Biomedical subjects

R Jove

Publications and source records attributed to R Jove.

At least 73 records · Page 4Linked to original sources

PubMed1995

Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction.

Raf-1 is a serine/threonine protein kinase that transduces signals from cell surface receptors to the nucleus. Interaction of Ras with a regulatory domain in the N-terminal half of Raf-1 is postulated to regulate Raf-1 protein kinase and signaling activities. To better understand molecular interactions of Ras with Raf-1 and regulation of the Raf-1 kinase, a panel of Raf-1 N-terminal mutants expressed in the baculovirus-insect cell system was used for mapping the precise region necessary for Ras interaction in the context of full-length, functional Raf-1 kinase. An 80-amino-acid sequence in Raf-1 between positions 53 and 132 was found to confer the ability to bind Ras protein in vitro and in infected insect cells. Deletion of residues 53 to 132 abolished Raf-1 kinase activation by Ras in insect cells, indicating that activation of the Raf-1 kinase by Ras requires the capacity to physically interact with Ras. By contrast, deletion of this Ras-binding site did not diminish activation of Raf-1 kinase by Src, implying that Src and Ras can activate Raf-1 through independent mechanisms. Significantly, Raf-1 mutants lacking the entire zinc finger motif or containing substitutions of two critical cysteine residues in the zinc finger retained the ability to bind Ras and to be activated by this interaction. Consistent with results obtained in the baculovirus-insect cell system, deletion of residues 53 to 132 but not mutations in the zinc finger motif abrogated the ability of kinase-inactive, dominant negative Raf-1 to block Ras-mediated signaling in Xenopus oocytes. Together, these results provide evidence that the direct physical interaction of Ras with Raf-1 amino acids 53 to 132 is required for activation of the Raf-1 kinase and signaling activities by Ras but not by Src. Furthermore, the adjacent zinc finger motif in Raf-1 is not essential either for interaction with Ras or for activation of the Raf-1 kinase.

Animals↗

PubMed1995

Proximal promoter region of the junB gene mediates attenuation of serum inducibility in Src-transformed cells.

Transcription of the junB gene is rapidly and transiently induced following stimulation of susceptible cells by growth factors in serum. Our previous studies demonstrated that serum inducibility of junB and other immediate-early genes is markedly attenuated in fibroblasts chronically transformed by the viral Src oncoprotein. Moreover, attenuation of junB induction occurs at the transcriptional level. To characterize further the molecular mechanisms of this attenuation, various full-length and recombinant junB constructs were transfected into normal and viral Src-transformed rat fibroblasts. A stable transfection system was used to faithfully reproduce regulation of the junB gene. Analyses of pooled populations of stably transfected cells demonstrate that sequences between -89 and +32 (relative to the transcriptional start site) are sufficient to confer both serum inducibility of the junB gene in normal cells and its attenuation in viral Src-transformed cells. By contrast, attenuation of c-fos serum inducibility by Src transformation involves element(s) distinct from regulatory elements identified previously in the c-fos promoter. These results identify a proximal promoter region of the junB gene that is involved in a novel negative regulation of its transcription.

Animals↗

PubMed1994

Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.

Amino Acid Sequence↗

PubMed1994

The native v-Raf.hsp90.p50 heterocomplex contains a novel immunophilin of the FK506 binding class.

We have recently reported that v-Raf forms a native heterocomplex with rat heat shock protein (hsp) 90 and a 50-kDa phosphoprotein (p50) in stably transfected 3Y1 fibroblasts (Stancato, L. F., Chow, Y-H., Hutchison, K. A., Perdew, G. H., Jove, R., and Pratt, W. B. (1993) J. Biol. Chem. 268, 21711-21716). Several members of the nuclear receptor family exist in heterocomplexes containing hsp90 and various members of the immunophilin protein family, including hsp56, an immunophilin of the FK506 binding class (Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458). In this work, we have asked if Raf is also associated with an immunophilin. We have immunoadsorbed v-Raf from stably transfected rat 3Y1 fibroblasts and show that the immunoadsorbed v-Raf.hsp90.p50 heterocomplex binds the immunosuppressant drug [3H]FK506. The binding is of high affinity (KD 82 nM) and specific in the sense that it is competed by nonradioactive FK506 and rapamycin but not by cyclosporin A. The [3H]FK506 binding activity is eliminated when the heterocomplex proteins are dissociated from v-Raf. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for the immunophilin association. We have shown that hsp90 and p50 do not bind FK506, and the v-Raf heterocomplex does not contain any of the established FK506-binding proteins. Thus, we propose that Raf is associated with a novel immunophilin.

