Src oncogene at 64B

EVOLUTIONARY HOMOLOGS part 1/3

Src oncogene at 42A, a second Drosophila Src homolog

In Drosophila, Src64 was considered a unique ortholog of the vertebrate c-src; however, more recent evidence has been shown to the contrary. The closest relative of vertebrate c-src found to date in Drosophila is not Dsrc64, but Dsrc41, a gene identified for the first time in this paper. In contrast to Src64, overexpression of wild-type Src41 causes little or no appreciable phenotypic change in Drosophila. Both gain-of-function and dominant-negative mutations of Src41 cause the formation of supernumerary R7-type neurons, suppressible by one-dose reduction of boss, sevenless, Ras1, or other genes involved in the Sev pathway. Dominant-negative mutant phenotypes are suppressed and enhanced, respectively, by increasing and decreasing the copy number of wild-type Src41. The colocalization of Src41 protein, actin fibers and DE-cadherin, as well as the Src41-dependent disorganization of actin fibers and putative adherens junctions in precluster cells, suggest that Src41 may be involved in the regulation of cytoskeleton organization and cell-cell contacts in developing ommatidia (Takahashi, 1996).

The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine kinase (RTK) signaling. An extragenic suppressor of Raf, Su(Raf)1, has been isolated that encodes a Drosophila Src family gene (Src42A) identical to the previously cloned DSrc41. Characterization of Src42A mutations shows that Src42A acts independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Src42A negatively regulates Egfr signaling during oogenesis and negatively regulates receptor tyrosine kinase signaling in the eye. For example Src42A suppresses the rough eye phenotype caused by expression of hyperactive Ras or Raf. Src42A mutation also leads to defects in head and tail morphology, tracheal development and wing morphogenesis. This study provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the RTK signaling cascade. A possible model for Src42A function is discussed (Lu, 1999).

The functional status of Ras, Raf, Mek, or Mapk proteins does not appear to alter the ability of wild-type Src42A to repress receptor tyrosine kinase signaling: this would favor a model in which Src42A defines a branch pathway parallel to the main Ras/Mapk cascade with an integration point downstream of Mapk. This model is consistent with the reduction of maternal Src42A activity, which can still enhance Torso receptor tyrosine kinase signaling in the absence of Ras1 protein. The manner in which Src42A acts to modulate receptor tyrosine kinase signaling is similar in two ways to another branch pathway component, Kinase suppress of Ras-1 (ksr-1: see Drosophila Kinase suppressor of ras), of C. elegans. (1) Src42A does not appear to dramatically alter RTK-mediated processes when mutated alone. For example, mitotic clones of Src42A mutant cells in the eye do not produce extra photoreceptor cells. (2) The negative role of Src42A is only revealed when the Ras/Mapk cascade is compromised or hyperactived. Recently, Therrien (1998) reported the isolation of Src42A as a suppressor of a dominant negative form of fly ksr in the eye. In attempts to understand more about Src42A, a genetic screen was performed that isolated loss-of-function mutation in Egfr, rolled, and a new gene, semang, as suppressors of Src42A mutants (Zhang, 1999). It is suggested that Src42A works together with other branch pathway modulators such as Ksr to regulate signal transduction downstream of Egfr and other RTKs. If each branch pathway modulator takes over only a part of the total regulatory power, it would explain why Src42A or ksr-1 shows mild phenotypes when mutated alone. However, the phenotypes of Src42A do not overlap with two other Drosophila Src family members, Src64 and Tec29, both of which are involved in ring canal development during oogenesis. It is tempting to speculate that Src42A is activated following the activation of Egfr RTK via a Ras-1 independent mechanism. The signal from the activated Src42A would then integrate, in a negative fashion, with that from the main Ras/MAPK pathway to determine the final readout of an RTK pathway (Lu, 1999).

kinase suppressor of Ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of Ksr is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of Ksr remains unresolved. To further elucidate the role of Ksr and to identify proteins that may be required for Ksr function, a dominant modifier screen was conducted in Drosophila based on a Ksr-dependent phenotype. Overexpression of the Ksr kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with Ksr might modify the Ksr-dependent phenotype. Approximately 185,000 mutagenized progeny were screened for dominant modifiers of the Ksr-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for Ksr in Ras1 signaling. Mutations in genes encoding known components of the Ras pathway were isolated in a screen for the 14-3-3epsilon, Dsor1/mek, rolled/mapk, pointed, yan, and ksr loci. Furthermore, due to the ability of dominant-negative KSR (KDN) to block RAS/MAPK-mediated signaling, mutations in genes expected to function upstream of ksr were also isolated. These included mutations in the Egfr, Star, Sos, and Ras1 loci. In addition, 4 additional complementation groups were identfied. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor (Therrien, 2000).

Complementation test results reveal that SK2-4 is allelic to Src42A, the closest Drosophila homolog of the Src kinase family. Intriguingly, genetic data (suppression of sE-KDN, enhancement of sev-Ras1^V12, and suppression of Raf^HM7) suggest an inhibitory role for Src42A in Ras1 signaling, which is the opposite of the described function of Src-like kinases in vertebrates. During the course of this work, Src42A was also identified by another laboratory as being a dominant suppressor of a hypomorphic allele of Raf (Raf^C110). Genetic and molecular characterization of different Src42A alleles clearly supports an inhibitory function for Src42A in different RTK-dependent signaling pathways. Therefore, the further elucidation of the molecular function of Src42A in Drosophila may unveil a novel mechanism of action for this family of nonreceptor tyrosine kinases (Therrien, 2000).

