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Specific kinase activities of r-src proteins. The specific activities of pp60"' and pp6or-"(r proteins for autophosphorylation and enolase phosphorylation were determined with protein bound with monoclonal antibody 327 (29). Cells were labeled with 100 ,uCi
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Analysis of the biological and biochemical activities of pp60recombinant-src proteins encoded by 12 carboxyl-terminal mutants
showed that a wide family of alternate src carboxyl termini permit complete transforming and kinase activities. src proteins
having carboxyl termini which are up to 10 amino acids longer than that of pp60c-src (17 amino acid...
Citations
... Auto-phosphorylation of Y416 stimulates kinase activity, while phosphorylation of Y527 inhibits kinase activity (Yaciuk and Shalloway, 1986;Gould and Hunter, 1988;Okada and Nakagawa, 1989;Cary et al., 2002;Sun et al., 2002). The result is a restrained form of the enzyme (closed conformation) (Figure 1.1 C) (Kefalas et al., 1995). ...
Src is a 60 kDa tyrosine kinase that is expressed in most of animal tissues. Src has three
splice variants, C-src, which is ubiquitously expressed, and N1- and N2-src, which are
neuronal specific splice variants. The srcs are differentially spliced at their SH3 domains,
therefore the hypothesis is that this splicing allows them to have different binding
partners and perform different roles in neurons. The aim of this project is to identify new
interactions for the three src splice variants in neurons and their possible functional roles.
The SH3 domains, kinase active truncated proteins ( 80) and kinase dead mutant full
length versions of the three splice variants of src were cloned from a rat brain cDNA
library into bacterial expression vectors. GST-pull downs from nerve terminal lysates
showed that different src splice variants had different binding partners. These partners
were identified by mass spectrometry and confirmed by western blotting. C-src binding
partners included dynamin, synapsin, and synaptojanin, while N2-src binding partners
included synaptophysin, Munc18-1, and NSF. The interaction between N2-src and Munc
18-1 was characterized further; however a number of in vitro interaction assays and
kinase assays showed that Munc 18-1 may not be a direct binding partner for N2-src or
substrate.
N1-src displayed a stimulation-dependent interaction with dynamin I. This was shown to
be phosphorylation-dependent in contrast to C-src binding. The major phosphorylation
sites on dynamin I, S774 and S778, were not involved in the regulation of N1-src
binding. The binding site for N1-src on dynamin I was different to C-src, with extensive
mutagenesis studies suggesting that the interaction site is at the tail of the dynamin I xa
splice variant, which has an additional two phosphorylation sites.
... In v-src the last aa are replaced by a tail of 12 aa generated as a result of recombination events between the 3P end of c-src and 39-bp sequence from the downstream noncoding region [5]. The changes at the carboxy-terminal aa of v-src do not a¡ect its kinase activity or cell transformation [6]. The function of this region is unclear. ...
The significant differences in the metastatic properties of hamster fibroblasts transformed by the Rous sarcoma virus (RSV) were associated with mutations in the v-src carboxy-terminal region. To identify the capacity of this region for protein-protein interaction the two-hybrid system was used. The cDNA clone (vseap1), producing the protein specifically bound with the v-src C-terminal part in yeast cells in vivo and in GST-fusion system in vitro was isolated. Vseap1 shared 68% of homology with stressful agents induced RNA-gadd7/adapt15. Two vseap1 specific messenger RNAs were identified: 0.9-kbp RNA expressed in all transformed cells and three times less in embryo fibroblasts; 3.1-kbp transcript was deleted in the cells with suppressed v-src activity and H2O2 resistance.
... These 19 residues of c-Src (515-533) are replaced with 12 novel amino acids in v-Src during retroviral capture (Takeya and Hanafusa, 1983) and this results in the ability of v-Src to transform fibroblasts (Jove and Hanafusa, 1987) and an elevated basal level of kinase activity compared to c-Src (Coussens et al., 1985). This suggested that the carboxy-terminus of Src was important for the regulation of its activity and studies on chimeric c-srdv-src genes and mutated v-src genes indicated that it was the absence of the carboxy-terminus of c-Src rather than the presence of the carboxy-terminus of v-Src, that was important for transformation (Iba et al., 1984;Yaciuk and Shalloway, 1986). It is now known that a critical tyrosine residue at position 527 in c-Src (absent from v-Src) is a major phosphorylation site , important for kinase repression. ...