3T3 Cells↗

PubMed1993

Tyrosine phosphorylation of Ras GTPase-activating protein stabilizes its association with p62 at membranes of v-Src transformed cells.

Ras GTPase-activating protein (GAP) regulates the activity of Ras proteins, which have key roles in signal transduction pathways downstream of oncogenic and receptor tyrosine kinases. Previous studies indicated that Tyr-457 of bovine GAP (Tyr-460 of human GAP) is the major site of phosphorylation by viral Src (v-Src) kinase and epidermal growth factor receptor. The finding that Tyr-457 in GAP is located immediately adjacent to Src homology 2 (SH2) and 3 (SH3) domains led us to investigate the possibility that this specific phosphorylation regulates protein-protein interactions involving GAP. For this purpose, we constructed a full-length GAP mutant containing a substitution of Phe-457 in place of Tyr-457. Both wild-type GAP and mutant GAPF457 were tagged with the KT3 epitope at the carboxyl terminus and were expressed in v-Src transformed rat fibroblasts. In vivo phosphorylation analyses established that GAPF457 was weakly phosphorylated on tyrosine and, as expected, lacked the phosphopeptide containing Tyr-457. Analysis of GAP-associated proteins in anti-KT3 immunoprecipitates showed that GAP stably associated with two major phosphoproteins, p62 and p190, which have been previously described. Significantly, association of p62 with GAPF457 was reduced approximately 3-fold compared with wild-type GAP. Subcellular fractionation experiments further demonstrated that Tyr-457 phosphorylation of GAP stabilized its association with p62 at cell membranes. Based on these findings, we propose that one role of tyrosine phosphorylation in GAP is to enhance its association with p62 at membranes, which in turn may contribute to regulation of Ras signal transduction pathways.

Amino Acid Sequence↗

PubMed1993

Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system.

Recently, we have demonstrated that the tyrosine kinase pp60v-src can undergo cell-free assembly into a heterocomplex with rabbit hsp90 and p50 when the immunoadsorbed protein is incubated with rabbit reticulocyte lysate (Hutchison, K. A., Brott, B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem 267, 2902-2908). Using a baculovirus system to express a high level of human c-Raf serine/threonine kinase in Sf9 insect cells, we show here that immunoadsorbed c-Raf undergoes similar lysate-mediated assembly into a heterocomplex with hsp90 and p50. As with pp60v-src and steroid receptors, binding of c-Raf to hsp90 occurs in an ATP-dependent and K(+)-dependent manner and the resulting heterocomplex is stabilized by molybdate. With a very rapid and gentle procedure of Sf9 cell cytosol preparation and c-Raf immunoadsorption, we show coimmunoadsorption of the insect homologue of hsp90. The same procedures permit detection of a native complex of v-Raf with rat hsp90 and p50 in stably transfected rat 3Y1 fibroblasts, and v-Raf is also assembled into a heterocomplex with rabbit hsp90 and p50 by reticulocyte lysate. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for both formation of the native heterocomplex in mouse NIH 3T3 cells and cell-free reconstitution of the heterocomplex by rabbit reticulocyte lysate. Although the native Raf-heat shock protein heterocomplex is less stable than native pp60v-src and glucocorticoid receptor heterocomplexes, by analogy with these proteins its detection may have important implications regarding the mechanism of Raf trafficking through the cytoplasm.

3T3 Cells↗

PubMed1993

Regulation of glucocorticoid receptor function through assembly of a receptor-heat shock protein complex.

Incubation of immunopurified, hormone-free mouse glucocorticoid receptors with rabbit reticulocyte lysate results in ATP-dependent and monovalent cation-dependent assembly of the GR into a heterocomplex with hsp90, hsp70, and hsp56. Heterocomplex assembly is accompanied by conversion of the receptor from a form that does not bind steroid to a high affinity steroid-binding conformation. Reticulocyte lysate also promotes ATP-dependent dissociation of unliganded receptors from a prebound receptor-DNA complex. Receptor released from DNA has been reconstituted into the heat shock protein heterocomplex and converted to the non-DNA-binding state. The reticulocyte lysate also reconstitutes pp60v-src into a heterocomplex containing hsp90 and p50, both of which are components of the native heterocomplex form of the tyrosine kinase in cytoplasm. Although the c-Raf-1 serine/threonine kinase has never been found in native association with hsp90, it can be assembled into a heat shock protein heterocomplex by the ATP-dependent system in reticulocyte lysate.

