Src oncogene at 64B


Src and the cytoskeleton

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src becomes activated upon EGF stimulation. c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell-cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend on the presence of the actin binding domain of the EGF-receptor, since in cells expressing EGF-receptors that lack this domain, EGF fails to induce an activation and a translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src (Van der Heyden, 1997).

The interaction of the non-receptor tyrosine kinase, Src, with the cytoskeleton of adhesion sites was studied in nerve growth cones isolated from fetal rat brain. Of particular interest is the role of protein tyrosine phosphatases in the regulation of Src-cytoskeleton binding. Growth cones contain a high level of protein tryrosine phosphatase activity, most of it membrane-associated and forming large, multimeric and wheat germ agglutinin-binding complexes. The receptor tyrosine phosphatase PTP(alpha) seems to be the most prevalent species among the membrane-associated enzymes. As seen by immunofluorescence, PTP(alpha) is present throughout the plasmalemma of the growth cone, including filopodia, and it forms a punctate pattern consistent with that of integrin beta1. For adhesion site analysis, isolated growth cones were plated either onto laminin (the neurite growth substratum) or kept in suspension. Plating growth cones on laminin triggers an 8-fold increase in Src binding to the adherent cytoskeleton. This effect is blocked completely with the protein tyrosine phosphatase inhibitor, vanadate. Growth cone plating also increases the association with adhesion sites of tyrosine phosphatase activity (14-fold) and of PTP(alpha) immunoreactivity (6-fold). Vanadate blocks the enzyme activity but not the recruitment of PTP(alpha) to the adhesion sites. These data suggest that integrin binding to laminin triggers the recruitment of PTP(alpha) (and perhaps other protein tyrosine phosphatases) to adhesion sites, resulting in de-phosphorylation of Src's tyr 527. As a result, Src unfolds, becomes kinase-active, and its SH2 domain can bind to an adhesion site protein. This implies a critical role for protein tyrosine phosphatase activity in the earliest phases of adhesion site assembly (Helmke, 1998).

Osteopontin binding to integrin alphav beta3 in osteoclasts stimulates gelsolin-associated phosphatidylinositol (PtdIns) 3-hydroxyl kinase (PI 3-kinase), and leads to increased levels of gelsolin-bound PtdIns 3,4-P2, PtdIns 4,5-P2, and PtdIns 3, 4,5-P3, as well as the uncapping of barbed end actin, and actin filament formation. Inhibition of PI 3-kinase activity by wortmannin blocks osteopontin stimulation of actin filament formation, suggesting that activation of gelsolin-associated PI 3-kinase is an important pathway in cytoskeletal regulation. To study the mechanism of gelsolin-associated PI 3-kinase activation, anti-gelsolin immunoprecipitates were analyzed for the association of protein kinases. c-Src co-immunoprecipitates with gelsolin, and osteopontin stimulates its activity. Elimination of osteopontin-stimulated Src activity associated with gelsolin through antisense oligodeoxynucleotides blocks the stimulation of PI 3-kinase activity associated with gelsolin and the gelsolin-dependent cytoskeletal reorganization induced by osteopontin, including increased F-actin levels. In addition, treatment of osteoclasts with antisense oligonucleotides to Src reduces bone resorption. These results demonstrate that osteopontin stimulates gelsolin-associated Src, leading to increased gelsolin-associated PI 3-kinase activity and PtdIns 3,4,5-P3 levels, which facilitate actin filament formation, osteoclast motility, and bone resorption (Chellaiah, 1998).

Intramolecular SH2 and SH3 interactions mediate enzymatic repression of the Src kinases. One mechanism of activation is disruption of these interactions by the formation of higher affinity SH2 and SH3 interactions with specific ligands. A consensus Src SH3-binding site residing upstream of the Src SH2-binding site in FAK can function as a ligand for the Src SH3 domain. Surface plasmon resonance experiments indicate that a FAK peptide containing both the Src SH2- and SH3-binding sites exhibits increased affinity for Src. The presence of both sites in vitro more potently activates c-Src. A FAK mutant (FAKPro-2) with substitutions destroying the SH3-binding site shows reduced binding to Src in vivo. This mutation also reduces Src-dependent tyrosine phosphorylation on the mutant itself and downstream substrates, such as paxillin. These observations suggest that an SH3-mediated interaction between Src-like kinases and FAK may be important for complex formation and downstream signaling in vivo (Thomas, 1998).

The v-Src oncoprotein perturbs the dynamic regulation of the cellular cytoskeletal and adhesion network by a mechanism that is poorly understood. The effects of a temperature-dependent v-Src protein were examined on the regulation of p190 RhoGAP (see Drosophila RhoGAP), a GTPase activating protein (GAP) that has been implicated in disruption of the organized actin cytoskeleton. Also addressed was the dependence of v-Src-induced stress fiber loss on inhibition of Rho activity. Activation of v-Src induces association of tyrosine phosphorylated p190 with p120(RasGAP) and stimulation of p120(RasGAP)-associated RhoGAP activity, although p120(RasGAP) itself is not a target for phosphorylation by v-Src in chicken embryo cells. These events require the catalytic activity of v-Src and are linked to loss of actin stress fibers during morphological transformation and not mitogenic signaling. Furthermore, these effects are rapidly reversible since switching off v-Src leads to dissociation of the p190/p120(RasGAP) complex, inactivation of p120(RasGAP)-associated RhoGAP activity and re-induction of actin stress fibers. In addition, transient transfection of Val14-RhoA, a constitutively active Rho protein that is insensitive to RhoGAPs, suppresses v-Src-induced stress fiber loss and cell transformation. Thus, an activated Src kinase requires the inactivation of Rho-mediated actin stress fiber assembly to induce its effects on actin disorganization. This work supports p190 as a strong candidate effector of v-Src-induced cytoskeletal disruption, most likely mediated by antagonism of the cellular function of Rho (Fincham, 1999).

While previous reports have indicated that the temporal mitogen- and integrin-induced activation of ERK is linked to its spatial organization, by stimulation of nuclear translocation, this study shows that ERK can also be targeted to sites of cellular attachment where it is present in its active form. Integrin engagement generates cellular signals leading to the recruitment of structural and signalling molecules which, in concert with rearrangements of the actin cytoskeleton, leads to the formation of focal adhesion complexes. Using antisera reactive either with total ERK or with phosphorylated/activated forms of ERK, in rat embryo fibroblasts and embryonic avian cells that express v-Src, it has been found that active ERK is targeted to newly forming focal adhesions after integrin engagement or activation of v-Src. UO126, an inhibitor of MAP kinase kinase 1 (MEK1), suppresses focal adhesion targeting of active ERK and cell spreading. Also, integrin engagement and v-Src induces myosin light chain kinase (MLCK)-dependent phosphorylation of myosin light chain downstream of the MEK/ERK pathway, and MLCK and myosin activities are required for the focal adhesion targeting of ERK. The translocation of active ERK to newly forming focal adhesions may direct specificity towards appropriate downstream targets that influence adhesion assembly. These findings support a role for ERK in the regulation of the adhesion/cytoskeletal network and provide an explanation for the role of ERK in cell motility (Fincham, 2000).

