Autophosphorylation of the Fes tyrosine kinase. Evidence for an intermolecular mechanism involving two kinase domain tyrosine residues

The human c-fes proto-oncogene encodes a cytoplasmic tyrosine kinase (Fes) that is associated with multiple hematopoietic cytokine receptors. Fes tyrosine autophosphorylation sites may regulate kinase activity and recruit downstream signaling proteins with SH2 domains. To localize the Fes autophosphorylation sites, full-length Fes and deletion mutants lacking either the unique N-terminal or SH2 domain were autophosphorylated in vitro and analyzed by CNBr cleavage. Identical phosphopeptides of 10 and 4 kDa were produced with all three proteins, localizing the tyrosine autophosphorylation sites to the C-terminal kinase domain. Substitution of kinase domain tyrosine residues 713 or 811 with phenylalanine resulted in a loss of the 10- and 4-kDa phosphopeptides, respectively, identifying these tyrosines as in vitro autophosphorylation sites. CNBr cleavage analysis of Fes isolated from 32PO4-labeled 293T cells showed that Tyr-713 and Tyr-811 are also autophosphorylated in vivo. Mutagenesis of Tyr-713 reduced both autophosphorylation of Tyr-811 and transphosphorylation of Bcr, a recently identified Fes substrate, supporting a major regulatory role for Tyr-713. Wild-type Fes transphosphorylated a kinase-inactive Fes mutant on Tyr-713 and Tyr-811, suggesting that Fes autophosphorylation occurs via an intermolecular mechanism analogous to receptor tyrosine kinases.     

Identification of Gas6 as a ligand for Mer, a neural cell adhesion molecule related receptor tyrosine kinase implicated in cellular transformation

Mer/Nyk/Eyk is an orphan receptor tyrosine kinase expressed at high levels in monocytes and cells derived from epithelial and reproductive tissues. Overexpression of Mer has been associated with lymphoid malignancies. Here we identify Gas6, the product of a growth arrest specific gene, as a ligand for Mer. Gas6 has previously been shown to activate both Axl and Rse/Tyro3, two other receptor tyrosine kinases in the same family as Mer. The apparent relative association and dissociation rate constants of Gas6 for soluble Axl, Rse/Tyro3 and Mer were compared using surface plasmon resonance. Gas6 was shown to induce rapid phosphorylation of Mer expressed in several different types of cells. We also observed a transient activation of p42 MAP kinase following activation of Mer by Gas6. Thus, Gas6 exerts its biological effects through multiple receptor tyrosine kinases.     

Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet/endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2

Recent studies have shown that the Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase, SHP-2, associates with the cytoplasmic domain of PECAM-1 as it becomes tyrosine-phosphorylated during platelet aggregation: a process that can be mimicked in part by small synthetic phosphopeptides corresponding to the cytoplasmic domain of PECAM-1 encompassing tyrosine residues Tyr-663 or Tyr-686. To further examine the molecular requirements for PECAM-1/SHP-2 interactions, we generated human embryonic kidney (HEK)-293 cell lines that stably expressed mutant forms of PECAM-1 harboring tyrosine to phenylalanine (Tyr --> Phe) mutations in the cytoplasmic domain. Y663F and Y686F forms of PECAM-1 were tyrosine-phosphorylated to a somewhat lesser extent than wild-type PECAM-1, and a doubly substituted Y663,686F form of PECAM-1 failed to become tyrosine-phosphorylated, suggesting that the PECAM-1 cytoplasmic domain tyrosine residues 596, 636 and 701 do not serve as substrates for cellular kinases. Interestingly, SHP-2 binding was lost when either Tyr-663 or Tyr-686 were changed to phenylalanine, indicating that both residues are required for SHP-2/PECAM-1 association. Although PECAM-1 phosphopeptides NSDVQpY663TEVQV and DTETVpY686SEVRK stimulated the catalytic activity of the phosphatase to a similar extent, surface plasmon resonance studies revealed that the Tyr-663-containing peptide had approximately 10-fold higher affinity for SHP-2 than did the Tyr-686 peptide. Finally, peptido-precipitation analysis showed that the NH2-terminal SH2 domain of SHP-2 reacted preferentially with the Tyr-663 PECAM-1 phosphopeptide, while the Tyr-686 phosphopeptide associated only with the COOH-terminal SH2 domain of the phosphatase. Together, these data provide a molecular model for PECAM-1/SHP-2 interactions that may shed light on the downstream events that follow PECAM-1-mediated interactions of vascular cells.     

