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[44] Use of degenerate oligonucleotide probes to identify clones that encode protein kinases
Abstract
Publisher Summary This chapter describes an alternative homology cloning approach that uses degenerate oligodeoxynucleotides ("oligos") as hybridization probes. The oligo probes are designed to recognize target sequences that encode short stretches of six to nine highly conserved amino acid residues found within the catalytic domains. Such probes are ideally suited for the purpose of identifying a wide variety of novel protein kinases from a single library screen. Virtually all of the codon possibilities for a conserved stretch can be included in the probe mixture, thereby assuring that many different protein kinase genes (cDNAs) are recognized. False positives are reduced by targeting two or three conserved stretches and selecting further characterization only those clones that are identified by all of the probe mixtures. This approach is successful in identifying both novel protein-serine/threonine kinases and novel protein-tyrosine kinases. Degenerate oligodeoxynucleotides can also be used to prime the polymerase chain reaction and thus identify novel protein kinases through selective gene amplification.
... With the exception of PKA, the other kinases involved in the regulation of capacitation are unknown. RT-PCR with degenerate oligonucleotide primers corresponding to conserved regions present in protein kinases is a powerful tool for studying the expression of these enzymes in a particular tissue or cell type [12]. Using this approach, we have cloned a cDNA encoding a putative protein kinase of the Ser/Thr protein kinase subfamily that is specifically expressed postmeiotically in mouse male germ cells. ...
... The DNA products were then analyzed on 2% agarose gels. The following degenerate primers were used to target conserved regions of tyrosine kinases: for the RT step, a degenerate antisense 20-mer was synthesized corresponding to the coding subregion IX common to the subfamily of tyrosine kinases [12] with the introduction of a 5Ј EcoRI consensus site (in bold) for subcloning after three random nucleotides (5Ј-CGTGGATCCA(A/T)AGGACCA(C/G)AC(A/G)TC-3Ј); for the PCR step, the same primer was used together with a degenerate sense 20mer corresponding to the subregion VIb common to the subfamily of tyrosine kinases [12], with the consensus site for EcoRI (in bold) introduced after four random nucleotides (5Ј-ATTCGGATCCAC(A/C)G(A/C/T/G)GA(C/T) (C/T)T-3Ј). After the PCR was completed, the amplified products were subcloned into a TOPO TA cloning vector (InVitrogen) according to the manufacturer's instructions; the EcoRI subcloning consensus sites were not used. ...
... The DNA products were then analyzed on 2% agarose gels. The following degenerate primers were used to target conserved regions of tyrosine kinases: for the RT step, a degenerate antisense 20-mer was synthesized corresponding to the coding subregion IX common to the subfamily of tyrosine kinases [12] with the introduction of a 5Ј EcoRI consensus site (in bold) for subcloning after three random nucleotides (5Ј-CGTGGATCCA(A/T)AGGACCA(C/G)AC(A/G)TC-3Ј); for the PCR step, the same primer was used together with a degenerate sense 20mer corresponding to the subregion VIb common to the subfamily of tyrosine kinases [12], with the consensus site for EcoRI (in bold) introduced after four random nucleotides (5Ј-ATTCGGATCCAC(A/C)G(A/C/T/G)GA(C/T) (C/T)T-3Ј). After the PCR was completed, the amplified products were subcloned into a TOPO TA cloning vector (InVitrogen) according to the manufacturer's instructions; the EcoRI subcloning consensus sites were not used. ...
Using reverse transcription–polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a novel member of the testis-specific serine/threonine kinases (STK) from mouse male mixed germ cell mRNA. This PCR fragment recognized a 1020-bp transcript in male germ cells by northern blot analysis and was used to clone a full-length cDNA from a mouse mixed germ cell cDNA library. This cDNA has an open reading frame of 804 bases encoding a protein of 268 amino acids. This novel gene is almost identical to Stk22c, encoding a recently described testis-specific protein kinase, except for base-pair deletions that result in a shift in the coding region and an alteration of 22 amino acids (residues 109–131). Due to its homology with Stk22c, we have called this protein kinase gene Stk22d. Northern blot analysis revealed that this protein kinase is developmentally expressed in testicular germ cells and is not present in brain, ovary, kidney, liver, or early embryonic cells. We then cloned the human homologue of this protein kinase gene (STK22C) and found it to be expressed exclusively in the testis. Fluorescence in situ hybridization with both the human and mouse cDNA clones revealed syntenic localization on chromosomes 1p34–p35 and 4E1, respectively.