Drosophila Src42 binding activity regulates RAF by a novel CNK-dependent derepression mechanism

Connector enhancer of KSR (CNK), an essential component of Drosophila receptor tyrosine kinase/mitogen-activated protein kinase pathways, negatively regulates RAF function. This bimodal property depends on the N-terminal region of CNK, which integrates RAS activity to stimulate RAF and a bipartite element, called the RAF-inhibitory region (RIR), which binds and inhibits RAF catalytic activity. The repressive effect of the RIR is counteracted by the ability of Src42 to associate, in an RTK-dependent manner, with a conserved region located immediately C-terminal to the RIR. Strikingly, several cnk loss-of-function alleles have mutations clustered in this area and provide evidence that these mutations impair Src42 binding. Surprisingly, the derepressing effect of Src42 does not appear to involve its catalytic function, but critically depends on the ability of its SH3 and SH2 domains to associate with CNK. Together, these findings suggest that the integration of RTK-induced RAS and Src42 signals by CNK as a two-component input is essential for RAF activation in Drosophila (Laberge, 2005).

RTK-induced activation of the small GTPase RAS was recognized early on as a critical event in RAF activation. RAS triggers plasma membrane anchoring of RAF through a direct contact between GTP-loaded RAS and RAF. However, this step is insufficient to induce RAF activation, but is a prerequisite for a complex series of regulatory events. For example, Ste20-like kinases and Src family kinases (SFKs) have been shown to collaborate with RAS in RTK-induced Raf-1 activation, owing to their ability to directly phosphorylate Raf-1 serine 338 (S338) and tyrosine 341 (Y341), respectively. However, these particular events are probably specific to Raf-1 as the equivalent S338 residue in B-RAF is constitutively phosphorylated, whereas the Y341-like residue is not conserved in B-RAF or in Drosophila and C. elegans RAF. Nonetheless, it remains possible that these kinases use different means to regulate RAF members. This would be consistent with genetic findings in Drosophila, which suggest that RAF is also regulated by an RTK-induced but RAS-independent pathway linked to SFKs (Laberge, 2005).

In addition to kinases and phosphatases regulating RAF activity, a number of apparently nonenzymatic proteins also modulate RAF function. One of these is Connector eNhancer of KSR (CNK), an evolutionarily conserved multidomain-containing protein originally identified in a KSR-dependent genetic screen in Drosophila. Genetic experiments in flies have indicated that CNK activity is required downstream of RAS, but upstream of RAF, thus suggesting that CNK regulates RAF activity. In agreement with this interpretation, CNK was found to interact directly with the catalytic domain of RAF and to modulate its function. The role of CNK, however, is probably not restricted to the MAPK pathway. Indeed, although mammalian CNK proteins have also been found to modulate the RAS/MAPK pathway, recent studies have indicated that they also control other events, including membrane/cytoskeletal rearrangement, Rho-mediated SRF transcriptional activity and RASSF1A-induced cell death. Given their ability to influence distinct signaling events, it is possible that CNK proteins act as signal integrators to orchestrate crosstalks between pathways (Laberge, 2005).

Intriguingly, although CNK activity is vital for RAS/MAPK signaling in Drosophila, it has opposite effects on RAF function. A structure/function analysis revealed that two domains (SAM and CRIC) located in the N-terminal region of CNK are integrating RAS signals, enabling RAF to phosphorylate MEK. However, the ability of CNK to associate with the RAF catalytic domain was mapped to a short bipartite element, named the RAF-inhibitory region (RIR), that strongly antagonizes MEK phosphorylation by RAF. Surprisingly, the RIR exerts its effect even in the presence of RAS signals, hence resulting in lower RAS-induced MAPK signaling output (Laberge, 2005).

The inhibitory effect of the RIR is relieved by an RTK-induced SFK signal. Specifically, a region located immediately C-terminal to the RIR including tyrosine 1163 (Y1163) is essential for CNK's positive function in vivo and for Sevenless (Sev) RTK-dependent MAPK activation. Upon SEV expression, one of the two SFKs found in Drosophila, Src42, associates and mediates (through the Y1163 region of CNK) RTK positive effects on the MAPK module. Remarkably, cnk loss-of-function mutations affecting the Y1163 region are fully compensated by inactivation of the RIR, thereby arguing that the Y1163 region is integrating the RTK-induced Src42 signal to counteract the RIR inhibitory function. Unexpectedly, genetic and molecular evidence has revealed that it is not Src42 catalytic function per se, but rather its binding capacity that is the key event in this process. Taken together, these results provide compelling evidence that CNK regulates RAF function by integrating both RAS and Src42 signals elicited by an RTK (Laberge, 2005).