Animals↗

PubMed1993

Neurofibromatosis type 1 gene product (neurofibromin) associates with microtubules.

The neurofibromatosis type 1 (NF1) gene was recently identified by positional cloning and found to encode a protein with structural and functional homology to mammalian and yeast GTPase-activating proteins (GAPs). Using antibodies directed against the NF1 gene product, a protein of approximately 250 kDa was identified and termed neurofibromin. Double-indirect immunofluorescent labeling with anti-neurofibromin and anti-tubulin antibodies demonstrates that neurofibromin associates with cytoplasmic microtubules. Immunoblotting of microtubule-enriched cytoplasmic fractions, using antibodies generated against neurofibromin, shows that neurofibromin copurifies with microtubules. When portions of neurofibromin are expressed in Sf9 insect cells they associate with polymerized microtubules; furthermore, the critical residues for this interaction reside within the GAP-related domain of neurofibromin. The unexpected association of neurofibromin with microtubules suggests that neurofibromin is involved in microtubule-mediated intracellular signal transduction pathways.

3T3 Cells↗

PubMed1993

Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways.

Immediate early genes involved in controlling cell proliferation are rapidly and transiently induced following stimulation of susceptible cells with serum. To study how oncoproteins regulate immediate early genes, we examined serum inducibility of these genes in cells transformed by various oncoproteins. We found that induction of the immediate early gene, c-fos, by serum stimulation was markedly attenuated in four independent cell lines stably transformed by the v-Src tyrosine kinase. Cells chronically transformed by other oncoproteins implicated in tyrosine kinase signaling pathways, including v-Sis, v-Ras, and v-Raf, showed the same pattern of attenuation. In contrast, serum inducibility of c-fos was not attenuated in cells transformed by simian virus 40, which is thought to transform cells through a different pathway. Cell cycle analyses showed that proliferation of these transformed cell lines could be arrested effectively in 0.1% serum, demonstrating that the attenuation was not simply due to continuous cycling of transformed cells after serum deprivation. Moreover, serum inducibility of other immediate early genes, including c-jun, junB, egr-1, and NGFI-B, also was strikingly attenuated by these same oncoproteins. Nuclear run-on transcription assays established that this attenuation of serum inducibility occurred at the transcriptional level. Finally, flow cytometric analysis demonstrated that serum-starved v-Src-transformed cells were viable and able to progress into S phase of the cell cycle after serum stimulation, even though the induction of immediate early genes was greatly attenuated in these cells. Our results suggest that activation of immediate early genes is repressed by chronic stimulation of tyrosine kinase signaling pathways in transformed cells.

Animals↗

PubMed1992

Activated Src tyrosine kinase phosphorylates Tyr-457 of bovine GTPase-activating protein (GAP) in vitro and the corresponding residue of rat GAP in vivo.

GTPase-activating protein (GAP) is a key regulator of the cellular Ras protein, which is implicated in oncogenic signal transduction pathways downstream of the viral Src (v-Src) kinase. Previous studies demonstrated that v-Src induces tyrosine phosphorylation of GAP, suggesting that GAP may provide a biochemical link between v-Src and Ras signaling pathways. To determine the precise residues in GAP phosphorylated by Src kinases, we used a baculovirus/insect cell expression system for investigating in vitro phosphorylation of GAP. Phosphopeptide mapping analysis revealed that v-Src and normal cellular Src (c-Src) phosphorylate tyrosine residues in bovine GAP at one major site and one minor site in vitro. Significantly, the major site of GAP phosphorylation in vitro is also the major site of in vivo tyrosine phosphorylation of GAP in rat fibroblasts transformed by v-Src. Analyses of GAP deletion mutants and TrpE-GAP fusion proteins established that Tyr-457 of bovine GAP (and the corresponding residue of rat and human GAP) is the major site of tyrosine phosphorylation. Our results demonstrate that the v-Src kinase induces phosphorylation of the same tyrosine residue of GAP in vitro and in vivo, suggesting that GAP is a direct substrate of activated Src kinases in vivo. Because epidermal growth factor receptor phosphorylates the equivalent tyrosine residue in human GAP (Tyr-460), these findings are consistent with the hypothesis that specific phosphorylation of GAP at this site may have a physiologically important role in regulating mitogenic Ras signaling pathways.

