hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions

Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. We now report that hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding.     

Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain

To examine the interactions between Src homology,domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.     

Human immunodeficiency virus type 1 Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity.

It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.     

Identification of Molecular Switch Regulating Interactions of Janus Kinase 3 with Cytoskeletal Proteins

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Although mutations that abrogate Jak3 functions cause different immunological disorders, its constitutive activation leads to various types of cancer. Previously, we demonstrated that Jak3 interacted with actin-binding protein villin, thereby facilitating cytoskeletal remodeling and wound repair. In this study, we characterize the structural determinants that regulate the interactions between Jak3 and cytoskeletal proteins of the villin/gelsolin family. Functional reconstitution of kinase activity by recombinant full-length (wt) Jak3 using Jak3-wt or villin/gelsolin-wt as substrate showed that Jak3 autophosphorylation was the rate-limiting step during interactions between Jak3 and cytoskeletal proteins. Determination of kinetic parameters showed that phosphorylated (P) Jak3-wt binds to P-villin-wt with a dissociation constant (K_d) of 23 nm and a Hill''s coefficient of 3.7. Pairwise binding between Jak3 mutants and P-villin-wt showed that the FERM domain of Jak3 was sufficient for binding to P-villin-wt with a K_d of 40.0 nm. However, the SH2 domain of Jak3 prevented P-villin-wt from binding to the FERM domain of nonphosphorylated protein. We demonstrate that the intramolecular interaction between the FERM and SH2 domains of nonphosphorylated Jak3 prevented Jak3 from binding to villin and that tyrosine autophosphorylation of Jak3 at the SH2 domain decreased these intramolecular interactions and facilitated binding of the FERM domain to villin. Thus we demonstrate the molecular mechanism of interactions between Jak3 and cytoskeletal proteins where tyrosine phosphorylation of the SH2 domain acted as an intramolecular switch for the interactions between Jak3 and cytoskeletal proteins.     

A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.     

Autophosphorylation is required for high kinase activity and efficient transformation ability of proteins encoded by host range alleles of v-src.

pp60v-src is a nonreceptor protein tyrosine kinase that can transform both chicken and rodent fibroblasts. The src homology 2 (SH2) domain of this protein serves a critical role in the regulation of protein tyrosine kinase activity. The host range proteins pp60v-src-L, which contains a deletion of a highly conserved residue (Phe-172) in the SH2 domain, and pp60v-src-PPP, which contains a change from a Leu to a Phe at amino acid 186 in the SH2 domain, transform chicken but not rat cells and have slightly reduced kinase activity measured in vitro. The data presented here show that these altered proteins require autophosphorylation on Tyr-416 for high kinase activity and transforming ability. In the absence of autophosphorylation, there is a further decrease of at least threefold in in vitro kinase activity relative to the phosphorylated host range parental protein, no morphological transformation, a reduction in anchorage independent growth, and no disruption of the actin cytoskeleton. In addition, these SH2 mutations abolish the ability of the SH2 domain to bind a phosphorylated peptide that corresponds to the autophosphorylation site of pp60src. Thus, like mutant alleles of c-src encoding transformation competent proteins, and unlike v-src, transformation by pp60v-src-F172 delta and pp60v-src-L186F is dependent on phosphorylation of Y-416 for high kinase activity and transformation ability. The dependence of transformation on phosphotyrosine is not a reflection of an intramolecular interaction between the autophosphorylation site and the SH2 domains since purified SH2 domains are incapable of binding phosphorylated autophosphorylation site peptides in vitro.     

Inactivation of c-Yes tyrosine kinase by elevation of intracellular calcium levels.