This study shows that CNK integrates RAS and Src42 signals as a binary input, thereby allowing RAF to send signals to MEK. The RAS signal is received by the SAM and CRIC domains of CNK, which appears to enhance RAF catalytic function, whereas Src42 activity is integrated by the Y1163 region of CNK and seems to relieve the inhibitory effect that the RIR imposes on RAF's ability to phosphorylate MEK. Why would RAF activation depend on two distinct but corequired signals emitted by the same RTK? One possibility is that this requirement generates specificity downstream of an RTK. For example, only receptors that activate both RAS and Src42 would lead to activation of the MAPK module within discretely localized CNK complexes. Consequently, the combinatorial use of multifunctional signals might be a means to produce a specific output from generic signals (Laberge, 2005).

Intriguingly, despite the fact that the second Drosophila SFK, Src64, is naturally expressed in S2 cells, it does not act like Src42 in response to Sev, Egfr and InsR activation. Although the reason for this difference is not immediately clear, it was found that, unlike Tec29, overexpression of an Src64^YF variant is nonetheless capable of associating with CNK and inducing its tyrosine phosphorylation. It is thus possible that Src64 fulfills a similar role to Src42, but downstream of other RTKs or in response to other types of stimuli and that difference in either their subcellular localization, requirement for specific cofactors or additional regulatory events account for their distinct behavior (Laberge, 2005).

The mechanism by which the binding of Src42 to CNK deactivates the RIR is currently unknown and a number of scenarios can be envisioned. For example, it might induce a conformational change that suppresses the inhibitory effect that the RIR imposes on RAF catalytic activity. Alternatively, it is possible that Src42 binding displaces an inhibitory protein interacting with CNK or facilitates the relocalization of a CNK/RAF complex to a subcellular compartment that is required for RAS-dependent RAF activation. However, it is not believed that this mechanism involves displacing CNK away from RAF since neither Sev expression nor Src42 depletion alters the CNK/RAF interaction (Laberge, 2005).

Although several questions are left unanswered regarding the Src42/CNK association, collectively, the data suggest a subtle binding mode reminiscent of the mammalian Src/FAK interaction. Indeed, it appears that CNK is phosphorylated on the Y1163 residue not by Src42 itself, but either by the receptor or by another kinase and that this step generates a high-affinity binding site for the SH2 domain of Src42 thereby triggering its recruitment. This event is presumably not sufficient for a stable association and/or derepression of the RIR, but also requires the binding of the SH3 domain to an unidentified sequence element within CNK. The engagement of the SH3 and SH2 domains of Src42 on CNK would not only relieve the RIR's inhibitory effect, but would also derepress Src42 autoinhibited configuration and possibly orient favorably Src42 to phosphorylate one or a few specific tyrosine residues on CNK. This scenario is certainly plausible considering that CNK has a total of 39 tyrosine residues. This would explain why depletion of endogenous Src42 led to a reduction, but not a complete elimination, of SEV-induced CNK tyrosine phosphorylation or why the Y1163F mutation impairs CNK phosphorylation mediated by Src42^Y511F, since a disruption of the Src42/CNK association would prevent Src42 from phosphorylating the other sites. Although these Src42-dependent phosphorylated residues do not appear to play a role in activating the MAPK module, their concerted regulation suggests that CNK is coordinating signaling between the MAPK module and at least one other pathway (Laberge, 2005).

Dynamic localization of C. elegans TPR-GoLoco proteins mediates mitotic spindle orientation by extrinsic signaling via MES-1/Src

Cell divisions are sometimes oriented by extrinsic signals, by mechanisms that are poorly understood. Proteins containing TPR and GoLoco-domains (C. elegans GPR-1/2, Drosophila Pins, vertebrate LGN and AGS3) are candidates for mediating mitotic spindle orientation by extrinsic signals, but the mechanisms by which TPR-GoLoco proteins may localize in response to extrinsic cues are not well defined. The C. elegans TPR-GoLoco protein pair GPR-1/2 is enriched at a site of contact between two cells - the endomesodermal precursor EMS and the germline precursor P(2) - and both cells align their divisions toward this shared cell-cell contact. To determine whether GPR-1/2 is enriched at this site within both cells, mosaic embryos were generated with GPR-1/2 bearing a different fluorescent tag in different cells. It was surprising to find that GPR-1/2 distribution is symmetric in EMS, where GPR-1/2 had been proposed to function as an asymmetric cue for spindle orientation. Instead, GPR-1/2 is asymmetrically distributed only in P(2). A role for normal GPR-1/2 localization was demonstrated in P(2) division orientation. MES-1/Src signaling plays an instructive role in P(2) for asymmetric GPR-1/2 localization and normal spindle orientation. A model in which signaling localizes GPR-1/2 by locally inhibiting LET-99, a GPR-1/2 antagonist, was ruled out. Instead, asymmetric GPR-1/2 distribution is established by destabilization at one cell contact, diffusion, and trapping at another cell contact. Once the mitotic spindle of P(2) is oriented normally, microtubule-dependent removal of GPR-1/2 prevented excess accumulation, in an apparent negative-feedback loop. These results highlight the role of dynamic TPR-GoLoco protein localization as a key mediator of mitotic spindle alignment in response to instructive, external cues (Werts, 2011).

Alternative splicing of Src

The neural cell-specific N1 exon of the c-src pre-mRNA is negatively regulated in nonneural cells and positively regulated in neurons. Conserved intronic elements flanking N1 direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. These upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds (Chan, 1997).

