Requirements for localization of p130cas to focal adhesions.

p130cas (Cas) is an adapter protein that has an SH3 domain followed by multiple SH2 binding motifs in the substrate domain. It also contains a tyrosine residue and a proline-rich sequence near the C terminus, which are the binding sites for the SH2 and SH3 domains of Src kinase, respectively. Cas was originally identified as a major tyrosine-phosphorylated protein in v-Crk- and v-Src-transformed cells. Subsequently, Cas was shown to be inducibly tyrosine phosphorylated upon integrin stimulation; it is therefore regarded as one of the focal adhesion proteins. Using an immunofluorescence study, we examined the subcellular localization of Cas and determined the regions required for its localization to focal adhesions. In nontransformed cells, Cas was localized predominantly to the cytoplasm and partially to focal adhesions. However, in 527F-c-Src-transformed cells, Cas was localized mainly to podosomes, where the focal adhesion proteins are assembled. The localization of Cas to focal adhesions was also observed in cells expressing the kinase-negative 527F/295M-c-Src. A series of analyses with deletion mutants expressed in various cells revealed that the SH3 domain of Cas is necessary for its localization to focal adhesions in nontransformed cells while both the SH3 domain and the C-terminal Src binding domain of Cas are required in 527F-c-Src-transformed cells and fibronectin-stimulated cells. In addition, the localization of Cas to focal adhesions was abolished in Src-negative cells. These results demonstrate that the SH3 domain of Cas and the association of Cas with Src kinase play a pivotal role in the localization of Cas to focal adhesions.     

Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways

We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130(Cas) phosphorylation and stimulates focal adhesion kinase (FAK). However, the role of FAK and the related kinase, Pyk2, in Caco-2 spreading and migration is unclear. FAK- or Pyk2-specific siRNAs reduced protein levels by 90%. However, when detached cells were replated on collagen IV neither individual nor combined FAK and Pyk2 siRNAs affected the cell spreading rate. As combined FAK and Pyk2 siRNAs increased p130(Cas) protein levels, we cotransfected cells with 1 nm p130(Cas) siRNA to partially reduce p130(Cas) protein to control levels. Although p130(Cas) Tyr(P)(249) phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130(Cas) increased cell spreading by 20% compared to p130(Cas) siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130(Cas). FAK-binding mutant SH3 domain-deleted rat p130(Cas) was not phosphorylated after adhesion and, unlike full-length p130(Cas), did not restore spreading after human-specific p130(Cas) siRNA knockdown of endogenous p130(Cas). Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130(Cas) phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130(Cas).     

Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.     

Modulation of HeLa cells spreading by the non-receptor tyrosine kinase ACK-2

The CDC42 regulated non-receptor tyrosine kinase ACK-2 has been associated with integrin signaling. In this report, the effect of ACK-2 on the modulation of cell spreading and motility was examined. HeLa cells expressing epitope-tagged wild type ACK-2 showed a slower rate of spreading on fibronectin when compared with untransfected cells. An ACK-2 protein lacking its SH3 domain was still capable of modulating HeLa cell spreading suggesting that its tyrosine kinase activity is sufficient to induce the observed phenotype. The ACK-2 effect on the rate of cell spreading did not involve inhibition of integrin-mediated activation of PI-3K signaling, since it did not alter membrane translocation of a GFP-PH-AKT domain (AKT pleckstrin homology domain) used as a reporter for PI-3K products induced by cell adhesion. The ACK-2 effect appears to be upstream from the adapter protein CrkII, since co-expression of CrkII and ACK-2 results in a neutralization of ACK-2 mediated effects on HeLa cell spreading. Similarly, co-expression of p130Cas, which interacts with the adapter protein CrkII, with ACK-2, also results in a partial reversion of the ACK-2 effects on cell spreading. CrkII mediated reversal of the ACK-2 induced phenotype requires the activity of the small GTPase, Rap1. Co-expression of ACK-2 and CrkII with a dominant negative form of Rap1 reverses the neutralization by CrkII suggesting that CrkII mediated activation of Rap1 is required. However, an active form of Rap1 is not sufficient to reverse the ACK-2 phenotype by itself. A role for Rac1 in ACK-2 effects was also established. An activated Rac1 protein neutralized the ACK-2 mediated inhibition of cell spreading. A direct measurement of cell motility by either a modified Boyden chamber or wounding assay demonstrates that ACK-2 overexpression increases the motility of the cells. These results suggest that ACK-2 modulates HeLa cells spreading upstream of pathways regulated by CrkII and that ACK-2 may regulate cell motility by controlling the activation of small GTPases such as Rap1 and Rac1.     

AND-34/BCAR3 regulates adhesion-dependent p130Cas serine phosphorylation and breast cancer cell growth pattern

NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS-PAGE while NSP1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype.     

Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)

The focal adhesion adapter protein p130(cas) regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130(cas). AND-34/BCAR3, one of three NSP family members, binds the p130(cas) carboxyl terminus, adjacent to a bipartite p130(cas) Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130(cas). Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130(cas) complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130(cas) to bind the Src SH3 domain through an RPLPSPP motif in the p130(cas) SBD. Although our prior work identified phosphorylation of the serine within the p130(cas) RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130(cas). The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130(cas) complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130(cas) substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130(cas). Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130(cas) and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130(cas) complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130(cas) SBD.     

Two substrate-targeting sites in the Yersinia protein tyrosine phosphatase co-operate to promote bacterial virulence

YopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacterium Yersinia. Yersinia delivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C-terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N-terminal non-catalytic domain of YopH binds p130Cas in a phosphotyrosine-dependent manner and functions as a novel substrate-targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate-targeting site ('site 2') within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine-dependent manner, and co-operates with the N-terminal domain ('site 1') to promote efficient recognition of p130Cas by YopH in epithelial cells. The identification of two substrate-targeting sites in YopH that co-operate to promote epithelial cell detachment and bacterial virulence reinforces the importance of protein-protein interactions for determining protein tyrosine phosphatase specificity in vivo, and highlights the sophisticated nature of microbial pathogenicity factors.     

SH2-containing 5'-inositol phosphatase, SHIP2, regulates cytoskeleton organization and ligand-dependent down-regulation of the epidermal growth factor receptor

Phosphoinositide lipid second messengers are integral components of signaling pathways mediated by insulin, growth factors, and integrins. SHIP2 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate generated by the activated phosphatidylinositol 3'-kinase. SHIP2 down-regulates insulin signaling and is present at higher levels in diabetes and obesity. SHIP2 associates with p130Cas and filamin, regulators of cell adhesion/migration and cytoskeleton, influencing cell adhesion/spreading. Type I collagen specifically induces Src-mediated tyrosine phosphorylation of SHIP2. To better understand SHIP2 function, we employed RNA interference (RNAi) approach to silence the expression of the endogenous SHIP2 in HeLa cells. Suppression of SHIP2 levels caused severe F-actin deformities characterized by weak cortical actin and peripheral actin spikes. SHIP2 RNAi cells displayed cell-spreading defects involving a notable absence of focal contact structures and the formation of multiple slender membrane protrusions capped by actin spikes. Furthermore, decreased SHIP2 levels altered distribution of early endocytic antigen 1 (EEA1)-positive endocytic vesicles and of vesicles containing internalized epidermal growth factor (EGF) and transferrin. EGF treatment of SHIP2 RNAi cells led to the following: enhanced EGF receptor (EGFR) degradation; increased EGFR ubiquitination; and increased association of EGFR with c-Cbl ubiquitin ligase. Taken together, these experiments demonstrate that SHIP2 functions in the maintenance and dynamic remodeling of actin structures as well as in endocytosis, having a major impact on ligand-induced EGFR internalization and degradation. Accordingly, we suggest that, in HeLa cells, SHIP2 plays a distinct role in signaling pathways mediated by integrins and growth factor receptors.     

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in the Cas substrate domain. These tyrosine-phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas–Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-length Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects.     

CD36 signals to the actin cytoskeleton and regulates microglial migration via a p130Cas complex

The pattern recognition receptor CD36 initiates a signaling cascade that promotes microglial activation and recruitment to beta-amyloid deposits in the brain. In the present study we identify the focal adhesion-associated proteins p130Cas, Pyk2, and paxillin as novel members of the tyrosine kinase signaling pathway downstream of CD36 and show that assembly of this complex is essential for microglial migration. In primary microglia and macrophages exposed to beta-amyloid, the scaffolding protein p130Cas is rapidly tyrosine-phosphorylated and co-localizes with CD36 to membrane ruffles contemporaneous with F-actin polymerization. These beta-amyloid-stimulated events are not detected in CD36 null cells and are dependent on CD36 activation of Src family tyrosine kinases. Fyn, a Src kinase known to interact with CD36, co-precipitates with p130Cas and is an essential upstream intermediate in the signaling pathways leading to phosphorylation of the p130Cas substrate domain. Furthermore, the p130Cas-interacting kinase Pyk2 and the cytoskeletal adapter protein paxillin also demonstrate CD36-dependent phosphorylation, identifying these focal adhesion molecules as additional members of this beta-amyloid signaling cascade. Disruption of this p130Cas complex by small interfering RNA silencing inhibits p44/42 mitogen-activated protein kinase phosphorylation and microglial migration, illustrating the importance of this pathway in microglial activation and recruitment. Together, these data are the first to identify the signaling cascade that directly links CD36 to the actin cytoskeleton and, thus, implicates it in diverse processes such as cellular migration, adhesion, and phagocytosis.     

