Carcinoembryonic antigen-related cell adhesion molecule 1 inhibits proximal TCR signaling by targeting ZAP-70

The long cytoplasmic tail (CT) isoforms of carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1) are expressed on activated human T cells and possess two ITIM motifs in the CT. These isoforms of CEACAM1 are inhibitory for T cell responses initiated by the TCR/CD3 complex with the inhibition dependent upon the ITIMs of CEACAM1 and Src homology 2 domain-containing phosphatase 1 (SHP-1). However, the mechanism by which this inhibition occurs in T cells is unknown. We demonstrate here that the Src family kinase, Lck, and the ability of CEACAM1 to bind homophilically are required for the ITIM phosphorylation of CEACAM1 that is a prerequisite for CEACAM1 association with SHP-1. We further show that CEACAM1 associates with and recruits SHP-1 to the TCR/CD3 complex leading to decreased phosphorylation of CD3-zeta and ZAP-70 and consequently decreased activation of the elements downstream of ZAP-70. This is physiologically relevant because extinction of SHP-1 expression or blockade of homophilic binding by CEACAM1 using a Fab that specifically recognizes the homophilic binding region of human CEACAM1 increases the cytolytic function initiated by the TCR/CD3 complex. These studies show that long CT isoforms of CEACAM1 orchestrate an inhibitory program that abrogates extremely proximal events downstream of the TCR/CD3 complex by focusing on the activation of ZAP-70.     

RhoH regulates subcellular localization of ZAP-70 and Lck in T cell receptor signaling

RhoH is an hematopoietic-specific, GTPase-deficient Rho GTPase that plays a role in T development. We investigated the mechanisms of RhoH function in TCR signaling. We found that the association between Lck and CD3ζ was impaired in RhoH-deficient T cells, due to defective translocation of both Lck and ZAP-70 to the immunological synapse. RhoH with Lck and ZAP-70 localizes in the detergent-soluble membrane fraction where the complex is associated with CD3ζ phosphorylation. To determine if impaired translocation of ZAP-70 was a major determinant of defective T cell development, Rhoh(-/-) bone marrow cells were transduced with a chimeric myristoylation-tagged ZAP-70. Myr-ZAP-70 transduced cells partially reversed the in vivo defects of RhoH-associated thymic development and TCR signaling. Together, our results suggest that RhoH regulates TCR signaling via recruitment of ZAP-70 and Lck to CD3ζ in the immunological synapse. Thus, we define a new function for a RhoH GTPase as an adaptor molecule in TCR signaling pathway.     

Inhibition of ZAP-70 kinase activity via an analog-sensitive allele blocks T cell receptor and CD28 superagonist signaling

ZAP-70 is a cytoplasmic protein tyrosine kinase that is required for T cell antigen receptor (TCR) signaling. Both mice and humans deficient in ZAP-70 fail to develop functional T cells, thus demonstrating its necessity for T cell development and function. There is currently no highly specific, cell-permeable, small molecule inhibitor for ZAP-70; therefore, we generated a mutant ZAP-70 allele that retains kinase activity but is sensitive to inhibition by a mutant-specific inhibitor. We validated the chemical genetic inhibitor system in Jurkat T cell lines, where the inhibitor blocked ZAP-70-dependent TCR signaling in cells expressing the analog-sensitive allele. Interestingly, the inhibitor also ablated CD28 superagonist signaling, thereby demonstrating the utility of this system in dissecting the requirement for ZAP-70 in alternative mechanisms of T cell activation. Thus, we have developed the first specific chemical means of inhibiting ZAP-70 in cells, which serves as a valuable tool for studying the function of ZAP-70 in T cells.     

Superantigen-induced T cell:B cell conjugation is mediated by LFA-1 and requires signaling through Lck, but not ZAP-70.

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.     

Cutting edge: T cell migration regulated by CXCR4 chemokine receptor signaling to ZAP-70 tyrosine kinase

Chemokines regulate the homeostatic trafficking of lymphocytes and lymphocyte influx into sites of injury and inflammation. The signaling pathways by which chemokine receptors regulate lymphocyte migration remain incompletely characterized. We demonstrate that Jurkat T cells lacking the ZAP-70 tyrosine kinase exhibit reduced migration in response to the CXCR4 ligand CXCL12 when compared with wild-type Jurkat T cells. Expression of wild-type, but not kinase-inactive, ZAP-70 resulted in enhanced migration of ZAP-70-deficient Jurkat T cells. The tyrosine residue at position 292 in the interdomain B region of ZAP-70 exerts a negative regulatory effect on ZAP-70-dependent migration. Stimulation of Jurkat T cells with CXCL12 also resulted in ZAP-70-dependent tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) adapter protein. Although CXCL12-dependent migration of SLP-76-deficient Jurkat T cells was impaired, re-expression of SLP-76 did not enhance migration. These results suggest a novel function for ZAP-70, but not SLP-76, in CXCR4 chemokine receptor signaling in human T cells.     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

Talin-1 and kindlin-3 regulate alpha4beta1 integrin-mediated adhesion stabilization, but not G protein-coupled receptor-induced affinity upregulation