Receptor-mediated signal transduction requires the assembly of multimeric complexes of signalling proteins. A number of conserved protein domains, such as the SH2, SH3 and PH domains, are involved in mediating specific protein-protein interactions in such complexes. The identification of binding partners for these domains has added considerably to our understanding of signalling pathways and the aim of this work was to identify such binding proteins for the c-Src family of cytoplasmic protein tyrosine kinases, in haematopoietic cells. Affinity chromatography experiments were performed using domains from the c-Src family members as fusion proteins with glutathione S-transferase, to search for SH2 and SH3 binding proteins in the human monoblastic leukaemic cell line, U937. Protein microsequencing identified one of the SH3 binding proteins as WASP, the protein defective in Wiskott-Aldrich syndrome (WAS) and isolated X-linked thrombocytopenia. WAS is an X-linked recessive condition characterised by severe eczema, recurrent infections, thrombocytopenia, impaired humoral and cellular immunity and increased susceptibility to lymphoid malignancies. WASP bound preferentially in vitro to SH3 domains from c-Src family kinases and its expression was restricted to cells of haematopoietic lineages. WASP and various protein kinases were expressed in insect cells using the baculovirus expression system, and co-expression experiments demonstrated a specific interaction between WASP and Fyn in insect cells. A WASP-Fyn complex was also observed in U937 cells. Additional biochemical studies on the WASP-Fyn interaction revealed that WASP is tyrosine phosphorylated when co-expressed with Fyn in insect cells and under these conditions WASP could stimulate the tyrosine kinase activity of Fyn. In vivo phosphorylation of WASP was also observed in a human T-cell line, and data from U937 cells engineered to overexpress WASP, suggest that WASP may stimulate tyrosine kinase activity in the cell. Finally, analysis of the subcellular localisation of WASP in U937 cells using immunocytochemistry with confocal microscopy, showed co-localisation of WASP with Fyn and [alpha]-tubulin. Haematopoietic cells from individuals with WAS exhibit defects in cell morphology and signal transduction. Members of the c-Src family of protein tyrosine kinases, including Fyn are involved in a range of signalling pathways, including those regulating cytoskeletal structure. These data suggest that binding of Fyn to WASP may be a critical event in such signalling pathways in haematopoietic cells.
... Research Center viral collection . amino acids of v-src do not affect the kinase activity HET-SR was low metastatic, whereas HET-SR-1 and or cell transformation (Yaciuk and Shalloway, 1986), our HET-SR-8 are highly metastatic , results suggest that it is likely that the differences be-1992). All cells were maintained in Dulbecco's modified tween v-srcHM and v-srcLM in this site are responsible Eagle's medium supplemented with 5% fetal calf serum, for the differences in the metastatic activity. ...
Four different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70-200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). All cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-srcprotein in cell lines was not correlated with their metastatic potentialin vivo.Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-srcisoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and v-srcLM: 62 kDa. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-srcsequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the oncoprotein. Both v-srcvariants and chimeric v-srcwith mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretina cells were associated with the mutations in the carboxy-terminal region of the v-srconcogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM.
... First, as described above, deletion of the region C terminal to this residue does not result in loss of enzyme activity. However, truncation at Leu-516 or preceding residues abolishes both kinase activity and transformation ability (49,50). Moreover, Leu-516 is conserved in all Src family members as well as in all other protein-tyrosine kinases (in a few instances, a similar hydrophobic residue is present at the analogous position) (16). ...