Leukemia-associated mutations in SHIP1 inhibit its enzymatic activity, interaction with the GM-CSF receptor and Grb2, and its ability to inactivate PI3K/AKT signaling

The inositol 5-phosphatase SHIP1 is a negative regulator of the PI3K/AKT pathway, which is constitutively activated in 50-70% of acute myeloid leukemias (AML). Ten different missense mutations in SHIP1 have been described in 3% of AML patients suggesting a functional role of SHIP1 in AML. Here, we report the identification of two new SHIP1 mutations T162P and R225W that were detected in 2 and 1 out of 96 AML patients, respectively. The functional analysis of all 12 AML-associated SHIP1 mutations, one ALL-associated SHIP1 mutation (Q1076X) and a missense SNP (H1168Y) revealed that two mutations i.e. Y643H and P1039S abrogated the ability of SHIP1 to reduce constitutive PI3K/AKT signaling in Jurkat cells. The loss of function of SHIP1 mutant Y643H which is localized in the inositol phosphatase domain was due to a reduction of the specific activity by 84%. Because all other SHIP1 mutants had a normal enzymatic activity, we assumed that these SHIP1 mutants may be functionally impaired due to a loss of interaction with plasma membrane receptors or adapter proteins. In agreement with this model, we found that the SHIP1 mutant F28L located in the FLVR motif of the SH2 domain was incapable of binding tyrosine-phosphorylated proteins including the GM-CSF receptor and that the SHIP1 mutant Q1076X lost its ability to bind to the C-terminal SH3 domain of the adapter protein Grb2. In addition, SHIP1 mutant P1039S which does not reduce PI3K/AKT signaling anymore is located in a PXXP SH3 domain consensus binding motif suggesting that mutation of the conserved proline residue interferes with binding of SHIP1 to a so far unidentified SH3 domain containing protein. In summary, our data indicate that SHIP1 mutations detected in human leukemia patients impair the negative regulatory function of SHIP1 on PI3K/AKT signaling in leukemia cells either directly by reduced enzymatic activity or indirectly by disturbed protein interaction with tyrosine-phosphorylated membrane receptors or adapter proteins. These results further support a functional role of SHIP1 as tumor suppressor protein in the pathogenesis of AML.     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.     

Activation-induced bi-dentate interaction of SHIP and Shc in B lymphocytes

SHIP is a SH2 domain-containing inositol polyphosphatase that is selectively tyrosine phosphorylated and associated with the adapter protein Shc in B lymphocytes upon co-crosslinking surface immunoglobulin and FcγRIIB1. We previously observed that this stimulation condition is associated with a reduction in the interaction of Grb2 with phosphorylated Shc, an enhanced interaction of Shc with SHIP, and a block in the Ras signaling pathway. We proposed that the SH2 domain of SHIP competes with Grb2 in binding to phospho-Shc, resulting in a block in Ras signaling. To test this model, we examined the mode of SHIP–Shc interaction. Using recombinant Shc and SHIP interaction domains and purified Shc and SHIP phosphopeptides, we show that the interaction is bi-dentate such that the SH2 domain of SHIP recognizes phosphorylated Y317 and doubly-phosphorylated Y239/Y240 of Shc and the Shc PTB domain recognizes phosphorylated NPxpY motifs within SHIP. We observed no role for the Shc SH2 domain in the interaction. These findings are consistent with our earlier model that SHIP and Grb2 compete for binding to phospho-Shc and support the notion that, in addition to the hydrolysis of inositol phosphates and phospholipids, SHIP contributes to anti-proliferative biochemistry by blocking protein–protein interactions. J. Cell. Biochem. 67:32–42, 1997. © 1997 Wiley-Liss, Inc.     

A p130 Cas tyrosine phosphorylated substrate domain decoy disrupts v-Crk signaling

Background  

The adaptor protein p130 ^Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals.     

BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas

BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas^−/− murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance.     

Protein kinase C-delta is a negative regulator of antigen-induced mast cell degranulation

