phosphatase, PP1-arch, was purified approximately 1,000-fold from the extreme acidothermophilic archaeon Sulfolobus solfataricus (ATCC 35091). Purified preparations contained 40 to 70% of total protein as PP1-arch, as determined by assay-ing sodium dodecyl sulfate-polyacrylamide gels for protein phosphatase activity. The first 25 amino acids of the protein's sequence were identified, as well as an internal sequence spanning some 20 amino acids. Using this information, we cloned the gene for PP1-arch via the application of PCR and conventional cloning techniques. The gene for PP1-arch predicted a protein of 293 amino acids that bore striking resemblance to the members of the major family of protein-serine/threonine phosphatases from members of the domain Eucarya, the PP1/2A/2B superfamily. The core of the protein, spanning residues 4 to 275, possessed 29 to 31% identity with these eucaryal protein phosphatases. Of the 42 residues found to be absolutely conserved among the known eucaryal members of the PP1/2A/2B superfamily, 33 were present in PP1-arch. If highly conservative substitutions are included, this total reached 37. The great degree of sequence conservation between molecules from two distinct phylogenetic domains implies that the members of this enzyme superfamily had evolved as specialized, dedicated protein phosphatases prior to the divergence of members of the Archaea and Eucarya from one another.
The recombinant rat branched-chain alpha-ketoacid dehydrogenase kinase has been amplified from rat kidney cDNA, based on the previously reported rat cDNA sequence (Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M. J., and Harris, R. A. (1992) J. Biol. Chem. 267, 13127-13130). This kinase was expressed in Escherichia coli as a fusion protein with bacterial maltose-binding protein (MBP). Expression was improved by overexpression of chaperonins GroEL and GroES. The MBP-kinase, when reconstituted with lipoylated recombinant E2 (dihydrolipoyl transacylase), catalyzed phosphorylation of recombinant E1 (branched-chain alpha-ketoacid decarboxylase) with a kcat of 28.5 nmol of phosphate/min/nmol of MBP-kinase at 25 degrees C. Recombinant MBP-kinase alone demonstrated a slow rate of autophosphorylation with a kcat of 3.25 pmol of phosphate/min/nmol of kinase at 25 degrees C. Serine 22 of the kinase was identified as the possible site of autophosphorylation by Edman microsequencing analysis. Autophosphorylated kinase cannot transfer phosphate to E1, indicating that autophosphorylation of kinase is not an intermediate in ATP-dependent phosphorylation of E1. Therefore, despite the reported sequence similarity to prokaryotic histidine protein kinases, the mitochondrial rat branched-chain alpha-ketoacid dehydrogenase kinase apparently does not phosphorylate E1 via a histidine-mediated phosphotransfer reaction. Significant corrections to the published cDNA sequence of rat branched-chain alpha-ketoacid dehydrogenase kinase are included.
Protein-tyrosine phosphorylation has long been regarded as an exclusively eukaryotic phenomenon. Although some non-eukaryotes, mainly viruses, possess genes encoding protein-tyrosine kinases or protein-tyrosine phosphatases, these were probably appropriated from the eukaryotic hosts that constitute the sites of action of these enzymes. Herein we identify a gene, iphP, from the chromosome of the cyanobacterium Nostoc commune UTEX 584 that contains the His-Cys-Xaa-Ala-Gly-Xaa-Xaa-Arg sequence characteristic of known protein-tyrosine phosphatases. The expressed gene product, IphP, displayed protein-tyrosine phosphatase activity toward phosphotyrosine residues on reduced, carboxyamidomethylated, and maleylated lysozyme with optimum activity at pH 5.0. In addition, IphP dephosphorylated the phosphoseryl groups on casein that had been phosphorylated by the cAMP-dependent protein kinase. Cell lysates of N. commune probed with antibodies to phosphotyrosine indicated the presence of a tyrosine-phosphorylated protein of M(r) approximately 85 kDa. This tyrosine-phosphorylated protein was detected in cells grown in the presence of combined nitrogen but not in nitrogen-deficient media that induces the formation of differentiated N2-fixing cells (heterocysts). Together, these data suggest a role for protein-tyrosine phosphorylation in regulating cellular functions in this cyanobacterium. IphP is the first protein-tyrosine phosphatase to be discovered that is encoded by the chromosomal DNA of any prokaryote. Given the free-living nature of N. commune and the phylogenetic antiquity of the cyanobacteria, these findings suggest for the first time the existence of a protein-tyrosine phosphatase of genuine, unambiguous prokaryotic ancestry, thus raising fundamental questions as to the origin and role of tyrosine phosphorylation.