The mouse c-src gene contains a short neuron-specific exon, N1. To characterize the sequences that regulate N1 splicing, use was made of a heterologous gene, derived from the human beta-globin gene, containing a short internal exon that is usually skipped by the splicing machinery. Various fragments from the src gene were inserted into the globin substrate to measure their effects on the splicing of the test exon. These clones were transiently expressed in neuronal and nonneuronal cell lines, and the level of exon inclusion was measured by primer extension. Several sequences from the N1 exon region induce the splicing of the heterologous exon. The most powerful effect is seen with a sequence from the intron downstream of the N1 exon. This sequence acts as a strong splicing enhancer, activating splicing of the test exon when placed in the intron downstream. The enhancer is strongest in neuronal LA-N-5 cells but also activates splicing in nonneuronal HEK293 cells. Deletion and linker scanning mutagenesis indicate that the enhancer is made up of multiple smaller elements that must act in combination. One of these elements was identified as the sequence UGCAUG. Three copies of this element can strongly activate splicing of the test exon in LA-N-5 neuroblastoma cells. These component elements of the src splicing enhancer are also apparently involved in the splicing of other short cassette exons (Modafferi, 1997).

Src functional specificity

Hck and Src are members of the Src family of protein-tyrosine kinases that carry out distinct and overlapping functions in vivo. In an attempt to understand how Hck and Src can function both independently and in concert, a comparison has been made of (1) their in vitro substrate specificity and (2) the accessibility of their Src homology 2 (SH2) domain. Using several synthetic peptides, it has been demonstrated that Hck and Src recognize similar structural features in the substrate peptides; this suggests that both kinases have the intrinsic ability to carry out overlapping cellular functions by phosphorylating similar cellular proteins in vivo. Using a phosphotyrosine-containing peptide that has previously been shown to bind the SH2 domain of Src family kinases with high affinity, it was found that although Src can bind to the phosphopeptide, Hck shows no interaction. The inability of Hck to bind the phosphopeptide is not a result of a stable intramolecular interaction between its SH2 domain and C-terminal regulatory phosphotyrosine residue (Tyr-520), because most Hck molecules in the purified Hck preparation are not tyrosine-phosphorylated. In contrast to intact Hck, a recombinant truncation analog of Hck is able to bind the phosphopeptide with an affinity similar to that of the Src SH2 domain, suggesting that conformational constraints are imposed on intact Hck that limit accessibility of its SH2 domain to the phosphopeptide. Furthermore, the difference in SH2 domain accessibility is a potential mechanism that enables Src and Hck to perform their respective unique functions by (1) targeting them to different subcellular compartments, whereupon they phosphorylate different cellular proteins, and/or (2) facilitating direct binding to their cellular substrates (Sicilia, 1998).

Src and Ca2+ signaling

Changes in the concentration of extracellular calcium can affect the balance between proliferation and differentiation in several cell types, including keratinocytes, breast epithelial cells, and fibroblasts. This report demonstrates that elevation of extracellular calcium stimulates proliferation-associated signaling pathways in rat fibroblasts and implicates calcium-sensing receptors (CaR) as mediators of this response. Rat-1 fibroblasts express CaR mRNA and protein and respond to known agonists of the CaR with increased IP3 production and the release of intracellular calcium. Agonists of the CaR can stimulate increased c-SRC kinase activity and increased extracellular signal-regulated kinase 1/mitogen-activated protein kinase activity. Both of the increases in SRC activity and mitogen-activated protein kinase activation are blocked in the presence of R796W, a nonfunctional mutant of the CaR. Proliferation of wild-type Rat-1 cells is sensitive to changes in extracellular calcium, but either the expression of the nonfunctional CaR mutant or the inhibition of the calcium-dependent increase in SRC kinase activity blocks the proliferative response to calcium. These results provide evidence of a novel signal transduction pathway modulating the response of fibroblasts to extracellular calcium and imply that calcium-sensing receptors may play a role in regulating cell growth in response to extracellular calcium, in addition to their well known function in systemic calcium homeostasis (McNeil, 1998).

There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown. In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of approximately 60 and 125 kDa are observed in immunoblots using an anti-phosphotyrosine antibody and have been identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies. FAK co-immunoprecipitated with c-Src, and the phosphorylation of the c-Src.FAK complex is markedly enhanced by platelet-derived growth factor (PDGF) BB. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppresses Ca2+ currents by 60, 62, and 43%, respectively. In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody does not inhibit Ca2+ currents. Co-dialysis of anti-c-Src and anti-FAK antibodies inhibits Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone. PDGF-BB enhances Ca2+ currents by 43%, which are abolished by the anti-c-Src and anti-FAK antibodies. Neither the MEK inhibitor PD 098059 nor an anti-Ras antibody inhibits basal Ca2+ currents or PDGF-stimulated Ca2+ currents. The alpha1C subunit of the L-type Ca2+ channel co-immunoprecipitates with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel. These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF-induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells (Hu, 1998).

Tec (Burton's) non-receptor tyrosine kinases - Src interacting proteins

A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. Tec and Bmx are found to activate serum response factor (SRF), in synergy with constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where thrombin's effect is mediated by G12/13 proteins. Expression of Galpha12 and Galpha13 stimulates the autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which has been previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3 does not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed. Thus, Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Galpha12/13 (Mao, 1998).