... Mutagenic alteration of a non-conserved residue within subdomain IIII, Lys382, to alanine yielded an active protein kinase. This behavior confirmed that rSsoPK4 was the Phosphoamino acid analysis of revealed that rSsoPK4(284-635) phosphorylated itself as well as the aforementioned exogenous substrate proteins exclusively on threonine or threonine and serine residues (Figure 3), behavior consistent with the presence of a lysine in subdomain VIb [31]. Catalytic activity was greatest at pH 6.8. ...
The eukaryotic protein kinase (ePK) paradigm provides integral components for signal transduction cascades throughout nature. However, while so-called typical ePKs permeate the Eucarya and Bacteria, atypical ePKs dominate the kinomes of the Archaea. Intriguingly, the catalytic domains of the handful of deduced typical ePKs from the archaeon Sulfolobus solfataricus P2 exhibit significant resemblance to the protein kinases that phosphorylate translation initiation factor 2α (eIF2α) in response to cellular stresses. We cloned and expressed one of these archaeal eIF2α protein kinases, SsoPK4. SsoPK4 exhibited protein-serine/threonine kinase activity toward several proteins, including the S. solfataricus homolog of eIF2α, aIF2α. The activity of SsoPK4 was inhibited in vitro by 3ʹ,5ʹ-cyclic AMP (Ki of ~23 µM) and was activated by oxidized Coenzyme A, an indicator of oxidative stress in the Archaea. Activation enhanced the apparent affinity for protein substrates, Km, but had little effect on Vmax. Autophosphorylation activated SsoPK4 and rendered it insensitive to oxidized Coenzyme A.
... Der Vergleich der Aminosäuresequenzen des humanen und des murinen RIP ergab, dass die Kinase-(76% Identität) und die Deathdomänen (89% Identität) stärker konserviert sind als die Intermediärdomänen (56% Identität) (Stanger et al., 1995; Hsu et al., 1996). Die Kinasedomäne beinhaltet die Konsensussequenzen für die Kinasesubdomänen VI (DLKPEN) und VIII (GTLYYMAPE) von Serin/ Threoninkinasen (Hanks and Lindberg, 1991). Eine RIP-Mutante, in der das konservierte Lysin (K) an Position 45 in der Kinasesubdomäne II zu einem Arginin (N) mutiert wurde, zeigt keine Kinase-Aktivität mehr (Hsu et al., 1996). ...
In der vorliegenden Arbeit wurde die Bedeutung der zellulären Serin/Threonin-Kinase RIP1 für die HCMV-Infektion verifiziert. Die bereits in der Literatur beschriebene Hochregulation der Expression von RIP1 während der HCMV-Infektion, konnte auch in HFF-Zellen gezeigt werden. Hier konnte zum ersten Mal gezeigt werden, dass die Kinase-Aktivität von RIP1 für die HCMV-Replikation in HFF-Zellen notwendig war, da die Überexpression der Kinase-inaktiven RIP1-Mutante RIP K45R in HCMV-Replikations-Tests die Virusproduktion hemmen konnte. Weitere HCMV-Replikations-Tests mit RIP-Konstrukten zeigten, dass nicht alleine die Kinase-Aktivität von RIP1, und die damit verbundene Phosphorylierung möglicher viraler oder zellulärer Substrate, sondern wahrscheinlich auch die Bindung dieser Substrate an die Intermediär- und/oder die Deathdomäne, wichtig für die HCMV-Replikation war. Allerdings schien RIP1 nur zelltypspezifisch in HFF-Zellen notwendig für die HCMV-Replikation zu sein. Die Bedeutung von RIP1 für die HCMV-Infektion konnte nicht nur genetisch durch den Einsatz einer Kinase-inaktiven RIP1-Mutante, sondern auch chemisch mit Hilfe von RIP1-Kinase-Inhibitoren validiert werden. Interaktionen mit HCMV-viralen Proteinen, Effekte auf physiologische Zellfunktionen oder auf die Aktivierung von zellulären Signalwegen (z.B. MAP Kinase- und NFB-Signalwege) konnten als Ursachen für die Hemmung der HCMV-Replikation durch die Expression von RIP K45R weitgehend ausgeschlossen werden. Anhand von Sekretions-Experimenten konnte gezeigt werden, dass Überstände der mit RIP K45R transduzierten Zellen die HCMV-Replikation um ca. 50% hemmen konnten. In „cDNA-Arrays“, PCR-Analysen und Zytokin-ELISA-Tests konnten vor allen Dingen antivirale Zytokine wie z.B. MIP-1, RANTES, Eotaxin, IL-6 und IL-8 identifiziert werden, deren Transkription und Sekretion durch RIP1 gehemmt wurde. Von den identifizierten Zytokinen konnten IL-8 und RANTES die HCMV-Replikation