Histidine kinases are part of the two-component signal transduction system responsible for eubacterial responses to diverse environmental signals. They have recently been detected in eukaryotes but their existence in the kingdom Archaea remains uncertain. Here we report the sequence and function of a histidine kinase (CheAH.s.) from Halobacterium salinarium, the first such transmitter in Archaea. The protein CheAH.s. (668 residues) has significant sequence identity with the CheA proteins known from eubacterial signal transduction (e.g. 34% identity with CheA from Bacillus subtilis). Antibodies were raised against CheAH.s. as expressed in Escherichia coli and were used in Western blotting to demonstrate the expression of cheAH.s. in H. salinarium. As has been observed for other halophilic proteins, CheAH.s. has a deviant electrophoretic migration, with an apparent molecular weight of 103 kDa on SDS-PAGE compared with a calculated molecular weight of 72 kDa. Deletion of a part of the cheAH.s. gene leads to loss of both chemotactic and phototactic responses in H. salinarium as measured by swarm plate assays, motion analysis and tethering experiments. This indicates that CheAH.s. plays a crucial role in chemical and light signal integration, presumably interacting with at least two phototransducers and a number of chemoreceptors.
Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of
the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory
responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine
kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes
recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.
Ethylene behaves as a hormone in plants, regulating such aspects of growth and development as fruit ripening, flower senescence,
and abscission. Ethylene insensitivity is conferred by dominant mutations in the ETR1 gene early in the ethylene signal transduction
pathway of Arabidopsis thaliana. The ETR1 gene was cloned by the method of chromosome walking. Each of the four known etr1
mutant alleles contains a missense mutation near the amino terminus of the predicted protein. Although the sequence of the
amino-terminal half of the deduced ETR1 protein appears to be novel, the carboxyl-terminal half is similar in sequence to
both components of the prokaryotic family of signal transducers known as the two-component systems. Thus, an early step in
ethylene signal transduction in plants may involve transfer of phosphate as in prokaryotic two-component systems. The dominant
etr1-1 mutant gene conferred ethylene insensitivity to wild-type Arabidopsis plants when introduced by transformation.
We recently reported molecular cloning of the branched chain alpha-ketoacid dehydrogenase kinase, the first mitochondrial protein kinase to be cloned (Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M. J., and Harris, R. A. (1992) J. Biol. Chem. 267, 13127-13130). From a search for proteins related to the branched chain alpha-ketoacid dehydrogenase kinase, a cDNA encoding the 434 amino acid residues corresponding to pyruvate dehydrogenase kinase has been cloned from a rat heart cDNA library. Evidence that the clone codes for pyruvate dehydrogenase kinase includes: (a) the deduced amino acid sequence is identical to the partial sequence of the kinase determined by direct sequencing; (b) expression of the cDNA in Escherichia coli resulted in synthesis of a protein that phosphorylated and inactivated the pyruvate dehydrogenase complex; (c) kinase activity of the recombinant protein is sensitive to inhibition by a specific inhibitor of pyruvate dehydrogenase kinase; and (d) antiserum raised against the recombinant protein recognized the protein subunit known to correspond to pyruvate dehydrogenase kinase in a highly purified preparation of the pyruvate dehydrogenase complex. Like the branched chain alpha-ketoacid dehydrogenase kinase, pyruvate dehydrogenase kinase lacks motifs usually associated with eukaryotic Ser/Thr-protein kinases. Considerable sequence similarity exists between these mitochondrial protein kinases and members of the prokaryotic histidine kinase family, a diverse set of sensing and response systems important in the regulation of bacterial processes. Thus, molecular cloning of these proteins establishes a new eukaryotic family of protein kinases that is related to a prokaryotic family of protein kinases.
