Src family protein tyrosine kinases and cellular signal transduction pathways.

Members of the Src family of protein tyrosine kinases are thought to be involved in signal transduction pathways that control growth and cellular architecture. In recent years it has been shown that they interact with receptor tyrosine kinases (such as the platelet-derived growth factor receptor) and with receptors that themselves lack intrinsic tyrosine kinase activity (such as the interleukin-2 receptor). In some cases they are required for mitogenic signalling by these receptors. They are also activated in response to stress and during mitosis.

A mitotic function for Src?

During mitosis, the activity of the c-Src protein tyrosine kinase increases. The tyrosine phosphorylation of a 68 kDa protein (Sam68) also increases at this time, and recent studies have shown that Src and Sam68 interact. Sam68 is highly related to p62, a RasGAP-associated protein, and has homology to RNA-binding proteins. The relationship between p62 and Sam68, and their roles in Src signalling, need to be clarified, but these findings suggest that Src may participate in regulating RNA processing during the cell cycle.

Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division.

The protein tyrosine kinase c-Src is transiently activated at the transition from the G2 phase to mitosis in the cell cycle of mammalian fibroblasts. Fyn and Yes, the other members of the Src family present in fibroblasts, were also found to be activated at mitosis. In cells microinjected with a neutralizing antibody specific for Src, Fyn, and Yes (anti-cst.1) during G2, cell division was inhibited by 75 percent. The block occurred before nuclear envelope breakdown. Antibodies specific for phosphatidylinositol-3 kinase alpha and phospholipase C-gamma 1 had no effect. Microinjection of the Src homology 2 (SH2) domain of Fyn was also inhibitory. Functional redundancy between members of the Src family was observed; a Src-specific antibody had no effect in NIH 3T3 cells but inhibited cell division in fibroblasts in which the only functional Src family kinase was Src itself. Thus, Src family kinases and proteins associating with their SH2 domains are required for entry into mitosis.

Endothelial cell transformation by polyomavirus middle T antigen in mice lacking Src-related kinases.

BACKGROUND: Expression of polyomavirus middle T antigen (PymT) rapidly induces endothelial tumors (hemangiomas) in mice, with an apparent single rate-limiting step. Because activation of Src-like kinases is thought to be an important component of PymT-induced transformation, we have analyzed the functional requirement for individual kinases in this process. This type of analysis has only recently become possible, with the generation of 'gene knock-out' mice lacking each of the kinase genes src, fyn and yes. RESULTS: Hemangiomas develop efficiently in newborn mice lacking either src, fyn or yes after inoculation with a PymT-transducing retrovirus. In src- and fyn-deficient mice, the kinetics of induction and the histological properties of the tumors were indistinguishable from those in wild-type mice. In contrast, a reduced number of tumors arose in yes-deficient mice, with a significantly longer latency period. Transformed endothelial cell lines derived from the induced hemangiomas, however, did not differ in their morphological and tumorigenic properties from cell lines established previously from wild-type mice. Biochemical analysis of complexes between PymT and the Src-related kinases in these cell lines suggests that the Yes kinase is responsible for a significant amount of the PymT-associated kinase activity in transformed endothelial cells. CONCLUSION: We have demonstrated that inactivation of a single tyrosine kinase of the Src family in endothelial cells does not abrogate PymT-induced hemangioma formation. As the remaining kinases do not compensate for the absence of a family member by elevated kinase activity, the loss--which affects the transformation process to varying degrees--can be studied in this model system. Our studies suggest that the PymT-Yes kinase complex plays a major role in the tumor-initiating action of PymT.

Src-like adaptor protein (Slap) is a negative regulator of mitogenesis.