The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. In fibroblasts, activation of Rho GTPases, RhoA, Rac1, and Cdc42 by different transmembrane receptors leads to distinct rearrangements of the actin cytoskeleton. Activation of RhoA stimulates actomyosin-based contractility, which leads to the assembly of actin stress fibers and focal adhesions found at the end of stress fibers. Rac1 activation leads to localized actin polymerization at the cell periphery, resulting in the formation of lamellipodia, while activation of Cdc42 results in the formation of fine actin-rich protrusions, known as filopodia. Rac1 and Cdc42 stimulate the assembly of focal complexes that are associated with lamellipodia and filopodia, respectively. They contain a number of the same proteins found in Rho-induced focal adhesions, including vinculin, paxillin, and focal adhesion kinase. It is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. Individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively. The adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. These findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites (Timpson, 2001).

The importance of the SH3 domain of Src family kinase Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, mass spectrometry was used to identify approximately 25 potential binding partners for the SH3 domain of Hck from the monocyte cell line U937. Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP). Focus was also placed on a novel interaction between Hck and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegans gene, ced-12. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. Using purified proteins, it was confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of Hck. Hck and ELMO1 interact in intact cells and ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck. The binding of ELMO1 to Hck is specifically dependent on the interaction of a polyproline motif with the SH3 domain of Hck. These results suggest that these proteins may be novel activators/effectors of Hck (Scott, 2002).

Src as an agent of receptors and other surface proteins

The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated. In vivo tyrosine phosphorylation sites of the Eph receptors EphA3, EphA4, and EphB2 in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase. Tyrosine-phosphorylated EphB2 is detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate. Interestingly, a form of Src with reduced electrophoretic mobility and increased tyrosine phosphorylation is detected in Cos cells expressing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src. Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrates that phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src. In contrast, binding of the carboxy-terminal SH2 domain of phospholipase Cgamma is not abolished upon mutation of EphB2's tyrosine 611. Phosphopeptide mapping of autophosphorylated full-length EphB2, and wild-type and tyrosine-to-phenylalanine mutants of the EphB2 cytoplasmic domain fused to LexA, show tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in EphB2. Mutational analysis also indicates that tyrosines 605 and 611 are important for EphB2 kinase activity. It is proposed that Src kinase is a downstream effector that mediates the neuron's response to Eph receptor activation (Zisch, 1998).

There is a requirement for the nonreceptor tyrosine kinase, cellular Src (c-Src), in epidermal growth factor (EGF)-induced mitogenesis, and a synergistic interaction between c-Src and EGF receptor (EGFR) in tumorigenesis. Although endocytic internalization of EGFR may be thought to attenuate EGF-stimulated signaling, recent evidence suggests that signaling through Ras can be amplified by repeated encounters of endosome-localized receptor. Shc.Grb2.Sos complexes with the plasma membrane, where Ras resides almost exclusively. Based on these reports, EGFR trafficking behavior was examined in a set of single and double c-Src/EGFR C3H10T1/2 overexpressors to determine if c-Src affects basal receptor half-life, ligand-induced internalization, and/or recycling. Overexpression of c-Src causes no change in EGFR half-life but does produce an increase in the internalization rate constant of EGF.EGFR complexes, when the endocytic apparatus is not stoichiometrically saturated; this effect of c-Src on EGFR endocytosis is negligible at high receptor occupancy in cells overexpressing the receptor. In neither case are EGFR recycling rate constants affected by c-Src. These data indicate a functional role for c-Src in receptor internalization, which in turn could alter some aspects of EGFR signaling related to mitogenesis and tumorigenesis (Ware, 1997).

Growth factor receptor tyrosine kinase (RTK)-activated signaling pathways are well established regulators of neuronal growth and development, but whether these signals provide mechanisms for acute modulation of neuronal activity is just beginning to be addressed. In pheochromocytoma (PC12) cells, acute application of ligands for both endogenous RTKs [trkA, basic FGF (bFGF) receptor, and epidermal growth factor (EGF) receptor] and ectopically expressed platelet-derived growth factor (PDGF) receptors rapidly inhibits whole-cell sodium channel currents, coincident with a hyperpolarizing shift in the voltage dependence of inactivation. Sodium channel inhibition by trkA and PDGF receptors is mutually occlusive, suggestive of a common signal transduction mechanism. Furthermore, specific inhibitors for trkA and PDGF RTK activities abrogate sodium channel inhibition in response to NGF and PDGF, respectively, showing that the intrinsic RTK activity of these receptors is necessary for sodium channel inhibition. Use of PDGF receptor mutants deficient for specific signaling activities demonstrates that this inhibition is dependent on RTK interaction with Src but not with other RTK-associated signaling molecules. Inhibition is also compromised in cells expressing dominant-negative Ras. These results suggest a possible mechanism for the acute physiological actions of RTKs, and they indicate regulatory functions for Ras and Src that may complement the roles of these signaling proteins in long-term neuronal regulation (Hilborn, 1998).

The c-Abl tyrosine kinase localizes to the cytoplasm and plasma membrane in addition to the nucleus. However, there is little information regarding a role for c-Abl in the cytoplasm/plasma membrane compartments. A membrane pool of c-Abl is activated by the growth factors PDGF and EGF in fibroblasts. The pattern and kinetics of activation are similar to growth factor activation of Src family kinases. To determine whether a link exists between activation of c-Abl and members of the Src family, c-Abl kinase activity was examined in cells that express oncogenic Src proteins. c-Abl kinase activity is increased by 10- to 20-fold in these cells, and Src and Fyn kinases are shown to directly phosphorylate c-Abl in vitro. Furthermore, overexpression of wild-type Src potentiates c-Abl activation by growth factors, and a kinase-inactive form of Src reduces this activation, showing that Abl activation by growth factors occurs at least in part via activation of Src kinases. Significantly, c-Abl has a functional role in the morphological response to PDGF. Whereas PDGF treatment of serum-starved wild-type mouse embryo fibroblasts results in distinct linear or circular/dorsal membrane ruffling, c-Abl-null cells demonstrate dramatically reduced ruffling in response to PDGF, which is rescued by physiological re-expression of c-Abl. These data identify c-Abl as a downstream target of activated receptor tyrosine kinases and Src family kinases, and show for the first time that c-Abl functions in the cellular response to growth factors (Plattner, 1999).