per se supprimieren. Am Modell des IL-8-Promotors konnte gezeigt werden, dass die Expression und Sekretion der Zytokine IL-8 und RANTES möglicherweise über die Interaktion der RIP1-Kinase- und Intermediärdomäne mit ihren Promotoren gehemmt werden könnte. Als These wurde diskutiert, dass die Expression der Kinase-inaktiven RIP-Mutante RIP K45R wahrscheinlich über einen „dominant-negativen“ Effekt auf endogenes RIP1 über einen bislang nicht geklärten Signalweg die Expression antiviraler Zytokine „aktivieren“ und so die HCMV-Replikation hemmen konnte. Die in der vorliegenden Arbeit gewonnenen Daten deuteten darauf hin, dass die Hochregulation von RIP1 während der HCMV-Infektion, die antivirale Immunantwort hemmt. Dieses Ergebnis macht die zelluläre RIP1-Kinase zu einem Kandidaten für die Entwicklung spezifischer Inhibitoren, die den Vorteil hätten, dass sie nicht zur Resistenzbildung beitragen In this thesis, the importance of the cellular serine/threonine-proteinkinase RIP1 was verified during HCMV infection. RIP1 was upregulated during HCMV infection in HFF-cells, as it was already described in literature. It is demonstrated here that forced expression of RIP K45R - a kinase inactive RIP1 mutant - could potently block HCMV replication in HFF cells. RIP1 was up regulated during HCMV infection in HFF cells, as it has already been described in literature. Further HCMV replication assays using different RIP1 constructs show that not only kinase activity but also substrate binding to intermediate- and deathdomain of RIP1 seem to be essential for HCMV replication. The effect of RIP1 during HCMV replication seem to be cell type specific in HFF cells. Experiments with chemical inhibitors of RIP1 could also demonstrate the importance of RIP1 for the HCMV infection. Other possible explanations of the effect of RIP1, like interactions with other HCMV proteins, effects on physiological cell functions or activation of cellular signaling pathways (e.g. MAP kinase- and NFB-signaling pathways) has been excluded. By secretions-experiments it could be proved that supernatants of the RIP K45R transducted cells suppress the HCMV replication by 50%. Especially antiviral cytokines such as MIP-1, RANTES, Eotaxin, IL-6 and IL8 has been identified by cDNA arrays, PCR-analysis and cytokine EILSA assays. The transcription and secretion of these antiviral cytokines has been impeded by RIP1. IL-8 and RANTES could suppress the HCMV replication per se. With the model of the IL-8 promoter it could be demonstrated that expression and suppression of the cytokines might be impeded via interaction of the RIP1 kinase- and intermediate domain with their promotors. The assumption has been discussed that RIP K45R probably “activates” the expression of antiviral cytokines via a “dominant-negative” effect on endogenous RIP1 via a so far unexplained signaling pathway. The data collected during the thesis suggest that the up regulation of RIP1 during the HCMV infection impedes the antiviral immune response. These results indicate RIP1 to be a candidate for the development of specific inhibitors, which would be advantageous because they avoid formation of resistance.
... Twenty nanograms of the poly(A) RNA was reverse transcribed at 37°C for 90 min with 400 U of M-MLV reverse transcriptase (Life Technologies, Gaithersburg, MD) in a 25-µl reaction mixture containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 3 mM MgCl 2 , 1 mM dNTPs, 5 µg of random hexamers, 20 U of RNasin (Promega, Madison, WI), and 1 mM dithiothreitol (DTT). Two microliters of the resultant first-strand cDNA was amplified with a pair of degenerate oligonucleotide primers corresponding to conserved amino acid sequences in subdomains VIb and VIII of the catalytic region of serine/threonine protein kinases (Hanks and Lindberg 1991) Pennincykx et al. 1996) in a 25-µl reaction mixture (1× Perkin Elmer PCR buffer I [PE Applied Biosystems, Foster City, CA], 200 µM dNTPs, and 20 pmol of each primer). The amplification conditions were 1 min at 94°C for denaturation, 2 min at 42°C for annealing, and 2 min at 72°C for synthesis for the P4/P5 primer pair. ...