Amino Acid Sequence↗

PubMed1992

The protein-protein complex between pp60v-src and hsp90 is stabilized by molybdate, vanadate, tungstate, and an endogenous cytosolic metal.

In a recent study demonstrating the cell-free reconstitution of the pp60v-src-hsp90 complex, we found that the tyrosine kinase-hsp90 complex is stabilized by molybdate (Hutchison, K. A., Brott, B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem. 267, 2902-2908). In this paper, we examine in detail the stabilization of this protein-protein interaction by transition metal oxyanions. The pp60v-src-hsp90 complex is stabilized by sodium molybdate with the same concentration dependence as the glucocorticoid receptor-hsp90 complex. As with the steroid receptor heterocomplexes, vanadate and tungstate also stabilize the pp60v-src-hsp90 interaction. Passage of cytosol through a Chelex-100 metal-chelating resin destabilizes the native pp60v-src-hsp90 complex, suggesting that the complex is normally stabilized by an endogenous metal factor. Readdition of either the heat-stable components of cytosol or a partially purified endogenous metal factor stabilizes the metal-depleted complex. Molybdate also stabilizes the presence of p50, a known hsp90-associated protein, in the pp60v-src heterocomplex. Given the identical effects of transition metal oxyanions on both pp60v-src- and steroid receptor-hsp90 complexes and the lack of any sequence homology between pp60v-src and the receptors, it seems very likely that it is the common component, hsp90, that contains the site of the metal interaction.

Animals↗

PubMed1992

Reconstitution of interactions between the Src tyrosine kinases and Ras GTPase-activating protein using a baculovirus expression system.

Ras GTPase-activating protein (GAP) has been implicated in mitogenic signal transduction downstream of oncogenic and receptor tyrosine kinases. Previous studies have suggested that GAP is phosphorylated by oncogenic viral Src (v-Src) and that GAP is associated with a complex containing normal cellular Src (c-Src) in vertebrate fibroblasts. To investigate molecular interactions between the Src kinases and GAP, we developed an in vitro system for reconstituting Src-GAP complexes. For this purpose, we constructed recombinant baculovirus vectors that direct expression of Rous sarcoma virus v-Src, chicken c-Src, and bovine GAP in infected Sf9 insect cells. In vitro reconstitution experiments using baculovirus-expressed proteins demonstrate that both v-Src and c-Src associate in complexes with GAP. In addition, in vitro and in vivo phosphorylation analyses indicate that GAP serves as a substrate for both the v-Src and c-Src tyrosine kinases. To determine which structural features of GAP are involved in interactions with the Src kinases, we constructed recombinant baculoviruses that encode deletion mutants of bovine GAP. Deletion of the GAP amino-terminal portion containing Src homology 2 regions, which are highly conserved structural motifs postulated to mediate interactions among proteins, diminishes GAP phosphorylation and association with Src. This reconstitution system should facilitate further studies of molecular interactions between the Src kinases and GAP.

Baculoviridae↗

PubMed1992

Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.

A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein (hsp90) has been used here to form complexes between the pp60src tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src, which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60v-src, of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60c-src, which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90. Moreover, the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60v-src multiprotein complex. The native and reconstituted pp60src-hsp90 complexes have similar thermal stability and, like steroid receptor heterocomplexes, they are stabilized by molybdate. As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes, the reconstituted pp60src multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.

Animals↗

PubMed1992

Regulation of the junB gene by v-src.

The proteins encoded by cellular and viral src genes are believed to be involved in the transmission of mitogenic signals, the nuclear recipients of which are largely unknown. In this work, we report that four different v-src-transformed cell lines from three different species possess elevated levels of junB transcripts. Transient expression of junB promoter-chloramphenicol acetyltransferase constructs in NIH 3T3 cells was used to demonstrate that the increase in junB transcripts was specifically associated with v-src expression and could not be recapitulated with a c-src, v-H-ras, or v-raf expression vector. Deletion mutants were used to localize the v-src-responsive region in the junB promoter to a 121-nucleotide region encompassing the CCAAT and TATAA elements. This region is distinct from one in the 5' untranslated region of the junB gene which is required to maintain its high-level basal expression. Point mutagenesis of the junB TATAA box completely abolished v-src responsiveness, suggesting that proteins which bind to this element are modified by src transformation. Several v-src and c-src mutants were used to demonstrate that elevated tyrosine kinase activity of src proteins is required for the observed effects on junB expression. Finally, homology between the TATAA box regions of junB and the unrelated but src-responsive gene 9E3/CEF-4 suggests that modulation of gene activity through proteins which bind to this region may be a recurrent, although not exclusive, theme in src transforming action. Our results suggest that src proteins may modulate some nuclear effectors through pathways not involving cellular ras or raf gene products.