c-Src is a membrane-associated tyrosine kinase that can be activated by many types of extracellular signals, and can regulate the function of a variety of cellular protein substrates. Epidermal growth factor (EGF) and beta-adrenergic receptors activate c-Src by different mechanisms leading to the phosphorylation of distinct sets of c-Src substrates. In particular, it was found that EGF receptors, but not beta2-adrenergic receptors, activate c-Src by a Ral-GTPase-dependent mechanism. EGF has been known to activate c-Src, but the mechanism involved is not known. Therefore, to determine whether Ral activation by Ral-GEFs is required for EGF-induced c-Src activation, 293 cells were transiently transfected with either dnRal or a control vector. c-Src was then immunoprecipitated and its kinase activity measured in vitro. In control cells, EGF led to an ~2.7-fold increase in the activation of c-Src. However, in cells expressing dnRal, c-Src activation upon exposure to EGF is reduced by ~75%. This value is an underestimate of the true inhibitory effect of the protein since only ~70% of cells were transfected with dnRal while all of the cells were stimulated with EGF. These findings were confirmed in 3Y1(EFGR) cells, where the stable expression of inhibitory RalA28N also suppresses EGF-induced activation of c-Src by ~75%. Importantly, EGF activation of Erk is not blocked in the same cells, demonstrating clear specificity in the effects of Ral on EGF receptor signaling. Also, c-Src activated by EGF treatment or expression of constitutively activated Ral-GTPase leads to tyrosine phosphorylation of Stat3 and cortactin, but not Shc or subsequent Erk activation. In contrast, c-Src activated by isoproterenol leads to tyrosine phosphorylation of Shc and subsequent Erk activation, but not tyrosine phosphorylation of cortactin or Stat3. These results identify a role for Ral-GTPases in the activation of c-Src by EGF receptors and the coupling of EGF to transcription through Stat3 and the actin cytoskeleton through cortactin. They also show that c-Src kinase activity can be used differently by individual extracellular stimuli, possibly contributing to their ability to generate unique cellular responses (Goi, 2000).

The semaphorins/collapsins constitute a family of genes unified by the presence of a "semaphorin domain" that has been conserved through metazoan evolution. The semaphorin family comprises both secreted and transmembrane molecules and is thought to be made up of ligands for as yet unidentified receptors. The functions are not known, with the exception of those of sema III (also referred as sem D and collapsin 1), D-sema I, and D-sema II, which have been shown to be involved in axonal pathfinding. A mouse semaphorin cDNA, termed Sema VIb is described. Although Sema VIb contains the extracellular semaphorin domain, it lacks both the immunoglobulin domain and thrombospondin repeats, which are present in other described vertebrate (but not invertebrate) transmembrane semaphorins. During development Sema VIb mRNA is expressed in subregions of the nervous system and is particularly prominent in muscle. In adulthood, Sema VIb mRNA is expressed ubiquitously. The cytoplasmic domain of Sema VIb contains several proline-rich potential SH3 domain binding sites. Using an in vitro binding assay, it is shown that Sema VIb binds specifically the SH3 domain of the protooncogene c-src. In transfected COS cells Sema VIb coimmunoprecipitates with c-src. These results, along with evidence that Sema VIb can form dimers, suggest that the semaphorin family not only serves as ligands but may include members, especially those which are transmembrane, which serve as receptors, triggering intracellular signaling via an src-related cascade (Eckhardt, 1997).

PECAM-1 is an adhesion molecule expressed on hemopoietic and endothelial cells. PECAM-1 becomes tyrosine-phosphorylated in response to a variety of physiological stimuli. Furthermore, tyrosine-phosphorylated PECAM-1 associates with SHP-2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase expressed ubiquitously. In light of the significance of tyrosine protein phosphorylation as a regulatory mechanism, an investigation was carried out of the nature and impact of the protein-tyrosine kinases (PTKs) mediating PECAM-1 tyrosine phosphorylation. Through reconstitution experiments in COS-1 cells, it was determined that mouse PECAM-1 can be tyrosine-phosphorylated by Src-related PTKs and Csk-related PTKs (see Drosophila Csk), but not by other kinases such as Syk, Itk, and Pyk2. Using site-directed mutagenesis and peptide phosphorylation studies, it was found that these PTKs are efficient at phosphorylating Tyr-686, but not Tyr-663, of PECAM-1. Src-related enzymes also phosphorylate mouse PECAM-1 at one or more yet to be identified sites. Phosphorylation of PECAM-1 by Src or Csk family kinases is sufficient to trigger its association with SHP-2. Moreover, phosphorylation is able to promote binding of PECAM-1 to SHP-1, a SHP-2-related protein-tyrosine phosphatase expressed in hemopoietic cells. Taken together, these findings indicate that the Src and Csk families of kinases are strong candidates for mediating tyrosine phosphorylation of PECAM-1 and triggering its association with SH2 domain-containing phosphatases under physiological circumstances (Cao, 1998).

N-syndecan (syndecan-3 - see Drosophila Syndecan) is a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and is suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. N18 neuroblastoma cells were transfected with a rat N-syndecan cDNA and it was shown that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. It is suggested that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway (Kinnunen, 1998).

SHPS-1 is a receptor-like glycoprotein that undergoes tyrosine phosphorylation and binds SHP-2, a Src homology 2 domain containing protein tyrosine phosphatase, in response to various mitogens. Cell adhesion to extracellular matrix proteins, such as fibronectin and laminin, also induce the tyrosine phosphorylation of SHPS-1 and its association with SHP-2. These responses are markedly reduced in cells overexpressing the Csk kinase or in cells that lack focal adhesion kinase or the Src family kinases Src or Fyn. However, unlike Src, focal adhesion kinase does not catalyze phosphorylation of the cytoplasmic domain of SHPS-1 in vitro. Overexpression of a catalytically inactive SHP-2 markedly inhibits activation of mitogen-activated protein (MAP) kinase in response to fibronectin stimulation without affecting the extent of tyrosine phosphorylation of focal adhesion kinase or its interaction with the docking protein Grb2. Overexpression of wild-type SHPS-1 does not enhance fibronectin-induced activation of MAP kinase. These results indicate that the binding of integrins to the extracellular matrix induces tyrosine phosphorylation of SHPS-1 and its association with SHP-2, and that such phosphorylation of SHPS-1 requires both focal adhesion kinase and an Src family kinase. In addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, SHP-2 may play an important role, partly through its interaction with SHPS-1, in the activation of MAP kinase in response to the engagement of integrins by the extracellular matrix (Tsuda, 1998).

Cell motility on extracellular-matrix (ECM) substrates depends on the regulated generation of force against the substrate through integrins. Integrin-mediated traction forces can be selectively modulated by the tyrosine kinase Src. In Src-deficient fibroblasts, cell spreading on the ECM component vitronectin is inhibited, while the strengthening of linkages between integrin vitronectin receptors and the force-generating cytoskeleton in response to substrate rigidity is dramatically increased. In contrast, Src deficiency has no detectable effects on fibronectin-receptor function. Finally, truncated Src (lacking the kinase domain) co-localizes to focal-adhesion sites with alphav but not with beta1 integrins. These data are consistent with a selective, functional interaction between Src and the vitronectin receptor. This interaction takes place at an integrin-cytoskeleton interface to regulate cell spreading and migration (Felsenfeld, 1999).

The precise site of action of Src and its location in the signaling cascade that regulates vitronectin-receptor function remain unclear. The Src-family kinases Fyn and Src bind the focal-adhesion kinase (FAK), allowing an indirect interaction between these kinases and the cytoplasmic tail of the integrin beta-subunit. Fyn may also selectively associate with the fibronectin receptor through an indirect association with the integrin alpha5 subunit. However, these pathways have been identified on the basis of biochemical association and changes in kinase activity downstream of integrin activation ('outside-in' signaling), in contrast to the modulation of integrin function by Src described here (Felsenfeld, 1999).