Viroids--covalently closed, circular RNA molecules in the size range of 250 to 450 nucleotides-are the smallest known infectious agents and cause a number of diseases of crop plants. Viroids do not encode proteins and replicate within the nucleus without a helper virus. In many cases, viroid infection results in symptoms of stunting, epinasty, and vein clearing. In our study of the molecular basis of the response of tomato cv. Rutgers to infection by Potato spindle tuber viroid (PSTVd), we have identified a specific protein kinase gene, pkv, that is transcriptionally activated in plants infected with either the intermediate or severe strain of PSTVd, at a lower level in plants inoculated with a mild strain, and not detectable in mock-inoculated plants. A full-length copy of the gene encoding the 55-kDa PKV (protein kinase viroid)-induced protein has been isolated and sequence analysis revealed significant homologies to cyclic nucleotide-dependent protein kinases. Although the sequence motifs in the catalytic domain suggest that it is a serine/threonine protein kinase, the recombinant PKV protein autophosphorylates in vitro on serine and tyrosine residues, suggesting that it is a putative member of the class of dual-specificity protein kinases.
... Eucaryal organisms also contain a handful of eukaryotic protein kinases lacking a lysine or arginine residue within the catalytic loop. Two of these, specifically PID261\BUD32 [79] and Rio1p [80], both from yeast, have been shown to possess protein serine\threonine kinase activity. PKN6, a eukaryotic protein kinase from the bacterium Myxococcus xanthus, autophosphorylates itself, despite the absence of a lysine or an arginine residue in its catalytic loop [81]. ...
Protein phosphorylation/dephosphorylation has long been considered a recent addition to Nature's regulatory arsenal. Early studies indicated that this molecular regulatory mechanism existed only in higher eukaryotes, suggesting that protein phosphorylation/dephosphorylation had emerged to meet the particular signal-transduction requirements of multicellular organisms. Although it has since become apparent that simple eukaryotes and even bacteria are sites of protein phosphorylation/dephosphorylation, the perception widely persists that this molecular regulatory mechanism emerged late in evolution, i.e. after the divergence of the contemporary phylogenetic domains. Only highly developed cells, it was reasoned, could afford the high 'overhead' costs inherent in the acquisition of dedicated protein kinases and protein phosphatases. The advent of genome sequencing has provided an opportunity to exploit Nature's phylogenetic diversity as a vehicle for critically examining this hypothesis. In tracing the origins and evolution of protein phosphorylation/dephosphorylation, the members of the Archaea, the so-called 'third domain of life', will play a critical role. Whereas several studies have demonstrated that archaeal proteins are subject to modification by covalent phosphorylation, relatively little is known concerning the identities of the proteins affected, the impact on their functional properties, or the enzymes that catalyse these events. However, examination of several archaeal genomes has revealed the widespread presence of several ostensibly 'eukaryotic' and 'bacterial' protein kinase and protein phosphatase paradigms. Similar findings of 'phylogenetic trespass' in members of the Eucarya (eukaryotes) and the Bacteria suggest that this versatile molecular regulatory mechanism emerged at an unexpectedly early point in development of 'life as we know it'.
... In most eukaryotic protein kinases, the presence of a lysine residue of subdomain VIb, i.e., Asp-Xaa-Lys-Xaa-Xaa-Asn, is diagnostic of a protein-serine/threonine kinase, while the presence of an arginine, i.e., Asp-Xaa-Arg-Xaa-Xaa-Asn or Asp-Xaa-Xaa-Xaa-Arg-Asn, is indicative of a protein-tyrosine kinase (21). However, the lack of an arginine or lysine in subdomain VIb does not preclude the possibility that SsoPK2 is a protein kinase. ...
The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the “eukaryotic” protein kinase superfamily.
sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations
(5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins,
including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine
residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of
eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two
areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second
N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be
uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the
enzyme. Autophosphorylation was detected only at temperatures ≥60°C, whereas phosphorylation of exogenous proteins was detectable
at 37°C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser548, with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.
... These included the univer-sally conserved glycine (subdomain I), lysine (subdomain II), and glutamate residues (subdomain III) of the ATP-binding domain, the universally conserved Asp-Xaa 4 -Asn sequence of the catalytic loop (subdomain VIb), the universally conserved aspartate (subdomain VII) that participates in binding the divalent metal ion cofactor, and the highly conserved aspartate (subdomain IX) and arginine (subdomain XI) that form a salt bridge stabilizing the conformation of the C-terminal catalytic peptide-substrate binding domain (Fig. 3). The presumed subdomain VIb region did not, however, contain either the lysine residue that is generally indicative of specificity for serine and threonine residues or the arginine that correlates with selectivity for tyrosine (21). It should be noted, however, that functionally competent eukaryotic protein kinases lacking either of these basic amino acids also have been characterized from both yeast (3,12,13,58) and bacteria (64). ...