We have observed that soluble extracts from the extreme acidothermophilic archaebacterium Sulfolobus solfataricus contained protein phosphatase activity that was greatly stimulated by the divalent metal ions Mn2+, Mg2+, Ni2+, or Co2+. This activity apparently arose from a single enzyme since (a) stimulation by these divalent metal ions was not additive and (b) protein phosphatase activity eluted as a single peak from both a DE52 ion-exchange column and a Sephadex G-100 gel filtration column. Its apparent molecular mass was approximately 28,000 daltons. The enzyme dephosphorylated a variety of phosphoserine-containing substrates including casein, histone H2a, phosphorylase kinase, or glycogen phosphorylase. The enzyme would not dephosphorylate either histone H1 or a number of phosphotyrosine-containing compounds. It removed only half the phosphate bound to histone H2b, which is phosphorylated at two sites by the cAMP-dependent protein kinase. Protein phosphatase activity was inhibited by EDTA, Cu2+, Zn2+, NaF, inorganic phosphate, or pyrophosphate; but was unaffected by other potential activators and inhibitors such as microcystin, okadaic acid, vanadate, polyamines, or sulfhydryl modifying reagents. This enzyme represents the first protein phosphatase to be identified in any member of the third and oldest phylogenetic kingdom in nature, the archaebacteria.
We describe the isolation and characterization of a gene (ptpA) from Streptomyces coelicolor A3(2) that codes for a protein with a deduced M(r) of 17,690 containing significant amino acid sequence identity with mammalian and prokaryotic small, acidic phosphotyrosine protein phosphatases (PTPases). After expression of S. coelicolor ptpA in Escherichia coli with a pT7-7-based vector system, PtpA was purified to homogeneity as a fusion protein containing five extra amino acids. The purified fusion enzyme catalyzed the removal of phosphate from p-nitrophenylphosphate (PNPP), phosphotyrosine (PY), and a commercial phosphopeptide containing a single phosphotyrosine residue but did not cleave phosphoserine or phosphothreonine. The pH optima for PNPP and PY hydrolysis by PtpA were 6.0 and 6.5, respectively. The Km values for hydrolysis of PNPP and PY by PtpA were 0.75 mM (pH 6.0, 37 degrees C) and 2.7 mM (pH 6.5, 37 degrees C), respectively. Hydrolysis of PNPP by S. coelicolor PtpA were 0.75 mM (pH 6.0, 37 degrees C) and 2.7 mM (pH 6.5, 37 degrees C), respectively. Hydrolysis of PNPP by S. coelicolor PtpA was competitively inhibited by dephostatin with a Ki of 1.64 microM; the known PTPase inhibitors phenylarsine oxide, sodium vanadate, and iodoacetate also inhibited enzyme activity. Apparent homologs of ptpA were detected in other streptomycetes by Southern hybridization; the biological functions of PtpA and its putative homologs in streptomycetes are not yet known.
Addition of acetate to a stationary phase culture of Escherichia coli in glycerol mineral salts medium containing phosphorus-32-labeled orthophosphate results in rapid loss of isocitrate dehydrogenase activity and concomitant incorporation of phosphorus-32 into the enzyme. This is the first example of protein phosphorylation in a bacterium in which the endogenous substrate for the protein kinase has been identified.
Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.
The amino acid sequences of mammalian protein phosphatase 1 and 2A were compared pairwise with every sequence in the National Biomedical Research Foundation protein sequence database using an exhaustive searching programme [Coulson et al., Comp. J. 30 (1987) 420–424], The N-terminal half of the protein encoded by an open reading frame, orf221, in bacteriophage λ (nt 43224–43886 in the map of Daniels et al. [in Hendrix et al. (Eds.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 519–676] shows 35% identity to either protein phosphatase 1 or 2A in this region. If conservative replacements are included the overall homology rises to 49%. A gene in φ80 also shows 35% identity with the mammalian protein phosphatases. The results indicate that orf221 of phage λ and the homologous φ80 gene may encode protein phosphatases. The possible roles of protein phosphorylation in the propagation of bacteriophage are discussed.