Expression of CYP71B7, a cytochrome P450 expressed sequence Tag from Arabidopsis thaliana

c-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNEYTAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-16 through Lys-46, YTPENPTEPVNTSAGHYGVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.     

Identification of TrkB autophosphorylation sites and evidence that phospholipase C-gamma 1 is a substrate of the TrkB receptor

The TrkB receptor protein-tyrosine kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. In response to brain-derived neurotrophic factor and neurotrophin-3 treatment, TrkB expressed exogenously in Rat-2 cells is rapidly phosphorylated on tyrosine residues. At least 2 regions of TrkB contain phosphorylated tyrosines. The major sites of autophosphorylation are in the region containing Tyr-670, Tyr-674, and Tyr-675, which lies in the kinase domain and corresponds by sequence homology to the Tyr-416 autophosphorylation site in p60c-Src. Tyr-785, which lies just to the COOH-terminal side of the kinase domain in a relatively short tail characteristic of the Trk family of protein-tyrosine kinase receptors, is also phosphorylated in response to neurotrophin-3 treatment. The sequence around Tyr-785 fits a consensus sequence for binding phospholipase C-gamma 1. The simplest interpretation of these results is that, in response to neurotrophin binding, at least two and perhaps all three of the tyrosines in the Tyr-670/674/675 region are autophosphorylated independently, and Tyr-785 is autophosphorylated in vivo. Following activation of TrkB, phospholipase C-gamma 1 is phosphorylated on Tyr-783, Tyr-771, and Tyr-1254. Phospholipase C-gamma 1 also forms a complex with TrkB in response to neurotrophin-3 treatment, consistent with the possibility that one of the TrkB autophosphorylation sites provides a binding site for the phospholipase C-gamma 1 SH2 domains, as is the case for other receptor protein-tyrosine kinases. We conclude that phospholipase C-gamma 1 is directly phosphorylated by TrkB. Since phosphorylation of Tyr-783 and Tyr-1254 results in activation of phospholipase C-gamma 1, we predict that neurotrophin-3 leads to activation of phospholipase C-gamma 1 following binding to TrkB in Rat-2 cells.     

Enhancement of the transient-evoked otoacoustic emission produced by the addition of a pure tone in the guinea pig

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase implicated in cell-matrix interaction and integrin signaling. It is well established that Tyr-397 is the FAK autophosphorylation site and Tyr-407, -576/577, -861, and -925 are the sites on murine FAK that are mediated by Src family kinases. To study how FAK is regulated by tyrosine phosphatase(s), cells overexpressing chicken FAK are treated with sodium vanadate. Both the phosphotyrosine content and the enzymatic activity of FAK are increased in response to vanadate. Interestingly, sustained FAK Tyr-576/577 and -863 phosphorylations are detected in vanadate-treated FAK overexpressors and are dependent on FAK autophosphorylation. Further analysis of sodium vanadate-treated FAK overexpressors reveals that the enhanced FAK kinase activity parallels its elevated Tyr-576/577 phosphorylation. Thus, we conclude that Src-mediated FAK phosphorylation is regulated by a tyrosine phosphatase(s) and may be of physioligical significance.     