These results indicate that Src may act at or near the cytoplasmic membrane. First, the vitronectin-dependent localization of Src to stable pools on the bottom of the cell and the co-localization of Src with alphav but not beta1 integrins is consistent with the selective association of Src and vitronectin receptor at the membrane. Moreover, Src co-precipitates with vitronectin receptor but not fibronectin receptor in whole-cell lysates, indicating a selective association that is consistent with the functional specificity shown in this study. Finally, force generation through the vitronectin receptor and fibronectin receptor is likely to involve many of the same proteins, including actin/myosin and structural components of the focal-adhesion complex such as vinculin, talin and others. Therefore, it is inferred that Src acts at or near the cytoplasmic tail of the vitronectin receptor, at the point at which the pathways transducing force through vitronectin and fibonectin receptors diverge. The ability of cells to recognize and respond to the rigidity of the cellular environment implies the existence of a force-sensing apparatus within the cell. These results indicate that Src forms a regulatory component of the complex that links integrins to the cytoskeleton (Felsenfeld, 1999).

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, it has been found that a molecular complex comprising Src, Pyk2 (a calcium-dependent tyrosine kinase that is the predominant FAK family member in the adhesion structures of osteoclasts), and Cbl functions to regulate cell adhesion and motility. The activation of integrin alphavß3 induces the [Ca2+]i-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Deletion of c-Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alphavß3 integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src-/- mice (Sanjay, 2001).

Cyclic attachment and detachment of individual podosomes is required as cells, particularly highly motile cells such as macrophages and osteoclasts, migrate over a substratum. Pyk2, Src, and Cbl appear to play a pivotal role in these processes. Deletion of Src significantly alters the initial distribution of podosomes in osteoclasts, eventually leading to the formation of focal adhesion-like structures. This shift from podosome to focal adhesion correlates with a decrease in the formation and motility of lamellipodia and in cell migration, although it is not possible to determine which comes first. Adhesion to vitronectin or activation of the alphavß3 receptor induces an increase in intracellular calcium that does not depend on the presence of Src. The formation of a kinase-rich trimolecular complex results in the Pyk2- and Src-dependent phosphorylation of Cbl. Therefore, Pyk2, Src, and Cbl are all involved in the 'outside-in' alphavß3 integrin signaling which results in podosome assembly. In transfected 293-VnR cells, the PTB domain of Cbl binds to Tyr 416 in the activation loop of the Src kinase domain, and this interaction downregulates both Src kinase activity and integrin-mediated adhesion. Thus, Cbl might also be crucial in 'inside-out' signaling, playing a key role in podosome detachment and subsequent disassembly. In agreement with this hypothesis, deletion of the gene encoding Cbl, like the deletion of the gene encoding Src, leads to a significant, albeit smaller, decrease in osteoclast migration. This series of events could form the basis for the cyclic attachment-detachment of single adhesion sites at the leading edge of lamellipodia in motile cells, and thereby participates in the assembly-disassembly of individual podosomes, thereby ensuring cell adhesion while still allowing cell motility (Sanjay, 2001).

Src family kinases (SFKs) have been implicated as important regulators of ligand-induced cellular responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been impeded by extensive redundancy between family members. Mouse embryos were generated harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and tested for their ability to respond to growth factors or plating on extracellular matrix. While Src, Yes and Fyn are largely dispensable for platelet-derived growth factor (PDGF)-induced signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix proteins. Fibronectin-induced tyrosine phosphorylation of focal adhesion proteins, including the focal adhesion kinase FAK, is nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent with previous reports demonstrating the importance of FAK for cell migration, SYF cells display reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling by extracellular matrix-coupled receptors (Klinghoffer, 1999).

Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, it has been shown that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization, and coincides with Rho-mediated cytoskeletal remodeling. However, gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use an Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication (Postma, 1998).

The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors results in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment is mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, act as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the desensitized receptor functions as a critical structural component of a mitogenic signaling complex (Luttrell, 1999).

alphaß1 integrins have been implicated in the survival, spreading, and migration of cells and tissues. To explore the underlying biology, conditions were identified where primary ß1 null keratinocytes adhere, proliferate, and display robust alphavß6 integrin-induced, peripheral focal contacts associated with elaborate stress fibers. Mechanistically, this appears to be due to reduced FAK and Src and elevated RhoA and Rock activities. Visualization on a genetic background of GFPactin shows that ß1 null keratinocytes spread, but do so aberrantly, and when induced to migrate from skin explants in vitro, the cells are not able to rapidly reorient their actin cytoskeleton toward the polarized movement. As judged by RFPzyxin/GFPactin videomicroscopy, the alphavß6-actin network does not undergo efficient turnover. Without the ability to remodel their integrin-actin network efficiently, alphaß1-deficient keratinocytes cannot respond dynamically to their environment and polarize movements (Raghavan, 2003).

The results underscore a novel and distinct role for alphaß1 integrins in regulating this equilibrium in focal adhesion dynamics. Not surprisingly, three well-known regulators of focal contacts, FAK, RhoA, and Rock, appear to be at the heart of this regulation. As judged by immunofluorescence with purportedly specific phospho-FAK Abs, activated FAK localizes to the focal contacts of ß1 null keratinocytes. By this criterion, the underlying defects in focal contact turnover and in overall FAK and Src activities are not attributable to a defect in targeting FAK to alphavß6 focal contacts, and indeed, ligand-engaged alphavß6 can bind and activate FAK. Rather, in the absence of ß1, alphavß6 appears unable on its own to activate FAK to the threshold levels needed to properly control focal adhesion-actin cytoskeletal dynamics. Irrespective of the precise underlying mechanism, the consequences to this imbalance are excessive adhesion and inefficient spreading (Raghavan, 2003).

Although tyrosine kinase inhibitors can block focal adhesion formation in some situations, a greater role for tyrosine phosphorylation has been found in focal adhesion turnover and cell motility. Thus, activated FAK negatively regulates RhoA activity, and FAK null fibroblasts express robust actin stress-fiber networks that can be dissipated by Rock inhibition. The ability of Rho and Rock inhibitors to disperse both stress fibers and associated focal contacts in ß1 null keratinocytes provides compelling evidence that a FAK-RhoA imbalance is at the root of the focal adhesion-cytoskeletal imbalance in these cells. Although more complicated mechanisms are possible, the data are consistent with a model whereby in the absence of alphaß1 integrins, FAK/Src activation is not fully achieved, thereby diminishing p190RhoGAP phosphorylation, and yielding elevated RhoA/Rock activities (Raghavan, 2003).

Dynamic microtubules explore the peripheral (P) growth cone domain using F actin bundles as polymerization guides. Microtubule dynamics are necessary for growth cone guidance; however, mechanisms of microtubule reorganization during growth cone turning are not well understood. These issues are addressed by analyzing growth cone steering events in vitro, evoked by beads derivatized with the Ig superfamily cell adhesion protein apCAM. Pharmacological inhibition of microtubule assembly with low doses of taxol or vinblastine result in rapid clearance of microtubules from the P domain with little effect on central (C) axonal microtubules or actin-based motility. Early during target interactions, F actin assembly and activated Src, but few microtubules, are detected at apCAM bead binding sites. The majority of microtubules extend toward bead targets after F actin flow attenuation occurs. Microtubule extension during growth cone steering responses is strongly suppressed by dampening microtubule dynamics with low doses of taxol or vinblastine. These treatments also inhibit growth cone turning responses, as well as focal actin assembly and accumulation of active Src at bead binding sites. These results suggest that dynamic microtubules carry signals involved in regulating Src-dependent apCAM adhesion complexes involved in growth cone steering (Suter, 2004).