Sulfolobus solfataricus contains a membrane-associated protein kinase activity that displays a strong preference for threonine as the phospho-acceptor
amino acid residue. When a partially purified detergent extract of the membrane fraction from the archaeon S. solfataricus that had been enriched for this activity was incubated with [γ-32P]ATP, radiolabeled phosphate was incorporated into roughly a dozen polypeptides, several of which contained phosphothreonine.
One of the phosphothreonine-containing proteins was identified by mass peptide profiling as the product of open reading frame
[ORF] sso0469. Inspection of the DNA-derived amino acid sequence of the predicted protein product of ORF sso0469 revealed the presence of sequence characteristics faintly reminiscent of the “eukaryotic” protein kinase superfamily. ORF
sso0469 therefore was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein formed insoluble aggregates that could be dispersed using urea or detergents. The solubilized polypeptide
phosphorylated several exogenous proteins in vitro, including casein, myelin basic protein, and bovine serum albumin. Mutagenic
alteration of amino acids predicted to be essential for catalytic activity abolished or severely reduced catalytic activity.
Phosphorylation of exogenous substrates took place on serine and, occasionally, threonine. This new archaeal protein kinase
displayed no catalytic activity when GTP was substituted for ATP as the phospho-donor substrate, while Mn2+ was the preferred cofactor.
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.
Environmental effects and mitogens determine cell phenotype in eukaryotes mainly through MAPK pathways. However, MAPK signaling pathways in T. thermophila have not been studied comprehensively. This study aims to express recombinant MPK2, a MAPK from T. thermophila, in E. coli to characterize its kinase activity. MPK2 was cloned by RT-PCR using degenerate oligonucleotide primers and RACE method. The full-length cDNA of the MPK2 gene is 1705bp that includes 1281bp ORF coding for a putative protein of 426 amino acids having a mass of 50.2kDa. The putative MPK2 protein contains all eleven conserved subdomains that are characteristics of serine/threonine protein kinases, and a TDY motif, which is a putative dual phosphorylation site common in Protista. MPK2 displays highest 48% overall identity to human ERK5 (MAPK7). The expression vector pGEX4T-1-MPK2 was constructed by inserting the coding region of MPK2 cDNA into pGEX4T-1 after introducing the nine point mutations, and then transformed into E. coli BL21(DE3). Autophosphorylation of 76kDa GST-MPK2 at tyrosine residues was confirmed not only by Western blot using anti-phosphotyrosine monoclonal antibody but also by in vitro kinase assay. GST-MPK2 was also able to phosphorylate the artificial substrate myelin basic protein. This study concludes that the free-living unicellular protist T. thermophila MPK2 has commonly conserved MAPK enzyme features, possibly involved in the regulation of cell survival responding to abiotic or biotic stressors, and the production and movement of haploid gametic nuclei between pairs during conjugation.
Through specific interactions with members of the tumor necrosis receptor (TNFR) family, adapter molecules such as the serine/threonine (Ser/Thr) kinase RIP mediate divergent signaling pathways including NF-KB activation and cell death. In this study, we have identified and characterized a novel 61-kDa protein kinase related to RIP that is a component of both the TNFR-1 and the CD40 signaling complexes. Receptor interacting protein-2 (RIP2) contains an N-terminal domain with homology to Ser/Thr kinases and a C-terminal caspase activation and recruitment domain (CARD), a homophilic interaction motif that mediates the recruitment of caspase death proteases. Overexpression of RIPS signaled both NF-KB activation and cell death. Mutational analysis revealed the pro-apoptotic function of RIPS to be restricted to its C-terminal CARD domain, whereas the intact molecule was necessary for NF-KB activation. RIPS interacted with other members of the TNFR-1 signaling complex, including inhibitor of apoptosis protein cIAP1 and with members of the TNFR-associated factor (TRAF) family, specifically TRAF1, TRAF5, and TRAF6, but not with TRAF2, TRAF3, or TRAF4. These TRAF interactions mediate the recruitment of RIPS to receptor signaling complexes.
The expansive structural and functional range of the eukaryotic protein kinases is becoming increasingly more evident as new members of this family continue to be discovered. A classification scheme based on catalytic-domain sequence similarities groups together protein kinases that have similar overall structures, modes of regulation, and catalytic properties. This is particularly evident for the protein tyrosine kinases, many of which were first recognized during the past 2 years.