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal growth factor or endogenous platelet-derived growth factor receptors causes a rapid (less than 3 min) translocation of the cytosolic fluorescence to ruffle-like membrane structures. This response is not observed in cells expressing GFP only, and is completely inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004. Membrane-targeted PI 3-kinase also causes membrane localization of BtkPH-GFP that is slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain, which causes X-linked agammaglobulinemia, is introduced into the fluorescent construct, no translocation is observed after stimulation. In contrast, the E41K mutation, which confers transforming activity to native Btk, causes significant membrane localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type BtkPH-GFP, interferes with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P3 is likely to be an important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal organization of changes in this phospholipid in living cells (Varnai, 1999).

Src, Cbl and Abl form a ternary complex: Src interferes Abl phosphorylation of Cbl

The kinase activity of Abl is known to be regulated by a putative trans-acting inhibitor molecule interacting with the Src homology (SH) 3 domain of Abl. The kinase-deficient Src (SrcKD) directly inhibits the tyrosine phosphorylation of Cbl and other cellular proteins by Abl. Both the SH2 and SH3 domains of SrcKD are necessary for the suppressor activity toward the Abl kinase phosphorylating Cbl. To suppress the Cbl phosphorylation by Abl, the interaction between the SH3 domain of SrcKD and Cbl is required. This interaction between SrcKD and Cbl is regulated by a closed structure of Cbl. The binding of Abl to the extreme carboxyl-terminal region of Cbl unmasks the binding site of SrcKD to Cbl. This results in a ternary complex that inhibits the Abl-mediated phosphorylation of Cbl by steric hindrance. These results illustrate a mechanism by which the enzymatically inactive Src can exert a biological function in vivo (Shishido, 2000).

Cbl can form a closed structure that prevents the binding of Src to Cbl. The binding of Abl to Cbl is considered to trigger the structural changes in Cbl. In the case of other proteins that have a closed structure such as Src or Crk, the deletions of the C-terminal regions give rise to the formation of oncogenic proteins v-Src and v-Crk. Similarly, v-Cbl has a deletion of the C-terminal region of Cbl, including the proline-rich region of Cbl. These results suggest that the oncogenic activity residing in the N-terminal region of Cbl can be released because of a disruption of a closed structure of Cbl. In addition, Cbl has another oncogenic form, 70Z Cbl, which contains a 17-aa deletion between the N-terminal and the C-terminal regions. 70Z Cbl constitutively binds to Src although WT Cbl does not. This result also suggests that the constitutive conformational changes that open the structure of Cbl contribute to its oncogenic activation. The Cbl ring finger domain exhibits the ubiquitin ligase activity. The structural transition of Cbl induced by Abl might regulate the ligase activity because the ring finger domain is localized right next to the Src binding proline-rich region (Shishido, 2000).

In summary SrcKD directly inhibits Cbl phosphorylation by the Abl kinase. The analysis of mutants suggests that the complex of SrcKD and Cbl acts as an inhibitor of Abl. Although the physiological consequence of the interaction and the regulation of Abl, Cbl, and kinase-inactive form of WT Src is still unclear at the moment, it is possible that these interactions are important for the cytoskeleton reorganization. The kinase-activated Src activates Abl and the kinase-deficient Src reduces this activation in response to platelet-derived growth factor-regulating membrane ruffling. SrcKD regulates the cytoskeleton reorganization in osteoclasts, and Cbl has been found to act downstream of Src in these cells. The mechanism of Abl inhibition by SrcKD would not be limited to the inhibition of the WT Src kinase but would rather ensue from direct inhibition of the Abl kinase by SrcKD. Thus, this study suggests caution in interpreting data using a kinase-deficient molecule as a dominant negative to study the kinase activity (Shishido, 2000 and references therein).

p190 RhoGAP is a Src substrate

The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. The potent Rho regulatory protein p190 RhoGAP (GTPase-activating protein) has been identified as the principal Src substrate detected in the developing and mature nervous system. Mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, these results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase (Brouns, 2001).

It is proposed that the engagement of neural adhesion molecules results in Src activation and subsequent p190 RhoGAP phosphorylation in the developing brain. Such a signaling pathway most probably influences the various Rho-mediated actin reorganization events required for neural development, including the axonal guidance and fasciculation processes that are disrupted in the p190 RhoGAP mutant mice. Moreover, the defects in the morphogenesis of neuroepithelial tissues observed in p190 RhoGAP mutant mice may reflect disruption of the same pathways (Brouns, 2001).

Previous studies have shown that integrin/cell adhesion molecule-dependent signal transduction pathways are mediated by the Src kinases. In cultured neurons, activation of L1 or NCAM results in a rapid induction of the tyrosine phosphorylation of several proteins, and neurons lacking the Src or Fyn kinase are defective for neurite outgrowth on L1 or NCAM substrates, respectively. Src and Fyn, as well as L1, control axon fasciculation in the central nervous system. Experiments using fibroblasts lacking Src family members have shown that integrin engagement leads to inactivation of Rho through a mechanism requiring Src kinases, and that Rho inhibition correlates with Src-dependent tyrosine phosphorylation of p190 RhoGAP. These findings indicate that p190 RhoGAP mediates Src-dependent adhesion signaling to the actin cytoskeleton, and that cytoskeletal modulation almost certainly involves the Rho GTPase (Brouns, 2001 and references therein).