Eicosapentaenoic acid enhances nitric oxide production by cultured human endothelial cells

Flt-1, a tyrosine kinase receptor for vascular endothelial growth factor (VEGF), plays important roles in the angiogenesis required for embryogenesis and in monocyte/macrophage migration. However, the signal transduction of Flt-1 is poorly understood due to its very weak tyrosine kinase activity. Therefore, we overexpressed Flt-1 in insect cells using the Baculovirus system in order to examine for autophosphorylation sites and association with adapter molecules such as phospholipase Cgamma-1 (PLCgamma). Tyr-1169 and Tyr-1213 on Flt-1 were found to be auto-phosphorylated, but only a phenylalanine mutant of Tyr-1169 strongly suppressed its association with PLCgamma. In Flt-1 overexpressing NIH3T3 cells, VEGF induced autophosphorylation of Flt-1, tyrosine-phosphorylation of PLCgamma and protein kinase C-dependent activation of MAP kinase. These results strongly suggest that Tyr-1169 on Flt-1 is a major binding site for PLCgamma and important for Flt-1 signal transduction within the cell.     

Absence of autophosphorylation site Y882 in the p185neu oncogene product correlates with a reduction of transforming potential

Autophosphorylation of type I receptor tyrosine kinases (RTKs) comprises one step in the signaling events mediated by erbB receptors such as p185neu and EGFR. Previous analysis of p185neu has indicated that there are at least five tyrosine autophosphorylation sites, Y882, Y1028, Y1143, Y1226/7 and Y1253, of which Y882 might be important because of its location in the kinase activity domain. We have specifically analysed the effect of a Y882F (phenylalanine substituted for tyrosine at position 882) mutation in the enzymatic active domain. We also deleted the carboxyl terminal 122 amino acids which contained three other autophosphorylation sites (TAPstop) and combined mutants of that deletion with Y882F (Y882F/APstop). Both in vitro and in vivo transformation assays showed that substitution of tyrosine882 by phenylalanine significantly decreased the transforming potential of activated, oncogenic p185neu, although no significant difference in the total phosphotyrosine levels of the mutant proteins were observed. To analyse mitogenic signaling in response to ligand, the intracellular domains of p185neu and Y882F were fused with the extracellular domain of the EGF receptor. The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were less responsive to EGF stimulation than those expressing EGFR/neu receptors. In vitro kinase assays demonstrated that abolishing the autophosphorylation site Y882 diminished the enzymatic tyrosine kinase activity of p185neu. These studies, taken together with the phenotypic inhibition observed with cells expressing Y882F, suggest that the tyrosine882 residue may be important for p185neu-mediated transformation by affecting the enzymatic kinase function of the p185neu receptor.     

Full activation of the platelet-derived growth factor beta-receptor kinase involves multiple events

Activation of receptor tyrosine kinases is thought to involve ligand-induced dimerization, which promotes receptor transphosphorylation and thereby increases the receptor's phosphotransferase activity. We used two platelet-derived growth factor beta-receptor (beta-PDGFR) mutants to identify events that are required for full engagement (autophosphorylation and activation of the kinase activity) of the beta-PDGFR kinase. The F79/81 receptor (Tyr to Phe substitution at 579 and 581 in the juxtamembrane domain of the receptor) was capable of only very modest ligand-dependent autophosphorylation and also failed to associate with numerous SH2 domain-containing proteins. Furthermore, stimulation with platelet-derived growth factor (PDGF) did not increase the kinase activity of the F79/81 mutant toward exogenous substrates. However, the F79/81 receptor had basal kinase activity and could be artificially stimulated by incubation with ATP. Because the low kinase activity of the F857 mutant (Tyr to Phe substitution at 857 in the putative activation loop) could not be increased by incubation with ATP, failure to phosphorylate Tyr-857 may be the reason why the F79/81 mutant has low kinase activity. Surprisingly, the F857 mutant underwent efficient PDGF-dependent autophosphorylation. Thus the PDGF-dependent increase in the kinase activity of the receptor is not required for autophosphorylation. We conclude that full activation of the beta-PDGFR kinase requires at least two, apparently distinct events.     