3T3 Cells↗

PubMed1992

Localization of the viral and cellular Src kinases to perinuclear vesicles in fibroblasts.

Previous studies have shown that the viral oncogene product, v-Src, is localized to a perinuclear structure. Here, we demonstrate by immunofluorescence analysis that the perinuclear structure corresponds to a local concentration of vesicles. Overexpressed normal cellular Src, c-Src, and a temperature-sensitive mutant of v-Src also are associated with these perinuclear vesicles. This perinuclear localization is observed in a variety of cell types using several different antibodies to Src, and it is independent of the fixation method. Immunoelectron ultracryomicroscopic analysis of rat fibroblast cells transformed by v-Src demonstrates an association of this protein with the limiting membranes of vesicles concentrated in the perinuclear region. These vesicles appear at the electron microscopic level to be multivesicular bodies on the basis of their size (0.3-1.0 microns in diameter), large electron-transparent lumens, and electron-dense vesicular inclusions. Morphometric analysis indicates that approximately 20% of the total cell v-Src protein is associated with these structures. This subpopulation of v-Src may have been recovered from the plasma membrane via the endocytotic pathway in a manner analogous to endocytosis of the epidermal growth factor receptor. Localization of the Src tyrosine kinases to these perinuclear endocytotic vesicles may be necessary for oncogenic transformation by v-Src and for normal functions of c-Src.

Animals↗

PubMed1991

GTPase-activating protein interactions with the viral and cellular Src kinases.

GTPase-activating protein (GAP), which regulates the activities of Ras proteins, is implicated in mitogenic signal transduction by growth-factor receptors and oncoproteins with tyrosine kinase activity. Oncogenic viral Src (p60v-src) encoded in Rous sarcoma virus possesses elevated tyrosine kinase activity compared with its nononcogenic normal homolog, cellular Src (p60c-src). To examine molecular interactions between GAP and the two Src kinases, immunoprecipitates of Src or GAP prepared from cell lystates were resolved by gel electrophoresis and analyzed by an immunoblot procedure with antibodies to GAP or Src used as probes. Results suggest that p60c-src is associated with a complex containing GAP in immunoprecipitates from lysates of normal rat and chicken cells. However, GAP is not phosphorylated in p60c-src immunoprecipitates subjected to in vitro kinase reactions. By contrast, GAP undergoes tyrosyl phosphorylation in vitro when immunoprecipitates of p60v-src prepared from transformed cell lysates are incubated with ATP. Our findings suggest that p60v-src and p60c-src associate with complexes containing GAP and provide a biochemical link between both kinases and GAP/Ras signal transduction pathways. These results are consistent with the hypothesis that GAP has a role in mediating normal functions of p60c-src as well as oncogenic activities of p60v-src.

Animals↗

PubMed1991

Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein.

GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.

Animals↗

PubMed1989

In vivo phosphorylation states and kinase activities of transforming p60c-src mutants.

To further investigate regulation of p60src by tyrosine phosphorylation, the in vivo phosphorylation states and kinase activities of transforming mutants derived from p60c-src were examined. One set of six in vitro recombinants encoded chimeric proteins containing the carboxyl terminus of chicken p60c-src (including Tyr 527) in combination with various isolated amino acid substitutions present in p60v-src. Another set of seven viral isolates were randomly selected from soft-agar colonies infected with replicating retroviruses that originally encoded normal p60c-src. Results show that mutations at multiple sites throughout amino- and carboxy-terminal domains are sufficient to alter the in vivo phosphorylation state and kinase activity of p60c-src. Moreover, all of the transforming p60c-src mutants exhibited elevated in vivo kinase activities, as assayed by immune blot analysis of cellular proteins using antibodies to phosphotyrosine, which correlated well with enhanced auto-phosphorylation at Tyr 416. By contrast, Tyr 527 phosphorylation in the transforming mutants varied from low to high levels that were correlated to a limited extent with levels of in vivo kinase activities. Taken together, results suggest that, in addition to complete dephosphorylation of Tyr 527, other mechanisms contribute to p60c-src activation.

Animals↗