A single type of reversible protein-phosphorylating system, the ATP-dependent protein kinase/phosphatase system, is employed in signal transduction in eukaryotes. By contrast, recent work has revealed that three types of protein-phosphorylating systems mediate signal transduction in bacteria. These systems are (1) classical protein kinase/phosphatase systems, (2) sensor-kinase/response-regulator systems, and (3) the multifaceted phosphoenolpyruvate-dependent phosphotransferase system. Physiological, structural, and mechanistic aspects of these three evolutionarily distinct systems are discussed in the papers of this written symposium. © 1993 Wiley-Liss, Inc.
A global regulatory protein, AfsR, involved in secondary metabolism, was found to be phosphorylated by a membraneassociated phosphokinase, named AfsK, of Streptomyces coelicolor A3(2) and S. lividans. The N-terminal portion of AfsK, deduced from the nucleotide (nt) sequence of the afsK gene, which was located downstream from the afsR gene, showed significant sequence similarity to the catalytic domain of eukaryotic Ser/Thr protein kinases (PKs). Consistent with this, experiments with AfsK produced by use of an Escherichia coli host-vector system revealed a self-catalyzed phosphate incorporation into both Ser and Tyr residues of AfsK. The recombinant AfsK phosphorylated the purified AfsR at both Ser and Thr residues. Disruption of the chromosomal afsK gene with the phage vector KC515 resulted in significant, but not complete, loss of actinorhodin production. This result implies the involvement of afsK in the regulation of secondary metabolism. The presence of an additional PK able to phosphorylate AfsR is predicted, because the afsK-disrupted strain still contained an activity able to phosphorylate Ser and Thr residues of AfsR. Southern hybridization experiments showed that nt sequences homologous to afsK, as well as afsR, were distributed among many Streptomyces spp. It is thus concluded that a signal transduction system similar to that found in higher organisms is involved in the regulation of secondary metabolism in the bacterial genus Streptomyces.
When growing E. coli cells in a minimal medium containing radioactive orthophosphate, several proteins appear to be significantly labeled after analysis by polyacrylamide gel electrophoresis and autoradiography : at least four in the soluble cellular fraction, five in crude ribosomes and one in salt-washed ribosomes. In all cases, phosphorylation occurs at the level of threonine and serine residues as shown by paper electrophoresis of acid hydrolysates of these proteins. It is concluded that bacteria do contain a protein kinase activity.
The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli. Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library. A full-length clone was obtained consisting of a 27-bp 5' noncoding region, an open reading frame encoding the expected 157 amino acid protein, and an extensive 3' nontranslated sequence. The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly(A)+ mRNA from bovine heart. The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E. coli. The expressed enzyme was soluble, and catalytically active and was readily isolated and purified. The recombinant protein had the expected Mr of 18,000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes. The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from the bovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated. A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction. Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate. By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.
PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.
Publisher Summary To help cope with the rapidly expanding protein kinase family, a database of the catalytic domain amino acid sequences has been established. This database would be a useful resource for the initial classification of novel protein kinases and for other studies that require extensive sequence comparisons. The catalytic domain database is updated frequently and often includes new sequences before they can be found in the Genbank/EMBL/PIR resources. More importantly, the availability of this large group of sequences in a single file saves investigators from the tedious task of collecting them themselves. This chapter describes the current makeup of the catalytic domain database and present two examples of its use: analysis and graphic display of conserved catalytic domain residues using conservation plots and classification of protein kinases by phylogenetic mapping. A prerequisite for both of these tasks is a multiple sequence alignment. The protein kinase catalytic domain database file can be obtained electronically over Internet using the standard network file transfer program (FTP). The database file PKINASES.IG contains brief descriptions of the kinases and references for the sequences. One hundred and seventeen distinct sequences had been entered by mid-February, 1990. Seventy-five of these are taken from protein-serine/threonine kinases and 42 from protein-tyrosine kinases. Sixty-eight of the sequences are from vertebrate species, 24 from yeasts (both budding and fission), 18 from Drosophila, two from nematode, and one each from Aplysia , Aspergillus , Hydra , bean plant ( Phaseolus ), and avian erythroblastosis virus S13. 43 of the 68 current vertebrate entries are taken from human sources and twenty two of the remaining vertebrate sequences derive from four other mammals: bovine, rabbit, rat, and mouse.