The Src-like adaptor protein (Slap) is a recently identified adaptor protein containing Src homology 3 (SH3) and SH2 domains. Slap is found in a wide range of cell types and was shown to interact with the Eck receptor tyrosine kinase in a yeast two-hybrid interaction screen [1]. Here, we found that Slap is expressed in NIH3T3 cells and could associate with the activated platelet-derived growth factor (PDGF) receptor. Using mutated versions of the PDGF receptor and phosphopeptide competition experiments, we determined that Slap has the highest affinity for the Src-binding site of the PDGF receptor. Our inability to produce cell lines that stably expressed Slap suggested that Slap inhibited cell growth. We further investigated this issue by transiently expressing Slap by microinjection. Overexpression of Slap by this method inhibited DNA synthesis induced by PDGF and serum, whereas overexpression of the adaptor proteins Grb2 and Shc did not. Finally, microinjection of a Slap antibody into NIH3T3 cells that had been stimulated with suboptimal doses of growth factors potentiated the effects of the growth factors. These data suggest that, unlike other adaptor proteins, Slap is a negative regulator of signalling initiated by growth factors.

A function for phosphatidylinositol 3-kinase beta (p85alpha-p110beta) in fibroblasts during mitogenesis: requirement for insulin- and lysophosphatidic acid-mediated signal transduction.

We have previously shown that phosphatidylinositol 3-kinase alpha (PI 3-Kalpha) (p85alpha-p110alpha) is required for DNA synthesis induced by various growth factors (S. Roche, M. Koegl, and S. A. Courtneidge, Proc. Natl. Acad. Sci. USA 91:9185-9189, 1994) in fibroblasts. In the present study, we have investigated the function of PI 3-Kbeta (p85alpha-p110beta) during mitogenesis. By using antibodies specific to p110beta we showed that PI 3-Kbeta is expressed in NIH 3T3 cells. PI 3-Kbeta and PI 3-Kalpha have common features: PI 3-Kbeta is tightly associated with a protein serine kinase that phosphorylates p85alpha, it interacts with the Src-middle T antigen complex and the activated platelet-derived growth factor (PDGF) receptor in fibroblasts in vivo, and it becomes tyrosine phosphorylated after PDGF stimulation. PI 3-Kbeta was also activated in Swiss 3T3 and Cos7 cells stimulated with lysophosphatidic acid (LPA), a mitogen that interacts with a heterotrimeric G protein-coupled receptor. In contrast PI 3-Kalpha was activated to a lesser extent in these cells. Microinjection of neutralizing antibodies specific for p110beta into quiescent fibroblasts inhibited DNA synthesis induced by both insulin and LPA but poorly affected PDGF receptor signaling. Therefore, PI 3-Kbeta plays an important role in transmitting the mitogenic response induced by some, but not all, growth factors. Finally, we show that while oncogenic V12Ras interacts with type I PI 3-Ks, it could induce DNA synthesis in the absence of active PI 3-Kalpha and PI 3-Kbeta, suggesting that Ras uses other effectors for DNA synthesis.

Immunological phenotype of lymphomas induced by avian leukosis viruses.

The production of immunoglobulin by six cell lines derived from bursal tumors induced by avian leukosis virus follows two general patterns: (i) three cell lines that have been extensively passaged in culture synthesize and secrete light chains only; (ii) three cell lines that are recently isolated produce and secrete monomeric immunoglobulin M in addition to free light chains. All six cell lines synthesize and secrete both glycosylated and unglycosylated forms of light chain. We conclude that the cell lines established from lymphomas induced by avian leukosis virus represent relatively mature, but possibly abnormal, stages in the development of chicken B-lymphocytes. The immunoglobulin M produced by the cell lines failed to form detectable immune complexes with avian leukosis virus. It therefore appears that the immunoglobulin M is not directed against viral antigens and that autogenous antigenic stimulus cannot account for the sustained growth of the neoplastic B-lymphocytes.

DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases.

The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.

SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling.