The activated form of the Lck tyrosine protein kinase in cells exposed to hydrogen peroxide is phosphorylated at both Tyr-394 and Tyr-505

Members of the Src family of non-receptor tyrosine protein kinases are known to be inhibited by the intramolecular association between a phosphorylated carboxyl-terminal tyrosine residue and the SH2 domain. We have previously shown that exposure of cells to H2O2 strongly activates Lck, a lymphocyte-specific Src family kinase, by inducing phosphorylation on Tyr-394, an absolutely conserved residue within the activation loop of the catalytic domain. Here we show that Lck that has been activated by H2O2 is simultaneously phosphorylated at both the carboxyl-terminal tyrosine (Tyr-505) and Tyr-394. Thus, dephosphorylation of Tyr-505 is not a prerequisite for either phosphorylation of Lck at Tyr-394 or catalytic activation of the kinase. These results indicate that activation of Lck by phosphorylation of Tyr-394 is dominant over any inhibition induced by phosphorylation of Tyr-505. We propose that these results may be extended to all Src family members.     

Phosphatidylinositol 3-kinase-gamma activates Bruton's tyrosine kinase in concert with Src family kinases

Bruton's tyrosine kinase (Btk) is essential for normal B lymphocyte development and function. The activity of Btk is partially regulated by transphosphorylation within its kinase domain by Src family kinases at residue Tyr-551 and subsequent autophosphorylation at Tyr-223. Activation correlates with Btk association with cellular membranes. Based on specific loss of function mutations, the Btk pleckstrin homology (PH) domain plays an essential role in this activation process. The Btk PH domain can bind in vitro to several lipid end products of the phosphatidylinositol 3-kinase (PI 3-kinase) family including phosphatidylinositol 3,4,5-trisphosphate. Activation of Btk as monitored by elevation of phosphotyrosine content and a cellular transformation response was dramatically enhanced by coexpressing a weakly activated allele of Src (E378G) and the two subunits of PI 3-kinase-gamma. This activation correlates with new sites of phosphorylation on Btk identified by two-dimensional phosphopeptide mapping. Activation of Btk was dependent on the catalytic activity of all three enzymes and an intact Btk PH domain and Src transphosphorylation site. These combined data define Btk as a downstream target of PI 3-kinase-gamma and Src family kinases.     

Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591-786

We compared the interaction between the insulin receptor (IR) and the IR substrate (IRS) proteins IRS-1 and IRS-2) using the yeast two-hybrid system. Both IRS proteins interact specifically with the cytoplasmic portion of the IR and the related insulin-like growth factor-I receptor, and these interactions require receptor tyrosine kinase activity. Alignment of IRS-1 and IRS-2 revealed two conserved domains at the NH2 terminus, called IH1PH and IH2PTB, which resemble a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, respectively. The IH2PTB binds to the phosphorylated NPXY motif (Tyr-960) in the activated insulin receptor, providing a specific mechanism for the interaction between the receptor and IRS-1. Although the IH2PTB of IRS-2 also interacts with the NPEY motif of the insulin receptor, it is not essential for the interaction between the insulin receptor and IRS-2 in the yeast two-hybrid system. IRS-2 contains another interaction domain between residues 591 and 786, which is absent in IRS-1. This IRS-2-specific domain is independent of the IH2PTB and does not require the NPEY motif; however, it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop (Tyr-1146, Tyr-1150, and Tyr-1151). Importantly, this novel domain mediates the association between IRS-2 and insulin receptor lacking the NPXY motif and may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances IRS-2 phosphorylation.     