We have previously shown that the c-Src tyrosine kinase is activated four- to fivefold when cultured keratinocytes differentiate following the elevation of intracellular calcium levels. In contrast to c-Src, another Src family tyrosine kinase, c-Yes, was rapidly inactivated in these same cells, despite its marked similarity in structure and enzymatic activity to c-Src. The inactivation of c-Yes was independent of the protein kinase C pathway, which is usually activated by elevation of intracellular calcium levels. The protein levels of c-Src and c-Yes were not altered, but the phosphotyrosine content of both proteins was greatly reduced. As has been demonstrated for c-Src, in vitro dephosphorylation of c-Yes by incubation with protein tyrosine phosphatases also resulted in its activation, not inactivation. In vitro reconstitution experiments showed that c-Yes can be inactivated by preincubation with a Ca(2+)-supplemented cell extract and that this inhibition was reversed by the addition of EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Gradient sedimentation of cell lysates showed that in cells treated with calcium and ionophore, c-Yes formed complexes with two distinct cellular proteins, whereas similar complexes were not seen in c-Src immunoprecipitates. One of these two proteins has the ability to inhibit c-Yes kinase activity in vitro. Finally, the Ca(2+)-dependent inactivation of c-Yes was observed in kidney tubular cells and fibroblasts, suggesting that the Ca(2+)-dependent regulation of c-Yes tyrosine kinase is not unique to keratinocytes. We postulate that c-Yes is inactivated through a Ca2+ -dependent association with cellular proteins, which seems to override its activation resulting from tyrosine dephosphorylation.     

Ribosomal protein L10 interacts with the SH3 domain and regulates GDNF-induced neurite growth in SH-SY-5y cells

The 24.5 kDa ribosomal protein L10 (RP-L10), which was encoded by QM gene, was known to interact with the SH3 domain of Yes kinase. Herein, we demonstrate that RP-L10 interacts with the SH3 domain of Src and activates the binding of the Nck1 adaptor protein with skeletal proteins such as the Wiskott-Aldrich Syndrome Protein (WASP) and WASP interacting protein (WIP) in neuroblastoma cell line, SH-SY-5y. The RP-L10 was associated with the SH3 domains of Src and Yes. It is shown that two different regions of RP-L10 are associated with the Src-SH3. The effect of ectopic RP-L10 expression on neuronal cell scaffolding was explored in cells transiently transfected with QM. SH-SY-5y human neuroblastoma cells transfected with QM were considerably more susceptible to neurite outgrowth induced by glial cell line-derived neurotrophic factor (GDNF). However, RP-L10 did not directly interact with actin assembly. Taken together, these results suggest that the RP-L10 may positively regulate the GDNF/Ret-mediated signaling of neurite outgrowth in the neuroblastoma cell line, SH-SY-5y.     

Interactions between the Fyn SH3-domain and adaptor protein Cbp/PAG derived ligands, effects on kinase activity and affinity

Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched domains is a transmembrane adaptor protein primarily involved in negative regulation of T-cell activation by recruitment of C-terminal Src kinase (Csk), a protein tyrosine kinase which represses Src kinase activity through C-terminal phosphorylation. Recruitment of Csk occurs via SH2-domain binding to PAG pTyr317, thus, the interaction is highly dependent on phosphorylation performed by the Src family kinase Fyn, which docks onto PAG using a dual-domain binding mode involving both SH3- and SH2-domains of Fyn. In this study, we investigated Fyn SH3-domain binding to 14-mer peptide ligands derived from Cbp/PAG-enriched microdomains sequence using biochemical, biophysical and computational techniques. Interaction kinetics and dissociation constants for the various ligands were determined by SPR. The local structural impact of ligand association has been evaluated using CD, and molecular modelling has been employed to investigate details of the interactions. We show that data from these investigations correlate with functional effects of ligand binding, assessed experimentally by kinase assays using full-length PAG proteins as substrates. The presented data demonstrate a potential method for modulation of Src family kinase tyrosine phosphorylation through minor changes of the substrate SH3-interacting motif.     

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.     

Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix.

p60v-src has been shown to associate with a detergent-insoluble cellular matrix containing cytoskeletal proteins, but p60c-src does not bind to this matrix. We analyzed the association of mutant src proteins with the matrix and found that mutants which lack an amino-terminal portion (residues 149 to 169) of the SH2 domain cannot bind to the matrix. Neither the SH3 region nor other portions of the SH2 region were required for association. We also tested protein kinase-defective mutants and chimeras of p60v-src and p60c-src. We found a strong correlation between the kinase activity of p60src and its association with the detergent-insoluble matrix. Double infection of kinase-defective and kinase-active mutants did not result in matrix binding of the kinase-defective src proteins. We also found that Tyr-416, the major site of autophosphorylation in p60v-src, was not required for matrix association.     