The cyclic heptapeptide, microcystin-LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with Ki values below 0.1 nM. Protein phosphatase 2B is inhibited 1000-fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin-LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin-LR to PP1. We discuss the possibility that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin-LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.
Yersinia is the genus of bacteria that is the causative agent in plague or the black death, and on several occasions this
organism has killed a significant portion of the world's population. An essential virulence determinant of Yersinia was shown
to be a protein tyrosine phosphatase. The recombinant 50-kilodalton Yersinia phosphatase had a specificity for removal of
phosphate from Tyr-containing as opposed to Ser/Thr-containing phosphopeptides and proteins. Site-directed mutagenesis was
used to show that the Yersinia phosphatase possesses an essential Cys residue required for catalysis. Amino acids surrounding
an essential Cys residue are highly conserved, as are other amino acids in the Yersinia and mammalian protein tyrosine phosphatases,
suggesting that they use a common catalytic mechanism.
Five protein serine/threonine phosphatases (PP) have been identified by cloning cDNA from mammalian and Drosophila libraries. These novel enzymes, which have not yet been detected by the techniques of protein chemistry and enzymology, are termed PPV, PP2Bw, PPX, PPY and PPZ. The complete amino acid sequences of PPX, PPY and PPZ and an almost complete sequence of PPV are presented. In the catalytic domain PPV and PPX are more similar to PP2A (57-69% identity) than PP1 (45-49% identity), while PPY and PPZ are more similar to PP1 (66-68% identity) than PP2A (44% identity). The cDNA for PP2Bw encodes a novel Ca2+/calmodulin-dependent protein phosphatase only 62% identical to PP2B in the catalytic domain. Approaches for determining the cellular functions of these protein phosphatases are discussed.
Phosphate esters and anhydrides dominate the living world but are seldom used as intermediates by organic chemists. Phosphoric
acid is specially adapted for its role in nucleic acids because it can link two nucleotides and still ionize; the resulting
negative charge serves both to stabilize the diesters against hydrolysis and to retain the molecules within a lipid membrane.
A similar explanation for stability and retention also holds for phosphates that are intermediary metabolites and for phosphates
that serve as energy sources. Phosphates with multiple negative charges can react by way of the monomeric metaphosphate ion
PO3- as an intermediate. No other residue appears to fulfill the multiple roles of phosphate in biochemistry. Stable, negatively
charged phosphates react under catalysis by enzymes; organic chemists, who can only rarely use enzymatic catalysis for their
reactions, need more highly reactive intermediates than phosphates.
The majority of the phosphotyrosine recovered from partial acid hydrolysates of 32P-labeled Escherichia coli is derived from a single prominent protein. We show here by biochemical, genetic, and immunological criteria that this protein is actually glutamine synthetase adenylylated (not phosphorylated) at tyrosine. Furthermore, all of the phosphotyrosine detectable in partial acid hydrolysates of 32P-labeled Salmonella typhimurium was eliminated in a strain deficient in both glutamine synthetase and uridylyltransferase, an enzyme which uridylylates the regulatory protein PII at a tyrosine residue. These results suggest that protein-tyrosine phosphorylation represents a rare modification in eubacterial cells.