All of these data are consistent with a model for a Src/p190 RhoGAP adhesion pathway, which seeks to link the observed defects in the developing brain, the in vivo analysis of protein tyrosine phosphorylation, and the results of neuroblastoma cell-culture studies. Significantly, brain defects seen in Src/Fyn mutant mice resemble those seen in p190 RhoGAP mutant mice. Of particular interest are the axon guidance and fasciculation defects described in the olfactory system of Src/Fyn double-mutant mice at E11.5: the olfactory nerve is significantly defasciculated and axons 'wander' from the normal trajectory (Brouns, 2001 and references therein).

These fasciculation defects are strongly reminiscent of the defasciculation of cranial nerves detected at E10.5 in the p190 RhoGAP-/- mice described in this study, and the wandering behavior of olfactory axons reflects a similar disruption of axon guidance as that detected in the E16.5 cerebral cortex of p190 RhoGAP-/- mice. Notably, the Src/Fyn double-mutant mice, like the p190 RhoGAP-/- mice, die perinatally with axon guidance and fasciculation defects. However, additional functional redundancy provided by the tyrosine kinase Yes (which also appears to phosphorylate p190 RhoGAP in brain) and the p190-RhoGAP-related p190-B protein, together with the fact that there are likely to be additional Src family substrates in brain, make it difficult to relate the phenotypes among these various knockout animals in an unequivocal manner. Thus, definitive confirmation of the proposed relationships between Src kinase activity, p190 RhoGAP function and adhesion-mediated morphogenetic events await further genetic analysis (Brouns, 2001).

In addition to adhesion molecules, many different secreted growth factors, including epidermal growth factor, can promote tyrosine phosphorylation of p190 RhoGAP, suggesting that signals transduced by the numerous brain-expressed receptor tyrosine kinases may be mediated by p190 RhoGAP as well. Such receptors have also been implicated in a variety of morphogenetic events during neural development. In addition, mice lacking the Eph family receptor, EphB2 (Nuk), exhibit an anterior commissure guidance defect very similar to that seen in p190 RhoGAP mutant mice, suggesting that Eph ligands might signal through p190 RhoGAP. Significantly, EphB2 has been observed to associate with the binding partner of p190 RhoGAP -- p120 RasGAP. The fact that p190 RhoGAP is expressed widely throughout the nervous system at all stages of development, and is highly tyrosine phosphorylated in all regions of the brain, suggests that it is probably a common mediator of several diverse extracellular signals that affect many morphogenetic processes by precisely modulating Rho GTPase activity (Brouns, 2001).

SRC-family kinase Fyn phosphorylates the cytoplasmic domain of nephrin and modulates its interaction with podocin

Visceral glomerular epithelial cells (GEC) are critical for normal permselectivity of the kidney. Nephrin is a molecule that is expressed specifically in GEC in a structure called the slit diaphragm and is required for normal morphology and permselectivity of GEC. However, the mechanisms of action of nephrin are not understood precisely. The intracellular domain of nephrin has six conserved tyrosine residues. It was hypothesized that these tyrosine residues are phosphorylated by Src-family kinases and that this phosphorylation modulates the function of nephrin. A transient transfection system was used to study the role of tyrosine phosphorylation of the cytoplasmic domain of nephrin in its function. When nephrin was co-transfected with Src-family kinases Fyn or Src in Cos-1 cells, nephrin was strongly tyrosine phosphorylated by Fyn and less so by Src. The results with tyrosine-to-phenylalanine mutations suggested that multiple tyrosine residues contribute to phosphorylation mediated by Src-family kinases. The intracellular domain of nephrin is known to interact with another slit diaphragm protein, podocin. When nephrin and podocin were transfected with Fyn, the interaction between nephrin and podocin was augmented significantly. Podocin was not tyrosine phosphorylated by Fyn; thus, the increased interaction is likely to be secondary to tyrosine phosphorylation of nephrin. Fyn also significantly augmented the activation of the AP-1 promoter induced by nephrin and podocin. In summary, Fyn phosphorylates the cytoplasmic domain of nephrin on tyrosine, leading to enhanced association with podocin and downstream signaling of nephrin (Li, 2004).

Src targets FAK

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, a tryptic phosphopeptide mapping approach was used to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. Four tyrosines, 397, 407, 576, and 577, have been identified that are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII -- a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. Maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. These results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex (Calalb, 1995).