Protein phosphorylation is known to occur in Archaea. However, knowledge of phosphorylation in the third domain of life is rather scarce. Homology-based searches of archaeal genome sequences reveals the absence of two-component systems in crenarchaeal genomes but the presence of eukaryotic-like protein kinases and protein phosphatases. Here, the influence of the offered carbon source (glucose versus tryptone) on the phospho-proteome of Sulfolobus solfataricus P2 was studied by precursor acquisition independent from ion count (PAcIFIC). In comparison to previous phospho-proteome studies, a high number of phosphorylation sites (1318) located on 690 phospho-peptides from 540 unique phospho-proteins were detected, thus increasing the number of currently known archaeal phospho-proteins from 80 to 621. Furthermore, a 25.8/20.6/53.6 Ser/Thr/Tyr percentage ratio with an unexpectedly high predominance of tyrosine phosphorylation was detected. Phospho-proteins in most functional classes (21 out of 26 arCOGs) were identified, suggesting an important regulatory role in S. solfataricus. Focusing on the central carbohydrate metabolism in response to the offered carbon source, significant changes were observed. The observed complex phosphorylation pattern hints at an important physiological function of protein phosphorylation in control of the central carbohydrate metabolism, which might particularly operate in channeling carbon flux into the respective metabolic pathways.
RIOK3 was initially characterized as a homolog of Aspergillus nidulans sudD and showed down-regulation at the invasive front of malignant melanomas, but the molecular mechanism remains elusive. Here, we report that overexpression of RIOK3 inhibits TNFalpha-induced NF-kappaB activation, but down-regulation of endogenous RIOK3 expression by siRNA potentiates it. A yeast two-hybrid experiment revealed that RIOK3 interacted with caspase-10, and further, a GST pull-down assay and endogenous coimmunoprecipitation validated the interaction. We subsequently showed that the interaction was mediated by the RIO domain of RIOK3 and each death effector domain of caspase-10. Interestingly, our data demonstrated that RIOK3 suppressed caspase-10-mediated NF-kappaB activation by competing RIP1 and NIK to bind to caspase-10. Importantly, the kinase activity of RIOK3 was confirmed to be relevant to NF-kappaB signaling. Taken together, our findings strongly suggest that RIOK3 negatively regulates NF-kappaB signaling pathway activated by TNFalpha dependent on its kinase activity and NF-kappaB signaling pathway activated by caspase-10 independent of its kinase activity.
Ligation of the extracellular domain of the cell surface receptor Fas/APO-1 (CD95) elicits a characteristic programmed death response in susceptible cells. Using a genetic selection based on protein-protein interaction in yeast, we have identified two gene products that associate with the intracellular domain of Fas: Fas itself, and a novel 74 kDa protein we have named RIP, for receptor interacting protein. RIP also interacts weakly with the p55 tumor necrosis factor receptor (TNFR1) intracellular domain, but not with a mutant version of Fas corresponding to the murine lprcg mutation. RIP contains an N-terminal region with homology to protein kinases and a C-terminal region containing a cytoplasmic motif (death domain) present in the Fas and TNFR1 intracellular domains. Transient overexpression of RIP causes transfected cells to undergo the morphological changes characteristic of apoptosis. Taken together, these properties indicate that RIP is a novel form of apoptosis-inducing protein.
Ca2+/calmodulin-dependent protein kinase I (CaM kinase I) was previously purified from bovine brain (Nairn, A. C., and Greengard, P. (1987) J. Biol. Chem. 262, 7273-7281) based on its ability to phosphorylate the synaptic vesicle protein, synapsin I at site 1. The cDNA for this protein kinase has now been cloned from both a rat and a bovine brain cDNA library and the complete amino acid sequence of rat CaM kinase I determined. The rat cDNA encoded a protein of 331 amino acids with a calculated M(r) of 37,545, and the encoded kinase was expressed in bacteria as a glutathione S-transferase fusion protein. The resulting fusion protein was purified by Sepharose-CaM affinity chromatography and shown to be totally dependent on Ca2+ and CaM for activity. Furthermore, the purified kinase phosphorylates synapsin I at the same site (site 1) as the endogenous brain enzyme. CaM kinase I is homologous to other known protein kinases and contains all nine invariant amino acids conserved in the catalytic domain of this class of enzymes. CaM kinase I was most identical to CaM kinase II both in the catalytic domain and in a short region at the COOH-terminal that is predicted to be the calmodulin-binding domain. CaM kinase I appeared to be encoded by a single gene. RNase protection assays detected the mRNA encoding CaM kinase I in all tissues examined. High concentrations of the kinase mRNA were found in all regions of the brain with frontal cortex showing the greatest level. CaM kinase I was autophosphorylated in a Ca2+/CaM-dependent manner at a threonyl residue (Thr-177) which is located at a position equivalent to that of the threonyl residue (Thr-197) autophosphorylated in cAMP-dependent protein kinase.