Regulation of mast cell degranulation is dependent on the subtle interplay of cellular signaling proteins. The Src homology 2 (SH2) domain-containing inositol-5'-phosphatase (SHIP), which acts as the gatekeeper of degranulation, binds via both its SH2 domain and its phosphorylated NPXY motifs to the adapter protein Shc via the latter's phosphorylated tyrosines and phosphotyrosine-binding domain, respectively. This theoretically leaves Shc's SH2 domain available to bind proteins, which might be part of the SHIP/Shc complex. In a search for such proteins, protein kinase C-delta (PKC-delta) was found to coprecipitate in mast cells with Shc and to interact with Shc's SH2 domain following antigen or pervanadate stimulation. Phosphorylation of PKC-delta's Y(332), most likely by Lyn, was found to be responsible for PKC-delta's binding to Shc's SH2 domain. Using PKC-delta(-/-) bone marrow-derived mast cells (BMMCs), we found that the antigen-induced tyrosine phosphorylation of Shc was similar to that in wild-type (WT) BMMCs while that of SHIP was significantly increased. Moreover, increased translocation of PKC-delta to the membrane, as well as phosphorylation at T505, was observed in SHIP(-/-) BMMCs, demonstrating that while PKC-delta regulates SHIP phosphorylation, SHIP regulates PKC-delta localization and activation. Interestingly, stimulation of PKC-delta(-/-) BMMCs with suboptimal doses of antigen yielded a more sustained calcium mobilization and a significantly higher level of degranulation than that of WT cells. Altogether, our data suggest that PKC-delta is a negative regulator of antigen-induced mast cell degranulation.     

SH2-containing inositol 5'-phosphatase inhibits transformation of Abelson murine leukemia virus

v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) transforms pre-B cells. Transformation requires the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is antagonized by SH2-containing inositol 5'-phosphatase (SHIP), raising the possibility that v-Abl modulates PI3K signaling through SHIP. Consistent with this, we show that v-Abl expression reduces levels of full-length p145 SHIP in a v-Abl kinase activity-dependent fashion. This event requires signals from the Abl SH2 domain but not the carboxyl terminus. Forced expression of full-length SHIP significantly reduces Ab-MLV pre-B-cell transformation. Therefore, reduction of SHIP protein by v-Abl is a critical component in Ab-MLV transformation.     

Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling.

SH2-containing inositol 5'-phosphatase (SHIP) plays a negative regulatory role in hematopoietic cells. We have now cloned the rat SHIP isozyme (SHIP2) cDNA from skeletal muscle, which is one of the most important target tissue of insulin action. Rat SHIP2 cDNA encodes a 1183-amino-acid protein that is 45% identical with rat SHIP. Rat SHIP2 contains an amino-terminal SH2 domain, a central 5'-phosphoinositol phosphatase activity domain, and a phosphotyrosine binding (PTB) consensus sequence and a proline-rich region at the carboxyl tail. Specific antibodies to SHIP2 were raised and the function of SHIP2 was studied by stably overexpressing rat SHIP2 in Rat1 fibroblasts expressing human insulin receptors (HIRc). Endogenous SHIP2 underwent insulin-mediated tyrosine phosphorylation and phosphorylation was markedly increased when SHIP2 was overexpressed. Although overexpression of SHIP2 did not affect insulin-induced tyrosine phosphorylation of the insulin receptor beta-subunit and Shc, subsequent association of Shc with Grb2 was inhibited, possibly by competition between the SH2 domains of SHIP2 and Grb2 for the Shc phosphotyrosine. As a result, insulin-stimulated MAP kinase activation was reduced in SHIP2-overexpressing cells. Insulin-induced tyrosine phosphorylation of IRS-1, IRS-1 association with the p85 subunit of PI3-kinase, and PI3-kinase activation were not affected by overexpression of SHIP2. Interestingly, although both PtdIns-(3,4,5)P3 and PtdIns(3,4)P2 have been implicated in the regulation of Akt activity in vitro, overexpression of SHIP2 inhibited insulin-induced Akt activation, presumably by its 5'-inositol phosphatase activity. Furthermore, insulin-induced thymidine incorporation was decreased by overexpression of SHIP2. These results indicate that SHIP2 plays a negative regulatory role in insulin-induced mitogenesis, and regulation of the Shc. Grb2 complex and of the downstream products of PI3-kinase provides possible mechanisms of SHIP2 action in insulin signaling.     

The two SH2-domain-containing inositol 5-phosphatases SHIP1 and SHIP2 are coexpressed in human T lymphocytes.

The activation of many hematopoietic cells via cytokine receptors, as well as B and T cell receptors, leads to the tyrosine phosphorylation of Shc and its association with both Grb2-Sos1 complexes and with a 145 kDa protein referred to as the SH2 containing inositol 5-phosphatase (SHIP1). In a search of putative 5-phosphatase isoenzymes, we have isolated a second SH2 domain containing inositol 5-phosphatase, referred to as (SHIP2). Both SHIP1 and SHIP2 are coexpressed in human T lymphocytes. This was shown at the protein level by Western blot analysis in transformed T cell lines and in peripheral blood T lymphocytes either unstimulated or after in vitro activation through TCR-CD3 complex. SHIP1 protein level was not modulated after activation of T lymphocytes, in contrast to SHIP2, which was increased after long-term stimulation. SHIP1 was tyrosine phosphorylated in resting naive T cells. This was not observed in the transformed T cell lines. T lymphocyte is therefore a model of coexpression of the two SH2-containing inositol 5-phosphatases SHIP1 and SHIP2.