Biochemical characterization of phospholipids, sulfatide and heparin as potent stimulators for autophosphorylation of GSK-3beta and the GSK-3beta-mediated phosphorylation of myelin basic protein in vitro

The stimulatory effects of SH (sulfatide and heparin) and two phospholipids (PI and PS) on autophosphorylation of GSK-3beta and the GSK-3beta-mediated phosphorylation of myelin basic protein (MBP) and two synthetic MBP peptides (M86 and M156) were comparatively examined in vitro. It was found that (i) both PI and SH highly stimulated the GSK-3beta-mediated phosphorylation of MBP, but not glycogen synthase, and two MBP peptides through their direct binding to these substrates and (ii) both PI and heparin, as compared with sulfatide, highly stimulated autophosphorylation of GSK-3beta. The K(m) value of MBP for GSK-3beta was highly reduced and the V(max) value was significantly increased in the presence of these acidic modulators, which augmented further phosphorylation of MBP by the kinase. Under our experimental condition, similar stimulatory effects of PI and heparin were observed with the GSK-3beta-mediated phosphorylation of tau protein (TP) in vitro. These results presented here suggest that these two phospholipids and SH may function as effective stimulators for autophosphorylation of GSK-3beta and for the GSK-3beta-mediated high phosphorylation of SH-binding proteins, including MBP and TP, in the highly accumulated levels of these acidic and sulfated modulators in the brain.     

Tyrosine 813 is a site of JAK2 autophosphorylation critical for activation of JAK2 by SH2-B beta

The tyrosine kinase Janus kinase 2 (JAK2) binds to the majority of the known members of the cytokine family of receptors. Ligand-receptor binding leads to activation of the associated JAK2 molecules, resulting in rapid autophosphorylation of multiple tyrosines within JAK2. Phosphotyrosines can then serve as docking sites for downstream JAK2 signaling molecules. Despite the importance of these phosphotyrosines in JAK2 function, only a few sites and binding partners have been identified. Using two-dimensional phosphopeptide mapping and a phosphospecific antibody, we identified tyrosine 813 as a site of JAK2 autophosphorylation of overexpressed JAK2 and endogenous JAK2 activated by growth hormone. Tyrosine 813 is contained within a YXXL sequence motif associated with several other identified JAK2 phosphorylation sites. We show that phosphorylation of tyrosine 813 is required for the SH2 domain-containing adapter protein SH2-B beta to bind JAK2 and to enhance the activity of JAK2 and STAT5B. The homologous tyrosine in JAK3, tyrosine 785, is autophosphorylated in response to interleukin-2 stimulation and is required for SH2-B beta to bind JAK3. Taken together these data strongly suggest that tyrosine 813 is a site of autophosphorylation in JAK2 and is the SH2-B beta-binding site within JAK2 that is required for SH2-B beta to enhance activation of JAK2.     

A major site of tyrosine phosphorylation within the SH2 domain of Fujinami sarcoma virus P130gag-fps is not required for protein-tyrosine kinase activity or transforming potential.

Phosphorylation of the major autophosphorylation site (Tyr-1073) within Fujinami sarcoma virus P130gag-fps activates both the intrinsic protein-tyrosine kinase activity and transforming potential of the protein. In this report, a second site of autophosphorylation Tyr-836 was identified. This tyrosine residue is found within a noncatalytic domain (SH2) of P130gag-fps that is required for full protein-kinase activity in both rat and chicken cells. Autophosphorylation of this tyrosine residue implies that the SH2 region lies near the active site in the catalytic domain in the native protein and thus possibly regulates its enzymatic activity. Four mutations have occurred within the SH2 domain between the c-fps and v-fps proteins. Tyr-836 is one of these changes, being a Cys in c-fps. Site-directed mutagenesis was used to investigate the function of this autophosphorylation site. Substitution of Tyr-836 with a Phe had no apparent effect on the transforming ability or protein-tyrosine kinase activity of P130gag-fps in rat-2 cells. Mutagenesis of both autophosphorylation sites (Tyr-1073 and Tyr-836) did not reveal any cooperation between these two phosphorylation sites. The implications of the changes within the SH2 region for v-fps function and activation of the c-fps oncogenic potential are discussed.     