Activation of signalling by fibroblast growth factor receptor leads to phosphorylation of the signalling attenuator human Sprouty 2 (hSpry2) on residue Y55. This event requires the presence of the signalling adaptor fibroblast growth factor receptor substrate 2 (FRS2). The phosphorylation of hSpry2 is therefore mediated by an intermediate kinase. Using a SRC family kinase-specific inhibitor and mutant cells, hSpry2 was shown to be a direct substrate for SRC family kinases, including SRC itself. Activation of SRC via fibroblast growth factor signalling is dependent upon FRS2 and fibroblast growth factor receptor kinase activity. SRC forms a complex with hSpry2 and this interaction is enhanced by hSpry2 phosphorylation. Phosphorylation of hSpry2 is required for hSpry2 to inhibit activation of the extracellular signal-regulated kinase pathway. These results show that recruitment of SRC to FRS2 leads to activation of signal attenuation pathways (X. Li, 2004).

The axon guidance cue netrin is importantly involved in neuronal development. DCC (deleted in colorectal cancer) is a functional receptor for netrin and mediates axon outgrowth and the steering response. Different regions of the intracellular domain of DCC directly interact with the tyrosine kinases Src and focal adhesion kinase (FAK). Netrin activates both FAK and Src and stimulates tyrosine phosphorylation of DCC. Inhibition of Src family kinases reduces DCC tyrosine phosphorylation and blocks both axon attraction and outgrowth of neurons in response to netrin. Mutation of the tyrosine phosphorylation residue in DCC abolishes its function of mediating netrin-induced axon attraction. On the basis of these observations, a model is suggested in which DCC functions as a kinase-coupled receptor, and FAK and Src act immediately downstream of DCC in netrin signaling (W. Li, 2004).

Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1-DCC signaling are still poorly characterized. This study shows that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1-dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1; this suggests an essential role of the Src kinases in Netrin-1-dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and Y1418 has been demonstrated to be crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1-induced axon outgrowth are impaired in Fyn(-/-) CN and spinal cord explants. It is proposed that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1 (Meriane, 2004).

Although netrins are an important family of neuronal guidance proteins, intracellular mechanisms that mediate netrin function are not well understood. This study shows that netrin-1 induces tyrosine phosphorylation of proteins including focal adhesion kinase (FAK) and the Src family kinase Fyn. Blockers of Src family kinases inhibit FAK phosphorylation and axon outgrowth and attraction by netrin. Dominant-negative FAK and Fyn mutants inhibit the attractive turning response to netrin. Axon outgrowth and attraction induced by netrin-1 are significantly reduced in neurons lacking the FAK gene. These results show the biochemical and functional links between netrin, a prototypical neuronal guidance cue, and FAK, a central player in intracellular signaling that is crucial for cell migration (Liu, 2004).

The molecular mechanisms underlying the elaboration of branched processes during the later stages of oligodendrocyte maturation are not well understood. This study describes a novel role for the chemotropic guidance cue netrin 1 and its receptor deleted in colorectal carcinoma (Dcc) in the remodeling of oligodendrocyte processes. Postmigratory, premyelinating oligodendrocytes express Dcc but not netrin 1, whereas mature myelinating oligodendrocytes express both. Netrin 1 promotes process extension by premyelinating oligodendrocytes in vitro and in vivo. Addition of netrin 1 to mature oligodendrocytes in vitro evoked a Dcc-dependent increase in process branching. Furthermore, expression of netrin 1 and Dcc by mature oligodendrocytes was required for the elaboration of myelin-like membrane sheets. Maturation of oligodendrocyte processes requires intracellular signaling mechanisms involving Fyn, focal adhesion kinase (FAK), neuronal Wiscott-Aldrich syndrome protein (N-WASP) and RhoA; however, the extracellular cues upstream of these proteins in oligodendrocytes are poorly defined. A requirement was identified for Src family kinase activity downstream of netrin-1-dependent process extension and branching. Using oligodendrocytes derived from Fyn knockout mice, Fyn was shown to be essential for netrin-1-induced increases in process branching. Netrin 1 binding to Dcc on mature oligodendrocytes recruits Fyn to a complex with the Dcc intracellular domain that includes FAK and N-WASP, resulting in the inhibition of RhoA and inducing process remodeling. These findings support a novel role for netrin 1 in promoting oligodendrocyte process branching and myelin-like membrane sheet formation. These essential steps in oligodendroglial maturation facilitate the detection of target axons, a key step towards myelination (Rajasekharan, 2009).

Src interacts with G-proteins and their associated proteins

Heterotrimeric G proteins transduce signals from cell surface receptors to modulate the activity of cellular effectors. Src, the product of the first characterized proto-oncogene and the first identified protein tyrosine kinase, plays a critical role in the signal transduction of G protein–coupled receptors. However, the mechanism of biochemical regulation of Src by G proteins is not known. Galphas and Galphai, but neither Galphaq, Galpha12 nor Gbetagamma, directly stimulate the kinase activity of downregulated c-Src. Galphas and Galphai similarly modulate Hck, another member of Src-family tyrosine kinases. Galphas and Galphai bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. These data demonstrate that the Src family tyrosine kinases are direct effectors of G proteins (Ma, 2000).

Evidence is presented that activated Galphas and Galphai, but neither Galphaq, Galpha12, nor Gbetagamma, directly regulate Src-family tyrosine kinases by activating the downregulated, C-terminal phosphorylated kinase. In Src-family tyrosine kinase knockout SYF cells, GalphasQ227L and GalphaiQ205L induced tyrosine phosphorylation of many of cellular proteins is severely reduced, suggesting that Src-family tyrosine kinases are the major mediator of Galphas- and Galphai-induced protein tyrosine phosphorylation in cells. Furthermore, the switch II region of Galphas is involved in interacting with Src. In general, G proteins, including heterotrimeric and Ras-family G proteins, use their effector domains to contact the downstream effectors. For Ras, the switch I region is a core effector domain essential for all effector interactions. The switch II region of Galphas and Galphai has been shown to be important in interacting with their common effector adenylyl cyclase. These data suggest that Galphas uses some residues such as I235 in the switch II region, and likely residues in other regions, to interact with the catalytic domain of Src. These studies significantly advance understanding of an important aspect of the cross-talk between G protein-coupled receptors and tyrosine kinases. Furthermore, these experiments provide a possible mechanism of action by which the gip2 mutant of Galphai and the gsp mutant of Galphas induce oncogenicity (Ma, 2000).