The use of small-molecule inhibitors to study molecular components of cellular signal transduction pathways provides a means of analysis complementary to currently used techniques, such as antisense, dominant-negative (interfering) mutants and constitutively activated mutants. We have identified and characterized a small-molecule inhibitor, SU6656, which exhibits selectivity for Src and other members of the Src family. A related inhibitor, SU6657, inhibits many kinases, including Src and the platelet-derived growth factor (PDGF) receptor. The use of SU6656 confirmed our previous findings that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH 3T3 fibroblasts. By comparing PDGF-stimulated tyrosine phosphorylation events in untreated and SU6656-treated cells, we found that some substrates (for example, c-Cbl, and protein kinase C delta) were Src family substrates whereas others (for example, phospholipase C-gamma) were not. One protein, the adaptor Shc, was a substrate for both Src family kinases (on tyrosines 239 and 240) and a distinct tyrosine kinase (on tyrosine 317, which is perhaps phosphorylated by the PDGF receptor itself). Microinjection experiments demonstrated that a Shc molecule carrying mutations of tyrosines 239 and 240, in conjunction with an SH2 domain mutation, interfered with PDGF-stimulated DNA synthesis. Deletion of the phosphotyrosine-binding domain also inhibited synthesis. These inhibitions were overcome by heterologous expression of Myc, supporting the hypothesis that Shc functions in the Src pathway. SU6656 should prove a useful additional tool for further dissecting the role of Src kinases in this and other signal transduction pathways.

A functional screen in yeast for regulators and antagonizers of heterologous protein tyrosine kinases.

Tyrosine phosphorylation exerts a pivotal role in cell regulation processes of higher eukaryotes. Tight control of the activity of protein tyrosine kinases is crucial for ordered phosphorylation to occur. We have developed a functional screen for tyrosine kinase regulators using c-Src, the first cellular protein tyrosine kinase described, as a prototype; and fission yeast, Schizosaccharomyces pombe, as a genetically amenable host system. Inducible expression of c-Src in fission yeast is lethal. We have screened human cDNA libraries for clones able to counteract the lethal effect of Src. Two different classes of cDNAs, which we called SAS for sequences antagonizing Src, were obtained. The first class encodes for the protein tyrosine kinase Csk, known to regulate Src activity through phosphorylation of the C-terminal tyrosine. The second class consists of clones encoding three different tyrosine phosphatases, counteracting Src action by dephosphorylation of Src substrates and by dephosphorylation of Src itself. The system described here can be applied to identify regulators of other heterologous tyrosine kinases, including receptor-type tyrosine kinases, which impair growth of S. pombe.

No requirement for src family kinases for PDGF signaling in fibroblasts expressing SV40 large T antigen.

A growing body of literature suggests that the ubiquitously expressed Src family kinases (Src, Fyn and Yes) are required for agents such as platelet-derived growth factor (PDGF) to stimulate DNA synthesis. Yet Klinghoffer and colleagues recently presented evidence that fibroblasts derived from mice null for Src, Fyn and Yes responded normally to PDGF (Klinghoffer et al., 1999, EMBO J., 18: 2459 - 2471). What is the reason for this discrepancy? We noted that Klinghoffer et al. (1999) used SV40 large T antigen (largeT) to facilitate derivation of cell lines from the embryos. We therefore tested the effect of largeT on PDGF receptor signaling. We found that expression of largeT overcame the inhibitory effects of interfering forms of both Ras (N17Ras) and Src (SrcK-). Furthermore, injection of SrcK- or the cst.1 antibody (which inhibits Src, Fyn and Yes) failed to inhibit PDGF-stimulated DNA synthesis in NIH3T3 cells expressing dominant negative p53, and fibroblasts derived from p53 null embryos. These data suggest firstly that caution should be used in interpretation of experiments conducted in cell lines expressing largeT, and secondly that the role of Src family kinases in growth factor signaling may be to oppose the effects of negative growth regulators such as p53. Oncogene (2000) 19, 2867 - 2869

Structural requirements for the efficient regulation of the Src protein tyrosine kinase by Csk.