Angiogenesis and synovial cell hyperplasia are characteristic features of rheumatoid arthritis (RA). Many growth and survival factors use receptors belonging to the tyrosine kinase family that share conserved motifs within the intracellular catalytic domains. To understand further the molecular basis of cellular hyperplasia in RA, we have used degenerate primers based on these motifs and RNA obtained from the synovium of a patient with RA to perform reverse transcriptase-polymerase chain reaction. We report detection of the receptor tyrosine kinase (RTK) Axl in RA synovium and we document the expression pattern of Axl in capillary endothelium, in vascular smooth muscle cells of arterioles and veins, and in a subset of synovial cells in RA synovial tissue. Gas6 (for growth arrest-specific gene 6), which is a ligand for Axl and is related to the coagulation factor protein S, was found in synovial fluid and tissue from patients with RA and osteoarthritis. Axl expression and function was studied in human umbilical vein endothelial cells (HUVECs). Gas6 bound to HUVECs; soluble Axl inhibited this binding. Exogenous Gas6 protected HUVECs from apoptosis in response to growth factor withdrawal and from TNFalpha-mediated cytotoxicity. These findings may reveal a new aspect of vascular physiology, which may also be relevant to formation and maintenance of the abnormal vasculature in the rheumatoid synovium.
RIP3 is a novel gene product containing a N-terminal kinase domain that shares extensive homology with the corresponding domain
in RIP (receptor-interacting protein) and RIP2. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and
recruitment domain, RIP3 has a unique C terminus. RIP3 binds RIP through its unique C-terminal segment and by virtue of this
interaction is recruited to the tumor necrosis factor (TNF) receptor-1 signaling complex. Previous studies have shown that
RIP mediates TNF-induced activation of the anti-apoptotic NF-κB pathway. RIP3, however, attenuates both RIP and TNF receptor-1-induced
NF-κB activation. Overexpression studies revealed RIP3 to be a potent inducer of apoptosis, capable of selectively binding
to large prodomain initiator caspases.
The rapid expansion of nucleotide sequence data available in public databases is revolutionizing biomedical research. These databases have a variety of uses, including the discovery of novel genes, identification of homologous genes, analysis of alternative splicing, chromosomal localization of genes, and detection of polymorphisms. Data sets such as the human transcript map will undoubtedly accelerate identification of candidate genes in positional-cloning approaches. Careful in silico analysis can significantly reduce the amount of lab work required. Approximately half of all human genes are represented in these databases; therefore, one need not wait for the entire human genome to be sequenced before performing genome-wide studies.
Identification of key regulatory kinases in the intestinal epithelium are useful to understand the molecular mechanisms that underlie proliferation and differentiation in cells found in this compartment. We used the polymerase chain reaction (PCR) to amplify the catalytic kinase domain of serine-threonine kinases by employing degenerate primers and then screened an intestinal crypt cDNA library to clone and sequence the open reading frame of a novel serine-threonine kinase. This was then further characterized by Northern blot analysis and RNA in situ hybridization. This kinase, designated intestinal cell kinase, harbors a dual phosphorylation site found in mitogen-activating protein (MAP) kinases that is important for kinase activity.
In bacteria, extracellular signals are generally transduced into cellular responses via a two-component system. However, genome sequence data have now revealed the presence of 'eukaryotic-like' protein kinases and phosphatases. Mycobacterium tuberculosis appears to be unique among bacteria in that its genome contains 11 members of a newly identified protein kinase family. These M. tuberculosis eukaryotic-like protein kinases could be key regulators of metabolic processes, including transcription, cell development and interactions with host cells.