There are previous reports indicating that some biological effects of Galphas and Galphai could not be explained by their opposing effects on adenylyl cyclases. Both activated mutants (GTPase deficient) of Galphas and Galphai are oncogenes, found in certain human tumors. Both Galphas and Galphai can transform cells and activate the mitogen-activated protein kinase (MAPK) pathways. Activated Galphai subunits induce a transformed phenotype in fibroblasts independent of inhibition of adenylyl cyclase. Also, Galphas and Galphai regulate adipogenesis in mouse 3T3-L1 cells and stem cell differentiation of F9 teratocarcinoma cells into primitive endoderm, which could not be explained by changes in intracellular cAMP. Furthermore, inhibition of magnesium uptake in S49 cells by isoproterenol or prostaglandin E1 has been shown to be Galphas-dependent, but cAMP and PKA-independent. Similarly, in differentiating wing epithelial cells of Drosophila, activation of Galphas leads to formation of wing blisters. This pathway has been genetically demonstrated to be independent of PKA. Recently, engagement of beta-adrenergic receptors initiate a Galphas-dependent, PKA-independent pathway leading to apoptosis in S49 cells. These reports, together with the observations presented here, clearly indicate that Galphas and Galphai can signal through novel transduction pathways, in addition to the classically defined cAMP second messenger system. The relative contribution of these different effector systems to the physiology of G proteins in organisms remains to be addressed (Ma, 2000).

c-Src kinase activity can be modulated by either tyrosine phosphorylation or conformational changes. Neither Galphas nor Galphai change the Tyr527 phosphorylation state, suggesting that autodephosphorylation of Tyr527 is not the regulatory mechanism used by G proteins. Galphas and Galphai do increase the autophosphorylation of Tyr416. Binding data showing that Galphas and Galphai interact with the catalytic domain of Src suggest that a conformational change model is likely. Indeed, a phosphotyrosine binding experiment has shown that G protein stimulation causes the release of phospho-Tyr527 from the SH2 domain, making it available for interacting with other proteins. In a similar manner, activation of Hck kinase activity by the Nef protein of human immunodeficiency virus-1 (HIV-1), comes about through a conformational change, which occurs in the absence of dephosphorylation of the tail Tyr527. Enzymatic kinetic measurement reveals that the major effect of Galphas and Galphai is to decrease the Km for the peptide substrate. These data suggest that G protein binding changes the conformation of c-Src and allows the peptide substrate easier access to the active site. This model is consistent with the structural data of Src-family tyrosine kinases. In the downregulated state, two intramolecular interactions stabilize the restrained conformation of the kinase domain. The activation loop forms an alpha helix that packs between the upper and lower lobes of the catalytic domain, thus blocking the peptide substrate binding site. In the active state, the activation loop swings away from the entrance of the catalytic cleft, allowing access of the substrate to the active site. This activation mode has also been observed in the activation of cyclin-dependent kinase (cdk). Cyclin binding to the catalytic domain repositions cdk's activation loop and permits access of substrates to the active site. It is proposed that G protein binding to the catalytic domain modulates the position and conformation of the activation loop, as well as other elements in the catalytic domain. This could lead to relief of steric hindrance at the entrance to the catalytic cleft; increased accessibility of the active site to substrates, and exposure of the side chain of Tyr416, making it a better substrate for autophosphorylation (the Tyr416 hydroxyl group is buried in the catalytic cleft in the downregulated form) and thus increased kinase activity. Regardless of the detailed chemical mechanism, these data demonstrate that c-Src and Hck are novel direct effectors of Galphas and Galphai proteins (Ma, 2000).

G-protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of G-protein-coupled receptors (GPCRs). GRK2 expression is altered in several pathological conditions, but the molecular mechanisms that modulate GRK2 cellular levels are largely unknown. GRK2 is degraded rapidly by the proteasome pathway. This process is enhanced by GPCR stimulation and is severely impaired in a GRK2 mutant that lacks kinase activity (GRK2-K220R). ß-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of ß-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with ß-arrestin mutants that display an impaired c-Src interaction. The presence of an inactive c-Src mutant or of tyrosine kinase inhibitors strongly inhibits co-transfected or endogenous GRK2 turnover, respectively, and a GRK2 mutant with impaired phosphorylation by c-Src shows a markedly retarded degradation. This pathway for the modulation of GRK2 protein stability puts forward a new feedback mechanism for regulating GRK2 levels and GPCR signaling (Penela, 2001).

These results are consistent with the notion that GRK2-dependent binding of ß-arrestin to GPCRs allows the recruitment of c-Src to the receptor signaling complex at the plasma membrane, leading to phosphorylation of GRK2 on tyrosine residues and its targeting for degradation. This model is in agreement with the rapid ß-arrestin and c-Src recruitment following ß2AR stimulation, and with the agonist-stimulated phosphorylation of GRK2 by c-Src. Under basal conditions, ß-arrestin recruitment to the plasma membrane would be promoted by the activated state of different endogenous GPCRs and/or by the reported basal activity of overexpressed ß2AR. In the presence of GPCR agonists, an acceleration of the GRK2 degradation rate is detected, consistent with a more efficient ß-arrestin and c-Src translocation to the receptor complex. Although detailed knowledge of the sequential assembly of these proteins in a multimolecular complex is lacking, and other molecular interactions may participate in c-Src binding to the receptor complex and GRK2 tyrosine phosphorylation, the proposed model is consistent with the co-immunoprecipitation of ß-arrestin and c-Src, of GRK2 and ß-arrestin and of GRK2 and c-Src. Disruption of the ß-arrestin-c-Src interaction with specific mutants or inhibition of the phosphorylation step by dominant-negative Src or a GRK2 mutant lacking critical phosphorylation sites results, as predicted by this model, in a marked reduction in GRK2 degradation (Penela, 2001).

v-SRC specifically regulates the nucleo-cytoplasmic delocalization of the major isoform of TEL (ETV6)

TEL is a frequent target of chromosomal translocations in human cancer and an alleged tumor suppressor gene. TEL encodes two isoforms: a major TEL-M1 isoform as well as TEL-M43, which lacks the first 42 amino acid residues of TEL-M1. Both isoforms are potent transcriptional repressors that can inhibit RAS-induced transformation. v-SRC protein-tyrosine kinase relieves the repressive activity of TEL-M1, an activity that is associated with the v-SRC-induced delocalization of TEL-M1 from the nucleus to the cytoplasm. TEL-M1 delocalization requires the kinase activity of v-SRC and is not induced by oncogenic RAS or AKT. Cytoplasmic delocalization of TEL-M1 in response to v-SRC critically depends upon its unique amino-terminal domain (SRCD domain) because (1) v-SRC does not inhibit the repressive properties of TEL-M43, nor does it affected TEL-M43 nuclear localization; (2) fusion of the first 52 amino acid residues of TEL-M1 to FLI-1, an ETS protein insensitive to v-SRC-induced delocalization, is sufficient to confer v-SRC-induced delocalization to this TEL/FLI-1 chimeric protein. The v-SRC-induced nucleo-cytoplasmic delocalization of TEL-M1 does not involve phosphorylation of the SRCD and does not require TEL self-association and repressive domains. Finally, enforced expression of the v-SRC-insensitive TEL-M43, but not of TEL-M1, inhibits v-SRC-induced transformation of NIH3T3 fibroblasts. These results identify a regulatory domain in TEL that specifically impinges on the subcellular localization of its major TEL-M1 isoform. Furthermore, they indicate that inhibition of TEL-M1 nuclear function is required for v-SRC to induce cellular transformation (Lopez, 2003).