Measurement of 6-MV X-ray surface dose when topical agents are applied prior to external beam irradiation

The Syk protein-tyrosine kinase is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. After cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues. We have previously reported that Syk expressed in COS cells is predominantly phosphorylated at both Tyr518 and Tyr519 at its putative autophosphorylation site. In this study, we have examined the role of each of these two residues for the catalytic activity of Syk in vitro and for the Syk-induced phosphorylation of cellular proteins in intact cells. Mutation of either residue had minor effects on the catalytic activity of Syk, and even the double mutant [F518, F519]Syk was about 60% as active as the wild-type enzyme. In intact cells, however, all three mutants consistently failed to induce the extensive tyrosine phosphorylation of cellular proteins typically observed with wild-type Syk. We have recently shown that the doubly phosphorylated Y518/Y519 site is also the site for association of Syk with the SH2 domain of the Lck kinase, which suggests that although phosphates at Y518/Y519 may enhance the catalytic activity of Syk, its interaction with Src family protein-tyrosine kinases is at least equally important for the induction of downstream substrate phosphorylation.     

Identification of Tyr-762 in the platelet-derived growth factor alpha-receptor as the binding site for Crk proteins

Tyr-762 is an autophosphorylation site in the human platelet-derived growth factor (PDGF) alpha-receptor. In order to investigate whether phosphorylated Tyr-762 serves as a docking site for downstream signal transduction molecules, affinity purification using an immobilized synthetic peptide containing phosphorylated Tyr-762 and its surrounding amino acid residues was performed. Proteins in HeLa cell lysate of molecular sizes 27, 38 and 40 kDa bound to the phosphorylated, but not to the unphosphorylated peptide. Analyses of partial amino acid sequences of the purified proteins indicated that they were identical to CrkI, CrkII and CrkL respectively. The wild-type PDGF alpha-receptor, when expressed in porcine aortic endothelial cells, formed complexes with CrkII and CrkL upon ligand stimulation, which was specifically inhibited by a synthetic peptide containing phosphorylated Tyr-762. Replacement of Tyr-762 with a phenylalanine residue in the PDGF alpha-receptor abrogated ligand-induced binding of Crk proteins. Tyrosine phosphorylation of CrkII and CrkL increased by 1.8- and 1.3-fold, respectively, upon ligand stimulation of the wild-type alpha-receptor. In contrast, the Y762F mutant PDGF alpha-receptor failed to induce tyrosine phosphorylation of Crk proteins. CrkII and CrkL constitutively formed complex with the guanine nucleotide exchange factor C3G, in unstimulated as well as PDGF-stimulated cells. Moreover, the activated wild-type PDGF alpha-receptor but not the Y762F mutant receptor was found in a C3G immunoprecipitate, suggesting that a ternary complex between the activated PDGF alpha-receptor, Crk and C3G was formed. DNA synthesis stimulated by PDGF-BB as well as PDGF-induced MAP kinase activation was similar in cells expressing wild-type and mutant receptors. Interestingly, the activated PDGF beta-receptor was found not to bind Crk proteins. Instead, Tyr-771 of the beta-receptor, which is localized at an analogous position to Tyr-762 in the alpha-receptor, binds RasGAP. RasGAP is not bound to the alpha-receptor. Thus, this region in the kinase inserts of the two receptors may be important for the divergency in signaling from the two PDGF receptors.     