Yes-associated protein and p53-binding protein-2 interact through their WW and SH3 domains

To understand the role of the Yes-associated protein (YAP), binding partners of its WW1 domain were isolated by a yeast two-hybrid screen. One of the interacting proteins was identified as p53-binding protein-2 (p53BP-2). YAP and p53BP-2 interacted in vitro and in vivo using their WW1 and SH3 domains, respectively. The YAP WW1 domain bound to the YPPPPY motif of p53BP-2, whereas the p53BP-2 SH3 domain interacted with the VPMRLR sequence of YAP, which is different from other known SH3 domain-binding motifs. By mutagenesis, we showed that this unusual SH3 domain interaction was due to the presence of three consecutive tryptophans located within the betaC strand of the SH3 domain. A point mutation within this triplet, W976R, restored the binding selectivity to the general consensus sequence for SH3 domains, the PXXP motif. A constitutively active form of c-Yes was observed to decrease the binding affinity between YAP and p53BP-2 using chloramphenicol acetyltransferase/enzyme-linked immunosorbent assay, whereas the overexpression of c-Yes did not modify this interaction. Since overexpression of an activated form of c-Yes resulted in tyrosine phosphorylation of p53BP-2, we propose that the p53BP-2 phosphorylation, possibly in the WW1 domain-binding motif, might negatively regulate the YAP.p53BP-2 complex.     

Involvement of the SH3 domain in Ca2+-mediated regulation of Src family kinases

When cells are treated with Ca(2+) and Ca(2+)-ionophore, c-Src kinase activity increases, whereas c-Yes kinase activity decreases. This opposite modulation can be reproduced in an in vitro reconstitution assay and is dependent on Ca(2+) and on soluble factors present in cell lysates. Since c-Src and c-Yes share a high degree of homology, with the exception of their N-terminal "unique" domains, their activity was thought to be coordinately regulated. To assess the mechanism of regulation we generated stable cell lines expressing eight different constructs containing wild type c-Src and c-Yes, as well as swaps of the unique domain alone, unique and Src homology 3 (SH3) domains together and the SH3 domain alone. Swapping of the unique domains was not sufficient to reverse the regulation of the chimeric molecules. On the other hand, chimeras containing swaps of the unique plus the SH3 domains displayed reverse regulation, implicating both domains in the regulation of kinase activity by Ca(2+). To rule out the participation of the unique domain, we used chimeric molecules with swapped SH3 domains only and found that the SH3 domain is necessary and sufficient to confer Ca(2+)-mediated regulation of Src and Yes tyrosine kinases.     

Biochemical properties of the Cdc42-associated tyrosine kinase ACK1. Substrate specificity, authphosphorylation, and interaction with Hck

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.     

Identification of rat cDNA encoding hck tyrosine kinase from megakaryocytes.

A rat homologue of hck tyrosine kinase cDNA from a rat megakaryocyte library contains 1911 nucleotides with an open reading frame encoding 503 amino acids. The rat hck had distinct amino acid residues from the mouse homologue exhibiting 97.6% identity. The sequence contains the SH2 and SH3 regions that interact with cytoplasmic signaling proteins, the kinase domain including the nucleotide binding site and the autophosphorylation site, and the C-terminal Tyr-499 known as a negative regulator.     

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.     

The two major imatinib resistance mutations E255K and T315I enhance the activity of BCR/ABL fusion kinase

The resistance to the tyrosine kinase inhibitor imatinib in BCR/ABL-positive leukemias is mostly associated with mutations in the kinase domain of BCR/ABL, which include the most prevalent mutations E255K and T315I. Intriguingly, these mutations have also been identified in some patients before imatinib treatment. Here we examined the effects of these mutations on the kinase activity of a BCR/ABL kinase domain construct that also contained the SH3 and SH2 domains. When expressed in COS7 cells, the BCR/ABL construct with either E255K or T315I exhibited not only the resistance to imatinib but also the increase in activity to induce autophosphorylation as well as tyrosine phosphorylation of various cellular proteins, which included STAT5. The mutant kinases also showed increased activities in in vitro kinase assays. These results raise a possibility that the major imatinib resistance mutations E255K and T315I may confer the growth advantage on leukemic cells to expand in the absence of selective pressure from imatinib treatment.