Infection of Escherichia coli with phage lambda gt10 resulted in the appearance of a protein phosphatase with activity towards 32P-labelled casein. Activity reached a maximum near the point of cell lysis and declined thereafter. The phosphatase was stimulated 30-fold by Mn2+, while Mg2+ and Ca2+ were much less effective. Activity was unaffected by inhibitors 1 and 2, okadaic acid, calmodulin and trifluoperazine, distinguishing it from the major serine/threonine-specific protein phosphatases of eukaryotic cells. The lambda phosphatase was also capable of dephosphorylating other substrates in the presence of Mn2+, although activity towards 32P-labelled phosphorylase was 10-fold lower, and activity towards phosphorylase kinase and glycogen synthase 25 50-fold lower than with casein. No casein phosphatase activity was present in either uninfected cells, or in E. coli infected with phage lambda gt11. Since lambda gt11 lacks part of the open reading frame (orf) 221, previously shown to encode a protein with sequence similarity to protein phosphatase-1 and protein phosphatase-2A of mammalian cells [Cohen, Collins, Coulson, Berndt & da Cruz e Silva (1988) Gene 69, 131-134], the results indicate that ORF221 is the protein phosphatase detected in cells infected with lambda gt10. Comparison of the sequence of ORF221 with other mammalian protein phosphatases defines three highly conserved regions which are likely to be essential for function. The first of these is deleted in lambda gt11.
The amino acid sequences of mammalian protein phosphatase 1 and 2A were compared pairwise with every sequence in the National Biomedical Research Foundation protein sequence database using an exhaustive searching programme [Coulson et al., Comp. J. 30 (1987) 420-424]. The N-terminal half of the protein encoded by an open reading frame, orf 221, in bacteriophage lambda (nt 43,224-43,886 in the map of Daniels et al. [in Hendrix et al. (Eds.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 519-676] shows 35% identity to either protein phosphatase 1 or 2A in this region. If conservative replacements are included the overall homology rises to 49%. A gene in phi 80 also shows 35% identity with the mammalian protein phosphatases. The results indicate that orf 221 of phage lambda and the homologous phi 80 gene may encode protein phosphatases. The possible roles of protein phosphorylation in the propagation of bacteriophage are discussed.
Addition of acetate to a stationary phase culture of Escherichia coli in glycerol mineral salts medium containing phosphorus-32-labeled
orthophosphate results in rapid loss of isocitrate dehydrogenase activity and concomitant incorporation of phosphorus-32 into
the enzyme. This is the first example of protein phosphorylation in a bacterium in which the endogenous substrate for the
protein kinase has been identified.
Primary sequence patterns based on known conserved sites in eukaryotic protein kinases were used to search for eukaryotic-like protein kinase sequences in a six-frame translation of the bacterial subsection of GenBank. This search identified a previously unrecognized eukaryotic-like protein kinase gene in three related methanogenic archaebacteria, Methanococcus vannielii, M. voltae, and M. thermolithotrophicus. The proposed coding sequences are located in orthologous open reading frames (ORFs): ORF547, ORF294, and ORF114, respectively. The C-terminus of the ORFs contains 9 of the 11 subdomains characteristically conserved within the eukaryotic protein kinase catalytic domain. The N-terminus of the ORFs is similar to a putative glycoprotease in Pasteurella haemolytica and its homologue in Escherichia coli, the orfX gene. This is the first report of a eukaryotic-like protein kinase sequence observed in Archaebacteria.
The substrates of ion- and lipid-stimulated protein kinase activity in extracts of Escherichia coli were purified by chromatography. Subsequent N-terminal sequencing suggests that these substrates include the following: a novel 80 kDa protein co-purifying with RNA polymerase but partially homologous to elongation factor G; a protein with an apparent molecular weight of 65 kDa identified as the ribosomal protein S1; and a 32 kDa protein identified as succinyl CoA synthetase, a key enzyme in the tricarboxylic acid cycle. The phosphorylation of these three proteins was markedly stimulated by the addition of manganese, and occurred on threonine, serine or tyrosine residues as indicated by the stability of the phosphoresidues during acid treatment. In addition, a calcium-stimulated protein of 70 kDa was identified as the heat-shock protein DnaK, and a 17 kDa lipid-stimulated phosphoprotein as nucleotide diphosphate kinase.