The ability of the focal adhesion kinase (FAK) to integrate signals from extracellular matrix and growth factor receptors requires the integrity of Tyr397, a major autophosphorylation site that mediates the Src homology 2-dependent binding of Src family kinases. However, the precise roles played by FAK in specific Src-induced pathways, especially as they relate to oncogenic transformation, remain unclear. The role of FAK in v-Src-induced oncogenic transformation was investigated by transducing temperature-sensitive v-Src (ts72v-Src) into p53-null FAK+/+ or FAK-/- mouse embryo fibroblasts (MEF). At the permissive temperature (PT), ts72v-Src induces abundant tyrosine phosphorylation, morphological transformation and cytoskeletal rearrangement in FAK-/- MEF, including the restoration of cell polarity, typical focal adhesion complexes, and longitudinal F-actin stress fibers. v-Src rescues the haptotactic (referring to cell-motility toward substratum-bound insolubilized extracellular matrix components), linear directional, and invasive motility defects of FAK-/- cells to levels found in FAK+/+ or FAK+/+-[ts72v-Src] cells, and, in the case of monolayer wound healing motility, there is an enhancement. Src activation failed to increase the high basal tyrosine phosphorylation of the Crk-associated substrate, CAS, found in FAK-/- MEF, indicating that CAS phosphorylation alone is insufficient to induce motility in the absence of FAK- or v-Src-induced cytoskeletal remodeling. Compared with FAK+/+[ts72v-Src] controls, FAK-/-[ts72v-Src] clones exhibited 7-10-fold higher anchorage-independent proliferation that could not be attributed to variations in either v-Src protein level or stability. Re-expression of FAK diminished the colony-forming activities of FAK-/-[ts72v-Src] without altering ts72v-Src expression levels, suggesting that FAK attenuates Src-induced anchorage independence. These data also indicate that the enhanced Pyk2 level found in FAK-/- MEF plays no role in v-Src-induced anchorage independence. Overall, these data indicate that FAK, although dispensable, attenuates v-Src-induced oncogenic transformation by modulating distinct signaling and cytoskeletal pathways (Moissoglu, 2003).

Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell proliferation, survival, migration, and invasion. To understand how FAK contributes to cell invasion, the regulation of matrix metalloproteinases (MMPs) by FAK was explored. v-Src-transformed cells activate a FAK-dependent mechanism that attenuates endocytosis of MT1-MMP. This in turn increases cell-surface expression of MT1-MMP and cellular degradation of extracellular matrix. Further, an interaction between FAK's second Pro-rich motif and endophilin A2's SH3 domain was identified. This interaction served as an autophosphorylation-dependent scaffold to allow Src phosphorylation of endophilin A2 at Tyr315. Tyr315 phosphorylation inhibits endophilin/dynamin interactions, and blockade of Tyr315 phosphorylation promotes endocytosis of MT1-MMP. Together, these results suggest a regulatory mechanism of cell invasion whereby FAK promotes cell-surface presentation of MT1-MMP by inhibiting endophilin A2-dependent endocytosis (Wu, 2005).

Structural basis for the recognition of c-Src by its inactivator Csk

The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. The crystal structure was determined of a complex between the kinase domains of Csk and c-Src at 2.9 Å resolution, revealing that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate binding site used by most tyrosine kinases to recognize substrates is destabilized in Csk by a deletion in the activation loop (Levenson, 2008).

Activation of Src by dephosphosphorylation

Protein tyrosine phosphatase alpha (PTPalpha) is believed to dephosphorylate the Src proto-oncogene at phosphotyrosine (pTyr)527, a critical negative-regulatory residue. It thereby activates Src, and PTPalpha overexpression neoplastically transforms NIH 3T3 cells. pTyr789 in PTPalpha is constitutively phosphorylated and binds Grb2, an interaction that may inhibit PTPalpha activity. This phosphorylation also specifically enables PTPalpha to dephosphorylate pTyr527. Tyr789 to Phe mutation abrogates PTPalpha-Src binding, dephosphorylation of pTyr527 (although not of other substrates), and neoplastic transformation by overexpressed PTPalpha in vivo. It is suggested that pTyr789 enables pTyr527 dephosphorylation by a pilot binding with the Src SH2 domain that displaces the intramolecular pTyr527-SH2 binding. Consistent with model predictions, it is found that excess SH2 domains can disrupt PTPalpha-Src binding and can block PTPalpha-mediated dephosphorylation and activation in proportion to their affinity for pTyr789. Moreover, as predicted by the model, catalytically defective PTPalpha has reduced Src binding in vivo. The displacement mechanism provides another potential control point for physiological regulation of Src-family signal transduction pathways (Zheng, 2000).

Src role in membrane traffic

Caveolae are plasma membrane specializations present in most cell types. Caveolin, a 22-kDa integral membrane protein, is a principal structural and regulatory component of caveolae membranes. Previous studies have demonstrated that caveolin co-purifies with lipid modified signaling molecules, including Galpha subunits (See Drosophila G protein salpha 60A), H-Ras, c-Src (see Drosophila (Src oncogene 1), and other related Src family tyrosine kinases. In addition, it has been shown that caveolin interacts directly with Galpha subunits and H-Ras, preferentially recognizing the inactive conformation of these molecules. However, it is not known whether caveolin interacts directly or indirectly with Src family tyrosine kinases. In this study, the structural and functional interaction of caveolin with Src family tyrosine kinases is examined. Caveolin interacts with wild-type Src (c-Src) but does not form a stable complex with mutationally activated Src (v-Src). Thus, it appears that caveolin prefers the inactive conformation of Src. Deletion mutagenesis indicates that the Src-interacting domain of caveolin is located within residues 82-101, a cytosolic membrane-proximal region of caveolin. A caveolin peptide derived from this region (residues 82-101) functionally suppressed the auto-activation of purified recombinant c-Src tyrosine kinase and Fyn, a related Src family tyrosine kinase. The effect of caveolin on c-Src activity was analyzed in vivo by transiently co-expressing full-length caveolin and c-Src tyrosine kinase in 293T cells. Co-expression with caveolin dramatically suppressed the tyrosine kinase activity of c-Src as measured via an immune complex kinase assay. Thus, it appears that caveolin structurally and functionally interacts with wild-type c-Src via caveolin residues 82-101. Besides interacting with Src family kinases, this cytosolic caveolin domain (residues 82-101) has the following unique features: (1) it is required to form multivalent homo-oligomers of caveolin. (2) it interacts with G-protein alpha-subunits and down-regulates their GTPase activity; (3) it binds to wild-type H-Ras and (4) it is membrane-proximal, suggesting that it may be involved in other potential protein-protein interactions. Thus, this 20-amino acid stretch of caveolin residues has been termed the caveolin scaffolding domain (Li, 1996).