Repression of the tyrosine kinase activity of the cellular src protein (pp60c-src) depends on the phosphorylation of a tyrosine
residue (Tyr-527) near the carboxy terminus. Tyr-527 is located 11 residues C terminal from the genetically defined end of
the kinase domain (Leu-516) and is therefore in a negative regulatory region. Because the precise sequence of amino acids
surrounding Tyr-527 appears to be unimportant for regulation, we hypothesized that the conformational constraints induced
by phosphorylated Tyr-527 may require the correct spacing between the kinase domain (Leu-516) and Tyr-527. In this report,
we show that deletions at residue 518 of two, four, or seven amino acids or insertions at this residue of two or four amino
acids activated the kinase activity and thus the transforming potential of pp60c-src. As is the case for the prototype transforming
variant, pp60527F, activation caused by these deletions or insertions was abolished when Tyr-416 (the autophosphorylation
site) was changed to phenylalanine. In comparison with wild-type pp60c-src, the src proteins containing the alterations at
residue 518 showed a lower phosphorylation state at Tyr-527 regardless of whether residue 416 was a tyrosine or a phenylalanine.
Mechanisms dealing with the importance of spacing between the kinase domain and Tyr-527 are discussed.
... This region was chosen because it does not include any amino acids required for activity by analogy with other PTKs and is therefore from a region unique to eck. The leucine at position 871 corresponds to the last residue believed to be required for activity (31). eck was immunoprecipitated from 3"S-labeled A431 cells and was Anti-eck antiserum was used to immunoprecipitate eck from A431 cell lysates as described in Materials and Methods. ...
A human epithelial (HeLa) cDNA library was screened with degenerate oligonucleotides designed to hybridize to highly conserved regions of protein-tyrosine kinases. One cDNA from this screen was shown to contain a putative protein-tyrosine kinase catalytic domain and subsequently used to isolate another cDNA from a human keratinocyte library that encompasses the entire coding region of a 976-amino-acid polypeptide. The predicted protein has an external domain of 534 amino acids with a presumptive N-terminal signal peptide, a transmembrane domain, and a cytoplasmic domain of 418 amino acids that includes a canonical protein-tyrosine kinase catalytic domain. Molecular phylogeny indicates that this protein kinase is closely related to eph and elk and that this receptor family is more closely related to the non-receptor protein-tyrosine kinase families than to other receptor protein-tyrosine kinases. Antibodies raised against a TrpE fusion protein immunoprecipitated a 130-kDa protein that became phosphorylated on tyrosine in immune complex kinase assays, indicating that this protein is a bona fide protein-tyrosine kinase. Analysis of RNA from 13 adult rat organs showed that the eck gene is expressed most highly in tissues that contain a high proportion of epithelial cells, e.g., skin, intestine, lung, and ovary. Several cell lines of epithelial origin were found to express the eck protein kinase at the protein and RNA levels. Immunohistochemical analysis of several rat organs also showed staining in epithelial cells. These observations prompted us to name this protein kinase eck, for epithelial cell kinase.
... The V3, V4, and V5 fragments of Recombinations were made at either BglI (codon 431) or PstI (codon 515) sites. psrcll (35), pMvsrc, and pMcsrc (9), pRvsrcA, pRcsrc, pRS21, pRPB5, and pMBB4 (26) have been described. Fragments from these plasmids were cross-ligated to create the other plasmids. ...
We show that Schmidt-Ruppin D pp60v-src kinase activity is reduced by a mutation previously shown to be associated with Schmidt-Ruppin A pp60v-src temperature sensitivity and that its reduced transforming activity is associated with a conformational change in the SH3 region. The evolutionary relationship of seven v-src strains was studied by using parsimony analysis.
... In contrast, the 28-kDa fragment was not precipitated by aZ (Fig. 3C) and seemed to be derived from the 29.5-kDa fragment by further digestion (Fig. 4), suggesting that it lacked the extreme carboxy terminus. Since the sequence specificity of pronase E is broad, it is not clear where the carboxy-proximal cleavage may lie, but the size difference of 1.5 kDa allows tentative placement of a pronase-sensitive site close to Leu-516, the last residue in the kinase domain (Fig. 3D) (39, 52, 54). Digestion with papain or proteinase K gave a subset of the fragments created by pronase E or thermolysin: mostly small fragments from both p60WT and p6OF527, with low yields of a 28-kDa fragment (Fig. 2). ...