Autophosphorylating histidine kinase and response-regulator domains constitute the building blocks of two-component signaling systems. These systems use a unique phosphotransfer chemistry to regulate many aspects of bacterial physiology. Homologous systems are now being found in eukaryotes. Despite their common mechanism of phosphotransfer, the two-component systems display an extensive diversity in the arrangement of their domains, and flexibility in their roles in different signal transduction circuits.
A 6.3-kb DNA fragment encoding two eukaryotic-type serine/threonine protein kinases (Ser/Thr PK) was cloned from Streptomyces coelicolor A3(2) by using a PCR product obtained with primers based on highly conserved regions of eukaryotic Ser/Thr PK. The nucleotide (nt) sequence of the essential 4.4-kb fragment contained two possible ORFs. One ORF (PkaA) contained 543 amino acids (aa), while another (PkaB) consisted of 417 aa. The N-terminal half of both proteins showed significant similarity with the catalytic domain of eukaryotic Ser/Thr PK. On the other hand, the C-terminal region of PkaA, but not of PkaB, is rich in Pro and Gln residues, indicating that PkaA works as a PK as well as a transcription factor. The pkaB gene was overexpressed in Escherichia coli, and the gene product (PkaB) was found to be phosphorylated mainly at Thr. The pkaA gene was also overexpressed in E. coli, and the gene product (PkaA) was found to be phosphorylated mainly at Thr and slightly at Ser. In the case of PkaA, at least 100 aa residues from the C terminus were not essential for the PK activity. When the PCR product was used as a probe, it hybridized to DNA fragments from all the Streptomyces species tested, indicating that these types of Ser/Thr PK are distributed ubiquitously and play significant physiological roles in the various species of Streptomyces.
A multiple sequence alignment of 44 serine/threonine-specific protein phosphatases has been performed. This reveals the position of a common conserved catalytic core, the location of invariant residues, insertions and deletions. The multiple alignment has been used to guide and improve a consensus secondary-structure prediction for the common catalytic core. The location of insertions and deletions has aided in defining the positions of surface loops and turns. The prediction suggests that the core protein phosphatase structure comprises two domains: the first has a single, beta sheet flanked by alpha helices, while the second is predominantly alpha helical. Knowledge of the core secondary structures provides a guide for the design of site-directed-mutagenesis experiments that will not disrupt the native phosphatase fold. A sequence similarity between eukaryotic serine/threonine protein phosphatases and the Escherichia coli diadenosine tetraphosphatase has been identified. This extends over the N-terminal 100 residues of bacteriophage phosphatases and E. coli diadenosine tetraphosphatase. Residues which are invariant amongst these classes are likely to be important in catalysis and protein folding. These include Arg92, Asn138, Asp59, Asp88, Gly58, Gly62, Gly87, Gly93, Gly137, His61, His139 and Val90 and fall into three clusters with the consensus sequences GD(IVTL)HG, GD(LYF)V(DA)RG and GNH, where brackets surround alternative amino acids. The first two consensus sequences are predicted to fall in the beta-alpha and beta-beta loops of a beta-alpha-beta-beta secondary-structure motif. This places the predicted phosphate-binding site at the N-terminus of the alpha helix, where phosphate binding may be stabilised by the alpha-helix dipole.
Convergence as a phenomenon in molecular evolution is an issue that confuses many discussions. Often the problem is that not enough care is taken to state exactly what kind of convergence one has in mind. Functional and mechanistic convergence are both common, and some structural convergence has probably occurred, but a convincing case for genuine sequence convergence has yet to be made.
In the prokaryotic two-component signal transduction systems, recognition of an environmental stimulus by a sensor molecule results in the activation of its histidine kinase domain and phosphorylation of a histidine residue within that domain. This phosphate group is then transferred to an aspartate residue in the receiver domain of a cognate response regulator molecule, resulting in the activation of its output function. Although a few eukaryotic proteins were identified recently that show sequence similarity to the prokaryotic sensors or response regulators, it has not been clear whether they constituted a part of a 'two-component' system. Here we describe a two-component system in Saccharomyces cerevisiae that regulates an osmosensing MAP kinase cascade.