Protein tyrosine kinases of the Src family are negatively regulated by phosphorylation in the C-terminal tail of the molecule. A different protein tyrosine kinase, Csk, is largely responsible for this regulation. The phosphorylated tail of c-Src engages with the SH2 domain in a conformation that is associated with low kinase activity and which involves stabilization by the SH3 domain. Inducible expression of c-Src in fission yeast is lethal unless Csk is coexpressed. Using this assay we present evidence that Src regulation by C-terminal phosphorylation does not require the myristylation signal or the unique domain at the N-terminus of the Src protein. Mutagenesis of the SH3 and SH2 domains of Csk show that neither are necessary in yeast or in vitro for efficient regulation of Src. Mutation of Tyr416 of Src, a site of autophosphorylation common to most protein tyrosine kinases, abolished the ability of Src to arrest growth of phosphorylate endogenous proteins. Tyr416 had the same effect on a shorter form of Src consisting of the kinase domain only, indicating that the mutation affects a property intrinsic to the catalytic domain. The residual activity of full-length Src mutated at Tyr416 is efficiently repressed by Csk action, suggesting that regulation by C-terminal phosphorylation does not act by preventing phosphorylation at Tyr416.

A variety of tumours induced by the middle T antigen of polyoma virus in a transgenic mouse family.

A transgenic mouse family expressing the middle T antigen of polyoma virus under control of the immunoglobulin heavy chain (IgE) enhancer showed the frequent occurrence of carcinomas in various organs, predominantly in females. Most frequently affected were the salivary and thyroid glands, but mammary tumours, liver haemangiomas and adenocarcinomas of unknown origin were also observed. In all tumours, the middle T antigen was found to be complexed with cellular tyrosine kinases. These results extend the range of tumour types associated with in vivo expression of middle T and with the subsequent deregulation of pp60c-src and related tyrosine kinases.

Association of Fyn with the activated platelet-derived growth factor receptor: requirements for binding and phosphorylation.

Three members of the Src family of tyrosine kinases [pp60c-src (Src), p59fyn (Fyn) and pp62c-yes (Yes)] are ubiquitously expressed, and are thus likely to have general roles in growth control. We have previously shown that, after addition of platelet-derived growth factor (PDGF) to quiescent cells, all three kinases become activated and associated with the PDGF receptor. We have now addressed the requirements for this association. First, we have used a baculovirus expression system to show that Fyn associates with the activated PDGF receptor in vitro in the absence of other proteins, demonstrating that the association between the two molecules is direct. Second, by generating cell lines expressing chimeric molecules consisting of Fyn sequences fused to a portion of beta-galactosidase, we found that the SH2 domain of Fyn is necessary for ligand-stimulated association with the PDGF receptor in vivo. Third, those fusion proteins that associated with the PDGF receptor also became phosphorylated in vivo following PDGF treatment, and in in vitro kinase assays, suggesting that the amino-terminal half of Fyn contains the sites of PDGF-stimulated phosphorylation. Partially purified, kinase-negative Fyn also became phosphorylated in the activated PDGF receptor complex in vitro, demonstrating that the PDGF receptor phosphorylates Fyn, rather than the novel phosphorylations occurring by autophosphorylation.

Fibroblast growth factor receptor-1 regulation of Src family kinases.

Fibroblast growth factors (FGFs) induce proliferation and differentiation of a wide variety of cells by stimulation of cell surface expressed high affinity-binding receptor tyrosine kinases. Members of the Src family of cytoplasmic tyrosine kinases are substrates for certain growth factor receptors. We have examined interactions between FGF receptor-1 (FGFR-1) and Src, Fyn and Yes. In lung capillary endothelial cells and murine fibroblasts, bFGF stimulation led to increased autophosphorylation of Src family members. In contrast, in porcine aortic endothelial cells (FGFR-1/PAE) and lung fibroblasts from chinese hamster (CCL 39), activation of FGFR caused reduced autophosphorylation of Src and Fyn. In neither case could complex-formation between Src members and FGFR be seen. Analysis of a panel of mutated FGFR-1 expressed in PAE cells showed that FGFR-1/Y766F mediated an increased autophosphorylation of Src members and upregulation of their kinase activities. Y766 in FGFR-1 has been shown to serve as a binding site for phospholipase C-gamma, which regulates Ca2+ fluxes and protein kinase C (PKC) activity. The negative effect on Src kinase activity upon FGFR stimulation was mimicked by activation of PKC in FGFR-1/PAE or CCL 39 cells using phorbol 12-myristate 13-acetate (PMA) and Src was phosphorylated in vitro by purified recombinant PKC alpha. Moreover, inhibition of PKC attenuated the bFGF induced decrease in autophosphorylation of Src family members. These data indicate a negative regulatory role for PKC on Src kinase activity in certain cell types.