The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding
of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms
of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there
are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src.
The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is
not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the
kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus,
and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and
suggest functions for the amino-terminal region.
... It seems reasonable that the protein binding site is located on the carboxylterminal end of the ATP-binding domain because the amino-terminus of the calmodulin-dependent protein kinase II is within 12 residues of the glycine triad [5]. The carboxyl-terminal boundary for the protein substrate-binding region is likely to be Arg 583 in PK-C because this is the most distal residue conserved in all protein kinases [5] and truncations near this residue in either the myosin light chain kinase [8] or pp60 src [9] retain their capacity to bind and phosphorylate substrates. Thus the ATP-binding and protein substratebinding properties for all protein kinases would be expected in a 240 amino acid segment between the June 1989 glycine triad and the distal conserved Arg. ...
A 29-residue synthetic peptide, Leu530-Leu-Tyr-Glu-Met-Leu-Ala-Gly-Gln-Ala-Pro-Phe-Glu-Gly-Glu-Asp -Glu-Asp- Glu-Leu-Phe-Gln-Ser-Ile-Met-Glu-His-Asn-Val-NH2(558), corresponding to part of the catalytic domain of protein kinase C, is a potent activator of the enzyme, with a Ka of approx. 10 microM. Activation was 59 +/- 4% of that observed with phosphatidylserine, predominantly due to an increased Vmax, partially calcium-dependent, observed with all three isoenzymes (alpha, beta, gamma), and resulted in autophosphorylation. It is proposed that the region between Gly528 and Arg583 is part of the protein substrate binding region of protein kinase C and synthetic peptide analogs of this region activate the enzyme by blocking the action of the enzyme's basic pseudosubstrate autoregulatory region.
... The reduced kinase activity of pp60c-s/c(Ams17) relative to pp60'-"'(F527) may have resulted from encroachment of the An577 mutation near a conserved domain required for protein stability and catalytic activity. Premature termination of pp60u-"" translation preceding Leu 516, the furthest downstream of the residues which are conserved between pp60'-"" and pp60"-"", has previously been shown to decrease in vivo protein stability and transforming activity; truncation just past Leu 516 was shown to cause a similar partial decrease in pp60"-"' kinase activity (Yaciuk and Shalloway, 1986). Cartwright et al. (7987) (the only prior report describing the kinase activity of a pp60'-"rc(4m577) carboxyl terminal mutant), have previously re-ported that pp60'-"rc(Ams17) has increased biological activity but not increased specific kinase activity. ...
pp60c-src kinase and transforming activities are negatively regulated by phosphorylation of Tyr 527, a residue 6 amino acids from its carboxyl terminus. Tyr 527 to Phe mutation has been shown to activate pp60c-src, yet pp60c-src(F527) is still less active than pp60v-src. To see if additional carboxyl terminal mutation can stimulate pp60c-src transforming activity to pp60v-src levels, we compared the properties of pp60c-src(Am517), a pp60c-src mutant in which the 17 carboxyl terminal amino acids were deleted, with those of pp60c-src(F527) and pp60c-src(F519), a protein in which the Tyr nearest to Tyr 527 was changed to Phe. Tyr 519 to Phe mutation did not affect pp60c-src activities while the Am517 mutation activated focus formation, growth in soft agarose, in vivo tumorigenicity and in vitro specific kinase activity to levels between those of pp60c-src and pp60c-src(F527). This contrasts with a previous study [Cartwright et al. (1987) Cell 49, 83-91] which reported that Am517 mutation enhances biological activities without enhanced kinase activity. These data support the hypotheses that (1) complete transformation by pp60c-src requires activation of its protein tyrosine kinase activity and (2) that downregulation by the pp60c-src carboxyl terminus is governed by phosphorylation of Tyr 527; additional changes beyond that needed to prevent this phosphorylation do not further enhance activity.