Many bacterial signaling pathways involve a two-component design. In these pathways, a sensor kinase, when activated by a signal, phosphorylates its own histidine, which then serves as a phosphoryl donor to an aspartate in a response regulator protein. The Sln1 protein of the yeast Saccharomyces cerevisiae has sequence similarities to both the histidine kinase and the response regulator proteins of bacteria. A missense mutation in SLN1 is lethal in the absence but not in the presence of the N-end rule pathway, a ubiquitin-dependent proteolytic system. The finding of SLN1 demonstrates that a mode of signal transduction similar to the bacterial two-component design operates in eukaryotes as well.
We have detected a protein phosphatase activity in soluble extracts from the halophilic archaeon Haloferax volcanii. This activity was markedly stimulated by the divalent metal ions Mn2+ and Cd2+. It dephosphorylated phosphoseryl residues in casein, mixed histones, and phosphorylase a, but not phosphotyrosyl residues in reduced, carboxyamidomethylated and maleylated lysozyme. This protein phosphatase activity was inhibited by NaF, Zn2+, vanadate, molybdate, inorganic phosphate, inorganic pyrophosphate, or p-nitrophenyl phosphate, or by treatment with diethylpyrocarbonate. Activity was unaffected by other potential inhibitors or activators such as polyamines, heparin, cyclic nucleotides, Ca2+/calmodulin, tartrate, tetramisole, okadaic acid, microcystin LR, or sulfhydryl-modifying agents. The functional similarities between this protein-serine phosphatase and that previously identified in another archaeon, the extreme acidothermophile Sulfolobus solfataricus, suggest the existence of a family of divalent metal ion-stimulated protein-serine phosphatases of extremely ancient origin in the Archaea.
Soluble extracts of the methanogenic archaeon, Methanosarcina thermophila TM-1, contained a divalent metal ion-stimulated protein-serine phosphatase activity. This activity was sensitive to micromolar concentrations of okadaic acid, microcystin-LR, or calyculin A, three compounds thought to be highly specific inhibitors of the type 1/2A/2B genetic superfamily of eukaryotic protein-serine/threonine phosphatases. The observation that each of these three chemically unrelated compounds inhibited this archaeal protein phosphatase activity suggests the existence of structural homology, and perhaps even common genetic ancestry, with the type 1/2A/2B superfamily of protein-serine/threonine phosphatases found in eukaryotic organisms.
Protein phosphorylation has been shown to occur in over fifty different bacterial species and, therefore, seems to be a universal device among prokaryotes. Most of the protein kinases responsible for this modification of proteins share the common property of using adenosine triphosphate as phosphoryl donor. However, they differ from one another in a number of structural and functional aspects. Namely, they exhibit a varying acceptor amino acid specificity and can be classified, on this basis, in three main groups: protein-histidine kinases, protein-serine/threonine kinases and protein-tyrosine kinases.
Phosphorylation of proteins catalysed by protein kinases is associated with central functions in growth and proliferation of the eukaryotic cell, and kinases are particularly important in the signal transduction pathways. Enterobacterial protein kinases are structurally and functionally different from eukaryotic protein kinases, and no prokaryotic kinase has so far been described implicating a direct role for this activity in virulence. Virulent Yersinia possess a common virulence plasmid that encodes a number of secreted proteins (Yops), of which YopH has protein-tyrosine phosphatase activity with a key function in the block of phagocytosis by the pathogen. Here we report that the virulence plasmid of Yersinia pseudotuberculosis encodes a secreted protein kinase (YpkA) with extensive homology to eukaryotic Ser/Thr protein kinases. Specific mutants of ypkA resulted in avirulent strains. Thus, YpkA is, to our knowledge, the first reported prokaryotic secreted protein kinase involved in pathogenicity, presumably by interfering with the signal transduction pathways of the target cell.