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

Csk phosphorylates Src family protein tyrosine kinases on a tyrosine residue near their C-terminus and downregulates their activity. We previously observed that this regulation requires a stoichiometric ratio of Csk:Src in a time-independent manner. In this report we examined this unusual kinetic behavior and found it to be caused by Src autophosphorylation. First, pre-incubation of Src with ATP-Mg led to time-dependent autophosphorylation of Src, activation of its kinase activity and loss of its ability to be inactivated by Csk. However, the autophosphorylated Src can still be phosphorylated by Csk. The SH2 binding site for phospho-Tyr of this hyperactive and doubly phosphorylated form of Src is not accessible. Second, dephosphorylation of autophosphorylated Src by protein tyrosine phosphatase 1B allowed Src to be inactivated by Csk. Third, protein tyrosine phosphatase 1B preferentially dephosphorylates the Src autophosphorylation site and allows for Src regulation by Csk. Finally, Yes, another member of the Src family, was also only partially inactivated when a sub-stoichiometric amount of Csk was used. Mutation of the tyrosine autophosphorylation site of Yes to a phenylalanine resulted in a mutant Yes enzyme that can be fully inactivated by a sub-stoichiometric amount of Csk in a time-dependent manner. These results demonstrate that Csk phosphorylation inactivates Src and Yes only when they are not previously autophosphorylated and Src autophosphorylation can block the inactivation by Csk phosphorylation. This conclusion suggests a dynamic model for the regulation of the Src family protein tyrosine kinases, which is discussed in the context of previously reported observations on the regulation of Src family protein tyrosine kinases.     

The type II transforming growth factor-beta receptor autophosphorylates not only on serine and threonine but also on tyrosine residues

The type I and type II receptors for transforming growth factor-beta (TGF-beta) are structurally related transmembrane serine/threonine kinases, which are able to physically interact with each other at the cell surface. To help define the initial events in TGF-beta signaling, we characterized the kinase activity of the type II TGF-beta receptor. A recombinant cytoplasmic domain of the receptor was purified from Escherichia coli and baculovirus-infected insect cells. Anti-phosphotyrosine Western blotting demonstrated that the type II receptor kinase can autophosphorylate on tyrosine. Following an in vitro kinase reaction, the autophosphorylation of the cytoplasmic domain and phosphorylation of exogenous substrate was shown by phosphoamino acid analysis to occur not only on serine and threonine but also on tyrosine. The dual kinase specificity of the receptor was also demonstrated using immunoprecipitated receptors expressed in mammalian cells and in vivo 32P labeling showed phosphorylation of the receptor on serine and tyrosine. In addition, the kinase activity of the cytoplasmic domain was inhibited by the tyrosine kinase inhibitor tyrphostin. Tryptic mapping and amino acid sequencing of in vitro autophosphorylated type II receptor cytoplasmic domain allowed the localization of the sites of tyrosine phosphorylation to positions 259, 336, and 424. Replacement of all three tyrosines with phenylalanines strongly inhibited the kinase activity of the receptor, suggesting that tyrosine autophosphorylation may play an autoregulatory role for the kinase activity of this receptor. These results demonstrate that the type II TGF-beta receptor can function as a dual specificity kinase and suggest a role for tyrosine autophosphorylation in TGF-beta receptor signaling.     

SH1 domain autophosphorylation of P210 BCR/ABL is required for transformation but not growth factor independence

P210 BCR/ABL is a chimeric oncogene implicated in the pathogenesis of chronic myelogenous leukemia. BCR sequences have been shown to be required for activation of the tyrosine kinase and transforming functions of BCR/ABL. In this work, we show that two other structural requirements for full transforming activity of P210 BCR/ABL include a functional tyrosine kinase and the presence of tyrosine 1294, a site of autophosphorylation within the tyrosine kinase domain. Replacement of tyrosine 1294 with phenylalanine (1294F) greatly diminishes the transforming activity of BCR/ABL without affecting the specific activity of the protein tyrosine kinase. Expression of an exogenous myc gene in fibroblasts partially complements the transforming capacity of mutant P210 BCR/ABL (1294F). Surprisingly, tyrosine 1294 is not required for efficient induction of growth factor-independence in hematopoietic cell lines by P210 BCR/ABL. These results suggest that autophosphorylation at tyrosine 1294 may be important for recognition and phosphorylation of cellular substrates in the pathway of transformation, but it is not critical for mediating the events which lead to growth factor independence.     

Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C

The product of the c-kit proto-oncogene, denoted Kit/SCF-R, encodes a tyrosine kinase receptor for stem cell factor (SCF). Kit/SCF-R induces proliferation, differentiation or migration of cells within the hematopoietic, gametogenic and melanogenic lineages at different developmental stages. We report here that protein kinase C (PKC) mediates phosphorylation of Kit/SCF-R on serine residues in response to SCF or PMA in intact cells. The phosphorylation inhibits SCF-induced tyrosine autophosphorylation of Kit/SCF-R. In vitro studies showed that PKC phosphorylated the Kit/SCF-R directly on serine residues and inhibited autophosphorylation of Kit/SCF-R, as well as its kinase activity towards an exogenous substrate. The PKC-induced phosphorylation did not affect Kit/SCF-R ligand binding affinity. Inhibition of PKC led to increased SCF-induced tyrosine autophosphorylation, as well as increased SCF-induced mitogenicity. In contrast, PKC was necessary for SCF-induced motility responses, including actin reorganization and chemotaxis. Our data suggest that PKC is involved in a negative feedback loop which regulates the Kit/SCF-R and that the activity of PKC determines whether the effect of SCF will be preferentially mitogenic or motogenic.     

Impact of mutations at different serine residues on the tyrosine kinase activity of the insulin receptor

Insulin binding to its receptor activates a cascade of signaling events which are initiated by tyrosine autophosphorylation of the receptor and activation of the tyrosine kinase activity towards the insulin receptor substrates. In addition to phosphorylation at tyrosine residues a serine phosphorylation of the insulin receptor is observed. Neither the functional significance of serine phosphorylation of the receptor nor the location of relevant regulatory sites has been determined exactly so far. We studied potential functions of serine residues in human insulin receptor (HIR) with respect to its ability to undergo insulin stimulated autophosphorylation. Using site directed mutagenesis of HIR we exchanged serine to alanine at 13 different positions in the HIR beta-subunit. Sites were chosen according to the criteria of known serine phosphorylation sites (1023/25, 1293/94, 1308/09), conserved positions in hIR, hIGF-1 receptor, hIRR, and dIR (962, 994, 1037, 1055, 1074/78, 1168, 1177/78/82, 1202, 1263, 1267). All HIR mutants were expressed in HEK 293 cells and basal and insulin stimulated autophosphorylation were determined. We found that the exchange of serine to alanine at position 994 and at position 1023/25 increased insulin stimulated receptor autophosphorylation significantly (147% +/- 12% and 129% +/- 6% of control, p < 0.01, n = 7), while all other exchanges did not significantly alter insulin stimulated HIR autophosphorylation. The data suggest that the serine residues at position 994 as well as 1023/25 might be part of inhibitory domains of the insulin receptor.     

Autophosphorylation of purified c-Src at its primary negative regulation site

The phosphorylating and transforming activities of c-Src are negatively regulated by phosphorylation at Tyr-527 near its carboxyl terminus. Previous studies have indicated that c-Src preferentially autophosphorylates Tyr-416, a residue in the middle of the catalytic domain, in vitro, and that Tyr-527 is phosphorylated by the carboxyl-terminal Src kinase, Csk. However, indirect evidence suggests that c-Src may also autophosphorylate Tyr-527 as part of a negative feedback loop. While some in vivo evidence suggests that Tyr-527 can be autophosphorylated in an intermolecular interaction, it has not previously been possible to directly demonstrate significant autophosphorylation in vitro. Here we show that c-Src purified from recombinant bacteria can autophosphorylate Tyr-527 to high levels in vitro when incubated with sufficiently high concentrations of ATP (KM(Mg2+/ATP) approximately equal to 20 microM) that are well above those that have been used previously. In vitro Tyr-527 autophosphorylation can occur both as an intra- and intermolecular interaction; higher enzyme concentrations are required for intermolecular Tyr-527 phosphorylation than for Tyr-416 autophosphorylation. These results support the possibility that, like G-proteins, c-Src can switch itself off in vivo by its own enzymatic activity.