Signaling downstream of Src

This study examines the molecular mechanism of erythropoietin-initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3-kinase (PI3-kinase). Antisense oligonucleotides against src but not lyn inhibit the formation of erythropoietin-dependent colonies derived from human bone marrow cells and erythropoietin-induced differentiation of K562 human erythroleukaemia cells. Antisense p85alpha oligonucleotide or LY294002, a selective inhibitor of PI3-kinase, independently inhibit the formation of erythropoietin-dependent colonies. In K562 cells, Src associates with PI3-kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibits the erythropoietin-induced activation of PI3-kinase and its association with erythropoietin receptor. PP1, a selective inhibitor of the Src family, reduced erythropoietin-induced tyrosine phosphorylation of erythropoietin receptor and its association with PI3-kinase in F-36P human erythroleukemia cells. The coexpression experiments and in vitro kinase assay further demonstrate that Src directly tyrosine-phosphorylates erythropoietin receptor, and associates with PI3-kinase. In vitro binding experiments have proven that glutathione S-transferase-p85alpha N- or C-terminal SH2 domains independently bind to erythropoietin receptor, which is tyrosine-phosphorylated by Src. Taken together, Src transduces the erythropoietin-induced erythroid differentiation signals by regulating PI3-kinase activity (Kubota, 2001).

Cell migration is a complex, highly regulated process that involves the continuous formation and disassembly of adhesions (adhesion turnover). Adhesion formation takes place at the leading edge of protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. Despite the importance of these processes in migration, the mechanisms that regulate adhesion formation and disassembly remain largely unknown. Quantitative assays have been developed to measure the rate of incorporation of molecules into adhesions and the departure of these proteins from adhesions. Using these assays, it has been shown that kinases and adaptor molecules, including focal adhesion kinase (FAK), Src, p130CAS (see CAS/CSE1 segregation protein), paxillin, extracellular signal-regulated kinase (ERK) and myosin light-chain kinase (MLCK) are critical for adhesion turnover at the cell front, a process central to migration (Webb, 2004).

Raf-1 is a regulator of cellular proliferation, differentiation and apoptosis. Activation of the Raf-1 kinase activity is tightly regulated and involves targeting to the membrane by Ras and phosphorylation by various kinases including the tyrosine kinase Src. The connector enhancer of Ksr1, CNK1 (See Drosophila), mediates Src-dependent tyrosine phosphorylation and activation of Raf-1. CNK1 binds preactivated Raf-1 and activated Src and forms a trimeric complex. CNK1 regulates the activation of Raf-1 by Src in a concentration-dependent manner typical for a scaffold protein. Down-regulation of endogenously expressed CNK1 by small inhibitory RNA interferes with Src-dependent activation of ERK. Thus, CNK1 allows a cross-talk between Src and Raf-1 and is essential for full activation of Raf-1 (Ziogas, 2005).

An unexpected role for the clock protein timeless in developmental apoptosis

Programmed cell death is critical not only in adult tissue homeostasis but for embryogenesis as well. One of the earliest steps in development, formation of the proamniotic cavity, involves coordinated apoptosis of embryonic cells. Recent work has demonstrated that c-Src protein-tyrosine kinase activity triggers differentiation of mouse embryonic stem (mES) cells to primitive ectoderm-like cells. In this report, Timeless (Tim), the mammalian ortholog of a Drosophila circadian rhythm protein, was identified as a binding partner and substrate for c-Src, and its role in the differentiation of mES cells was probed. To determine whether Tim is involved in ES cell differentiation, Tim protein levels were stably suppressed using shRNA. Tim-defective ES cell lines were then tested for embryoid body (EB) formation, which models early mammalian development. Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate. Cells retained within the centers of the failed cavities strongly expressed the pluripotency marker Oct4, suggesting that further development is arrested without Tim. Immunoblots revealed reduced basal Caspase activity in the Tim-defective EBs compared to wild-type controls. Furthermore, EBs formed from Tim-knockdown cells demonstrated resistance to staurosporine-induced apoptosis, consistent with a link between Tim and programmed cell death during cavitation. These data demonstrate a novel function for the clock protein Tim during a key stage of early development. Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate. As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent. These findings reveal a new function for Tim in the coordination of ES cell differentiation, and raise the intriguing possibility that circadian rhythms and early development may be intimately linked (O'Reilly, 2011).

Sonic hedgehog guides axons through a noncanonical, Src-family-kinase-dependent signaling pathway

Sonic hedgehog plays essential roles in developmental events such as cell fate specification and axon guidance. Shh induces cell fate specification through canonical Shh signaling, mediated by transcription. However, the mechanism by which Shh guides axons is unknown. To study this, an in vitro assay was developed for axon guidance, in which neurons can be imaged while responding to a defined gradient of a chemical cue. Axons of dissociated commissural neurons placed in a Shh gradient turned rapidly toward increasing concentrations of Shh. Consistent with this rapid response, attraction by Shh was shown not to require transcription. Instead, Shh stimulates the activity of Src family kinase (SFK) members in a Smoothened-dependent manner. Moreover, SFK activity is required for Shh-mediated guidance of commissural axons, but not for induction of Gli transcriptional reporter activity. Together, these results indicate that Shh acts via a rapidly acting, noncanonical signaling pathway to guide axons (Yam, 2009).

MIM and cortactin antagonism regulates ciliogenesis and hedgehog signaling: MIM promotes ciliogenesis by antagonizing Src-dependent phosphorylation of Cortactin

The regulation of protein tyrosine phosphorylation is an important aspect during the cell cycle. From G2-M transition to mitotic anaphase, phosphorylation of Tyr421, Tyr466 and Tyr482 of cortactin, an actin-filament associated protein, is dramatically induced. The phosphorylated cortactin is almost exclusively associated with centrosomes or spindle poles during mitosis. At G2-M transition prior to the breakdown of the nuclear envelope, two duplicated centrosomes migrate towards opposite ends of the nucleus to form the spindle poles. This centrosome-separation process and also the start of mitosis are inhibited or delayed by the depolymerization of actin filaments. Also inhibited is the separation of centrosomes when a truncated form of cortactin is expressed, whose C-terminus contains the tyrosine phosphorylation region but lacks the actin-binding domains. Mutations were introduced at the tyrosine phosphorylation sites in the truncated C-terminus of cortactin and it was found that the C-terminus could no longer interfere with centrosome separation process. This study shows that, cortactin phosphorylated at Tyr421, Tyr466 and Tyr482 mediates the actin-filament-driven centrosome separation at G2-M transition by providing a bridge between the centrosome and actin-filaments (Bershteyn, 2010).

The primary cilium is critical for transducing Sonic hedgehog (Shh) signaling, but the mechanisms of its transient assembly are poorly understood. Previously it has been shown that the actin regulatory protein Missing-in-Metastasis (MIM) regulates Shh signaling, but the nature of MIM's role was unknown. This study shows that MIM is required at the basal body of mesenchymal cells for cilia maintenance, Shh responsiveness, and de novo hair follicle formation. MIM knockdown results in increased Src kinase activity and subsequent hyperphosphorylation of the actin regulator Cortactin. Importantly, inhibition of Src or depletion of Cortactin compensates for the cilia defect in MIM knockdown cells, whereas overexpression of Src or phospho-mimetic Cortactin is sufficient to inhibit ciliogenesis. These results suggest that MIM promotes ciliogenesis by antagonizing Src-dependent phosphorylation of Cortactin and describe a mechanism linking regulation of the actin cytoskeleton with ciliogenesis and Shh signaling during tissue regeneration (Bershteyn, 2010).