The proto-oncogene c-Cbl is a negative regulator of DNA synthesis initiated by both receptor and cytoplasmic tyrosine kinases.

In C. elegans, genetic and biochemical data indicate that the Cbl homolog Sli-1 attenuates Let-23 (EGFR) signaling. To investigate whether c-Cbhl might have a role in mammalian growth factor-mediated mitogenic signaling, we microinjected NIH3T3 mouse fibroblasts with expression plasmids encoding wt and G306ECbl (a 'loss of function' mutant identified in C. elegans). We observed inhibition of PDGF BB- and EGF-induced DNA synthesis by wt Cbl but not the mutant. Microinjection of two different affinity purified polyclonal antisera against Cbl boosted a suboptimal PDGF-stimulated mitogenic response. The inhibition of both PDGF BB- and EGF-induced DNA synthesis by wt Cbl was reversed by co-expression with Myc but not with Fos. DNA synthesis initiated by constitutively activated Src was also blocked by Cbl expression, but curiously by the G306E mutant as well. These data are all consistent with the proposition that Cbl negatively affects mitogenic signaling in mammalian fibroblasts.

The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions.

The Src protein tyrosine kinase plays a critical role in a variety of signal transduction pathways. Strict regulation of its activity is necessary for proper signalling. We present here the crystal structure of chicken Src which is phosphorylated at Tyr527 and represents its least active form. Our structure, similar to the recently reported human Hck and Src structures, contains the SH3, SH2 and the kinase domains and the C-terminal regulatory tail but not the N-terminal unique domain. The SH3 domain uses its hydrophobic surface to coordinate the SH2-kinase linker such that residues Gln251 and Leu255 specifically interact with side chains in the beta2-beta3 and the alphaC-beta4 loops of the N-terminal lobe opposite of the kinase active site. This position of the SH3 domain and the coordination of the SH2-kinase linker also optimally places the SH2 domain such that the phosphorylated Tyr527 in the C-terminal tail interacts with the SH2 binding pocket. Analogous to Cdk2 kinase, the position of the Src alphaC-helix in the N-terminal lobe is swung out disrupting the position of the active site residues. Superposition of other protein kinases including human Hck and Src onto chicken Src indicate that the alphaC-helix position is affected by the relative position of the N-terminal lobe with respect to the C-terminal lobe of the kinase and that the presence of the SH3/SH2-kinase linker/N-terminal lobe interactions restricts the kinase lobes and alphaC-helix access to the active conformation. These superpositions also suggest that the highly conserved alphaC-beta4 loop restricts the conformational freedom of the N-terminal lobe by anchoring it to the C-terminal lobe. Finally, based on sequence alignments and conservation of hydrophobic residues in the Src SH2-kinase linker as well as in the alphaC-beta4 and beta2-beta3 loops, we propose that the Src-related kinases, Abl, Btk and Csk, share the same quaternary structure.

The discovery and validation of new drug targets in cancer.

Advances in our understanding of the signal transduction pathways involved in cellular growth control have provided several new strategies for cancer therapy. Recent advances now make it possible to develop selective inhibitors targeting genomic instability, the growth, survival, and invasion of the tumor, and its nourishment through the growth of new blood vessels.

The crystal structure of human CskSH3: structural diversity near the RT-Src and n-Src loop.

SH3 domains are modules occurring in diverse proteins, ranging from cytoskeletal proteins to signaling proteins, such as tyrosine kinases. The crystal structure of the SH3 domain of Csk (c-Src specific tyrosine kinase) has been refined at a resolution of 2.5 A, with an R-factor of 22.4%. The structure is very similar to the FynSH3 crystal structure. When comparing CskSH3 and FynSH3 it is seen that the structural and charge differences of the RT-Src loop and the n-Src loop, near the conserved Trp47, correlate with different binding properties of these SH3 domains. The structure comparison suggests that those glycines and acid residues which are very well conserved in the SH3 sequences are important for the stability of the SH3 fold.