Pathogenesis of Mycobacterium tuberculosis is closely connected to its survival and replication within the host. Some pathogenic bacteria employ protein kinases that interfere with the cellular signalling network of host cells and promote bacterial survival. In this study, the pknF and pknG genes, which encode two putative protein kinases of M. tuberculosis H(37)Rv, protein kinase F (PknF) and protein kinase G (PknG), respectively, were cloned and expressed in Escherichia coli. Purified PknF phosphorylated the peptide substrate myelin basic protein (MBP) at serine and threonine residues, while purified PknG phosphorylated only at serine residues. The activity of the two kinases was abrogated by mutation of the codon for the predicted ATP-binding-site lysine residue. Southern blot analysis revealed that homologues of the genes encoding the two kinases are present in M. tuberculosis H(37)Ra and Mycobacterium bovis BCG, but not in Mycobacterium smegmatis. Immunoblot analysis of various cellular fractions of M. tuberculosis H(37)Rv revealed that PknF is a transmembrane protein and that PknG is predominantly a cytosolic enzyme. The present study should aid in elucidating the role of these protein kinases in the pathogenesis of mycobacteria.
In this study, we report the cloning and characterization of a novel human Ste20-related kinase that we designated MST4. The 416 amino acid full-length MST4 contains an amino-terminal kinase domain, which is highly homologous to MST3 and SOK, and a unique carboxy-terminal domain. Northern blot analysis indicated that MST4 is highly expressed in placenta, thymus, and peripheral blood leukocytes. Wild-type but not kinase-dead MST4 can phosphorylate myelin basic protein in an in vitro kinase assay. MST4 specifically activates ERK but not JNK or p38 MAPK in transient transfected cells or in stable cell lines. Overexpression of dominant negative MEK1 or treatment with PD98059 abolishes MST4-induced ERK activity, whereas dominant-negative Ras or c-Raf-1 mutants failed to do so, indicating MST4 activates MEK1/ERK via a Ras/Raf-1 independent pathway. HeLa and Phoenix cell lines overexpressing wild-type, but not kinase-dead, MST4 exhibit increased growth rate and form aggressive soft-agar colonies. These phenotypes can be inhibited by PD98059. These results provide the first evidence that MST4 is biologically active in the activation of MEK/ERK pathway and in mediating cell growth and transformation.
Here we describe a differential display method for surveying the expression of most protein tyrosine kinases and applying it to cDNAs from human fetal and adult brains. The method involves two selective steps for processing the mRNA. At each step, degenerate oligonucleotide primers derived from highly conserved regions of the catalytic domain of the kinases are used. In the display with BstYI and BsiHKI digests of the cDNA, 65% and 59% of a total of 72 and 63 bands, respectively, represented fragments from a total of 27 different tyrosine kinases. The expression levels of the kinases in the display were comparable with those measured by RT-PCR. This method offers a relatively specific way to display differentially expressed gene families in any tissue and cell type.
Post-translational modifications of proteins control myriad biological functions. However, relatively few methods exist for the identification of the enzymes that catalyze these modifications. To expand this repertoire, we report a yeast genetic approach that enables the identification of protein tyrosine kinases (PTKs) from cDNA libraries. Yeasts were transformed with four vectors encoding: 1) a potentially universal PTK substrate fused to the LexA DNA binding domain, 2) the Grb2-SH2 domain fused to the B42 activation domain, 3) a fluorescent reporter gene controlled by LexA DNA sites, and 4) a Jurkat cDNA library. Transient expression of PTKs, such as the lymphocyte-specific kinase Fyn, resulted in phosphorylation of the DNA-bound substrate, recruitment of the Grb2-SH2 domain, and activation of the fluorescent reporter gene. This brief induction of protein expression circumvented the potential toxicity of PTKs to the yeast. Fluorescence activated cell sorting (FACS) enabled isolation of PTKs, and these enzymes were further characterized by flow cytometry and immunoblotting. This approach provides a potentially general method for the identification and evaluation of enzymes involved in the post-translational modification of proteins.
Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle whose activities are controlled by associated cyclins. PFTK1 shares limited homology to CDKs, but its ability to associate with any cyclins and its biological functions remain largely unknown. Here, we report the functional characterization of human PFTK1 as a CDK. PFTK1 specifically interacted with cyclin D3 (CCND3) and formed a ternary complex with the cell cycle inhibitor p21Cip1 in mammalian cells. We demonstrated that the kinase activity of PFTK1 depended on CCND3 and was negatively regulated by p21Cip1. Moreover, we identified the tumor suppressor Rb as a potential downstream substrate for the PFTK1/CCND3 complex. Importantly, knocking down PFTK1 expression by using siRNA caused cell cycle arrest at G1, whereas ectopic expression of PFTK1 promoted cell proliferation. Taken together, our data strongly suggest that PFTK1 acts as a CDK that regulates cell cycle progression and cell proliferation.
• cyclin D3
• p21
• retinoblastoma
• cell cycle
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