This study shows that MIM promotes ciliogenesis by inhibiting Src kinase activation during G1. Decreased levels of MIM lead to upregulation of activated Src and subsequent hyperphosphorylation of multiple actin-associated Src substrates including Cortactin (CTTN), which promotes increased F-actin branching and polymerization. Importantly, either inhibition of the Src catalytic domain or removal of CTTN is sufficient to restore ciliogenesis in the absence of MIM, suggesting that MIM regulates an intricate balance of actin regulatory factors that affect cilia dynamics, but is not uniquely required for ciliogenesis. Among the critical functional consequences of this deregulation in mesenchymal cells are failure to respond to Shh signaling and inability to induce hair growth. Collectively, these data reveal a mechanism that coordinates ciliogenesis with cell cycle progression and provide a strong connection between actin cytoskeletal regulators, ciliogenesis, and Shh signaling during tissue regeneration (Bershteyn, 2010).

Much like the developing node and the limbs, the hair follicles rely on primary cilia to transduce signals from Shh and other morphogens. Reduction in MIM levels in the dermal compartment leads to severe lack of hair and immature follicles that appear arrested at stage 2-4 of anagen, similar to the phenotypes observed upon dermal cilia ablation through conditional deletion of ciliary structural components Ift88 or Kif3A or in Shh mutant skin. The fact that MIM KD disrupts primary cilia in the regenerated dermal papillae provides additional evidence that the cilia defect forms the basis of the hair follicle phenotype and underscores the importance for dermal primary cilia in hair follicle development. Thus, the results support a key role for MIM in dermal cilia regulation and Shh signaling during de novo hair formation and demonstrate the utility of the hair regeneration system for rapid screening, identification, and analysis of in vivo, cell type-specific regulators of hair formation (Bershteyn, 2010).

The data point to a model whereby MIM promotes ciliogenesis by antagonizing Src activity during G1. MIM's inhibitory effect on Src is highlighted by the fact that multiple Src substrates, including other kinases, scaffolding proteins, and actin cytoskeleton regulators known to affect cell proliferation, adhesion and migration, become hyperphosphorylated upon MIM KD. This finding provides a potential basis for the frequent alteration of MIM levels in metastatic cancers, as Src is a well-known oncogene and the hyperphosphorylated substrates that were detected are all associated with decreased cell adhesion and increased cell motility. However, with respect to Src-dependent cilia disassembly, the Src substrate CTTN appears to be a key downstream effector, since CTTN KD impairs cilia resorption and removal of CTTN is sufficient to restore cilia in MIM KD cells (Bershteyn, 2010).

Several independent lines of evidence support the model that ectopic Src kinase activity during G1 inhibits ciliogenesis. First, the cilia defect in MIM KD cells can be rescued by several small molecule inhibitors of the SFK family. Second, overexpression of just p60 Src in Src/Yes/Fyn triple knockout SYF-/- MEFs potently inhibits ciliogenesis and completely abolishes any Shh responsiveness, despite the fact that most of the cells are in G1/G0. Third, transient expression of a constitutively active Src Y527F mutant reveals basal body localization and inhibits ciliogenesis in SYF-/-MEFs and primary dermal cells in a cell-autonomous manner. Finally, fourth: transiently expressed kinase dead Src K295R mutant functions as a dominant-negative with respect to p-CTTN and induces more and longer cilia in dermal cells. These data point to the powerful inhibitory actions of Src kinase and the critical need to restrain its activity during G1 (Bershteyn, 2010).

Emerging data support the idea that the basal body coordinates ciliogenesis with the cell cycle. This regulation is likely based on a dynamic cell-cycle-dependent equilibrium between factors that promote cilia formation after cytokinesis and factors that promote cilia disassembly prior to mitosis. MIM and CTTN appear to be two such factors, with MIM promoting cilia formation and Src-activated p-CTTN promoting cilia disassembly (Bershteyn, 2010).

Based on these results, a model is proposed whereby MIM antagonism of p-CTTN serves as a switch to regulate the timing of ciliogenesis and coordinate it with the cell cycle. Thus during G1/S, when the relative ratio of MIM to p-CTTN is high, cilia are maintained. As the cell cycle progresses toward G2/M, activation of Src leads to increased p-CTTN levels, shifting the ratio of MIM to p-CTTN to low and inducing cilia disassembly. When MIM is depleted, activation of Src leads to upregulation of p-CTTN during G1, artificially shifting the ratio of MIM to p-CTTN to resemble what it normally is during G2/M. Interestingly MIM-depleted cells display ectopic p-Src and p-CTTN in the cytosol and at the basal body, suggesting that Src and CTTN functions outside the basal body may also contribute to cilia maintenance. This is supported by the finding that inhibition of Src or depletion of CTTN restores cilia in MIM KD cells and underscores the critical relationship between MIM and Src activity (Bershteyn, 2010).

These findings combined with multiple lines of published data suggest that MIM and CTTN antagonism affects ciliogenesis by regulating the actin cytoskeleton. First of all, the antagonism between MIM and CTTN appears to be conserved in evolution and 'recycled' for multiple actin-dependent cellular processes including Drosophila border cells migration and clathrin-mediated endocytosis (Quinones, 2010). Moreover, high levels of MIM relative to CTTN were shown in vitro to inhibit CTTN mediated actin polymerization (Lin, 2005). In contrast, phosphorylation by Src is known to increase CTTN affinity and nucleating activity for F-actin (Ammer, 2008; Lua, 2005; Kruchten, 2008). Moreover, recent studies in NIH 3T3 and HeLa cells found that p-CTTN Y466/Y421 is enriched at the centrosomes during G2/M and mediates actin-dependent centrosome separation (Wang, 2008). Consistent with all of these data, increased levels and disorganized F-actin staining is seen in MIM KD cells that lack cilia and vice versa, reduced F-actin staining in CTTN KD cells that fail to disassemble their cilia. In addition, disruption of F-actin with polymerization inhibitors causes cilia elongation and prevents cilia disassembly. Thus, while F-actin may be required for initial basal body motility and membrane docking, the data suggest that F-actin needs to be cleared locally during G1 to allow cilia elongation and maintenance and then reformed later in the cell cycle to promote cilia disassembly. Interestingly, a recent screen for genes involved in ciliogenesis identified several regulators of actin dynamics and vesicle trafficking, and actin clearing by the centrosome has been shown to be required in other directed membrane events such as exocytosis of lytic granules. Moreover, Septin2, known to play a role in actin dynamics, appears to play a role in maintaining the ciliary protein diffusion barrier and proper signaling. Therefore, actin regulatory proteins localized at the basal body such as MIM and CTTN could provide a general mechanism for regulating directional plasma membrane remodeling and proper cilium-dependent signaling (Bershteyn, 2010).

Src, mitosis and meiosis

continued: Src oncogene at 64B Evolutionary homologs part 3/3 | back to part 1/3 |

Src oncogene at 64B: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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