The nonreceptor tyrosine kinase Src is expressed at a high level in cells that are specialized for regulated secretion, such as the neuron, and is concentrated on secretory vesicles or at the site of exocytosis. To investigate the possibility that Src may play a role in regulating membrane traffic, neuronal proteins were sought that interact with Src. The SH3 domain of Src, but not that of the splice variant N-Src, binds to three proteins from mouse synaptosomes or PC12 cells: dynamin, synapsin Ia, and synapsin Ib. Dynamin and the synapsins coprecipitate with Src from PC12 cell extracts, and they colocalize with a subset of Src in the PC12 cell by immunofluorescence. Neither dynamin nor the synapsins are phosphorylated by Src, suggesting that the interaction of these proteins serves to direct the kinase activity of Src toward other proteins in the vesicle population. In immunoprecipitates containing Src and dynamin, the clathrin adaptor protein alpha-adaptin is also found. The association of Src and synapsin suggests a role for Src in the life cycle of the synaptic vesicle. The identification of a complex containing Src, dynamin, and alpha-adaptin indicates that Src may play a more general role in membrane traffic as well (Foster-Barber, 1998).

Receptor-mediated endocytosis allows the specific removal of cell surface receptors and their cargo from the plasma membrane and targets them to endosomes, where they are sorted for downregulation or recycling. This process is initiated by recruitment of the receptor into a clathrin-coated pit at the plasma membrane, a structure formed by assembly of clathrin and adaptors into a protein lattice on the membrane's cytosolic face. Polymerization of clathrin into a hexagonal array provides a scaffold for organizing the adaptors, which recognize sequence motifs in the cytoplasmic domains of internalized receptors. A novel aspect of ligand-induced endocytosis of the epidermal growth factor receptor (EGFR) is described. Receptor signaling, upon ligand binding, stimulates modification and recruitment of clathrin. A partial explanation for the difference between constitutively endocytosed receptors and those whose endocytosis is ligand induced is that the adaptor recognition signal is constitutively accessible in the former, but cryptic in the latter, until ligand binding has occurred. For example, ligand binding to EGFR causes receptor tyrosine kinase activation and autophosphorylation. The implication of downstream signaling and effects on clathrin in several examples of ligand-induced endocytosis suggests a possible relationship between these processes, particularly in the case of the dramatic clathrin recruitment following receptor tyrosine kinase activation (Wilde, 1999).

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling (Wilde, 1999).

Tyrosine 1477 has been identified as the site of pp60c-src-mediated clathrin heavy chain (CHC) phosphorylation during ligand-induced endocytosis of EGFR. Tyrosine 1477 is conserved in the three mammalian CHC sequences that have been determined and in the CHC of D. melanogaster, D. discoidum, and S. cerevisiae. Structural analysis of the light chain-binding region of CHC confirms that tyrosine 1477 is solvent exposed and located near the predicted clathrin light chain-binding site. The clathrin light chain subunits negatively regulate spontaneous clathrin assembly, so that cellular clathrin assembly is adaptor dependent. It is possible that tyrosine phosphorylation of residue 1477 causes an increase in clathrin assembly by directly affecting CHC interactions or that it affects assembly indirectly by negating the inhibitory effects that light chains have on assembly. Alternatively, tyrosine phosphorylation of CHC could recruit a protein that might enhance assembly or enhance transport of clathrin to the cell periphery. Whether clathrin recruitment directly influences EGF uptake by increasing the local concentration of clathrin and promoting coated pit formation or whether the regulatory mechanism is more complex, possibly relating to changing the intracellular dynamics of clathrin, will be the focus of future studies. It should be pointed out that the phosphorylation and recruitment of clathrin represent only a subset of the molecular requirements for EGF endocytosis. In mouse fibroblasts, CHC phosphorylation is necessary and sufficient for clathrin recruitment in some cell lines (SV40 transformed) but not sufficient in others (3T3-like), implicating additional factors. In the case of a pathway as important as regulated endocytosis, it would not be surprising if, in different tissues, different SRC family kinases could mediate clathrin phosphorylation (Wilde, 1999).

Src and the cytoskeleton

continued: Src oncogene at 64B Evolutionary homologs part 2/3 | part 3/3 |

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