Chemokine/chemoattractant G protein-coupled receptors trigger an inside-out signaling network that rapidly activates integrins, a key step in inflammatory leukocyte recruitment. Integrins mediate leukocyte arrest and adhesion to endothelium through multivalent binding, and they transmit outside-in signals to stabilize adhesion and coordinate cell spreading and migration. In the present study, we used RNA interference in the U937 monocytic cell line to investigate the role of talin-1, kindlin-3, and α-actinin-1 in the fMLF- and SDF-1α-induced upregulation of α(4)β(1) integrin affinity and consequent adhesive events. Affinity upregulation of α(4)β(1) integrin was not impaired by small interfering RNA knockdown of talin-1, kindlin-3, or α-actinin-1. Only kindlin-3 knockdown increased flow-induced detachment from VCAM-1-coated surfaces in response to fluid flow, whereas knockdown of either talin-1 or kindlin-3 increased detachment from ICAM-1-coated surfaces. Biochemical analyses revealed that α(4)β(1) expression was highly enriched in U937 cell microridges and murine lymphocyte microvilli. Kindlin-3 was present throughout the cell, whereas talin-1 was largely excluded from microridges/microvilli. The subcellular colocalization of α(4)β(1) and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α(4)β(1) after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α(4)β(1)-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement.     

Regulation of integrin-mediated T cell adhesion by the transmembrane protein tyrosine phosphatase CD45.

The transmembrane protein tyrosine phosphatase CD45 is required for Ag receptor signal transduction in lymphocytes. Recently, a role for CD45 in the regulation of macrophage adhesion has been demonstrated as well. To investigate further the role of CD45 in the regulation of adhesion, we examined integrin-mediated adhesion to fibronectin of two T cell lines and their CD45-deficient variants. The absence of CD45 correlated with enhanced adhesion to fibronectin via integrin alpha5beta1 (VLA-5), but not alpha4beta1 (VLA-4) in both cell lines. Adhesion returned to normal levels upon transfection of wild-type CD45 into the CD45-deficient lines. Transfection of chimeric or mutant molecules expressing some, but not all, CD45 domains and activities demonstrated that both the transmembrane domain and the tyrosine phosphatase activity of CD45 were required for regulation of integrin-dependent adhesion, but the highly glycosylated extracellular domain was dispensable. In contrast, only a catalytically active CD45 cytoplasmic domain was required for TCR signaling. Transfectants that restored normal levels of adhesion to fibronectin coimmunoprecipitated with the transmembrane protein known as CD45-associated protein. These studies demonstrate a novel role for CD45 in adhesion regulation and suggest a possible function for its association with CD45-associated protein.     

Docking protein Gab2 is phosphorylated by ZAP-70 and negatively regulates T cell receptor signaling by recruitment of inhibitory molecules

To maintain various T cell responses and immune equilibrium, activation signals triggered by T cell antigen receptor (TCR) must be regulated by inhibitory signals. Gab2, an adaptor protein of the insulin receptor substrate-1 family, has been shown to be involved in the downstream signaling from cytokine receptors. We investigated the functional role of Gab2 in TCR-mediated signal transduction. Gab2 was phosphorylated by ZAP-70 and co-precipitated with phosphoproteins, such as ZAP-70, LAT, and CD3zeta, upon TCR stimulation. Overexpression of Gab2 in Jurkat cells or antigen-specific T cell hybridomas resulted in the inhibition of NF-AT activation, interleukin-2 production, and tyrosine phosphorylation. The structure-function relationship of Gab2 was analyzed by mutants of Gab2. The Gab2 mutants lacking SHP-2-binding sites mostly abrogated the inhibitory activity of Gab2, but its inhibitory function was restored by fusing to active SHP-2 as a chimeric protein. A mutant with defective phosphatidylinositol 3-kinase binding capacity also impaired the inhibitory activity, and the pleckstrin homology domain-deletion mutant revealed a crucial function of the pleckstrin homology domain for localization to the plasma membrane. These results suggest that Gab2 is a substrate of ZAP-70 and functions as a switch molecule toward inhibition of TCR signal transduction by mediating the recruitment of inhibitory molecules to the TCR signaling complex.     

Characterization of the signaling pathways regulating alpha2beta1 integrin-mediated events by a pharmacological approach

In certain instances of developing and adult organism, epithelial cells can change morphology and transform into mesenchymal-like type in order to move through the extracellular matrix. However, because of the multiplicity and complexity of signaling pathways that contribute to these processes, their molecular dissection has remained difficult. By using a pharmacological approach on the rat bladder carcinoma cell line NBT-II dispersion system, we have identified distinct signaling events for adhesion and motility in response to collagen, both activities depending on alpha2beta1 integrin. Treatment of cells with PKC inhibitors markedly impaired initial attachment on collagen without affecting the capacity of cells to move, suggesting that PKC activity is required for initial adhesion strength during cell translocation. Both adhesion and motility were diminished by tyrosine kinase inhibitors herbimycin and tyrphostin whereas tyrosine phosphatase inhibitors amplified cell scattering. The collagen-induced dispersion was insensitive to genistein which we previously showed to abrogate growth factor-induced scattering, thus demonstrating inducer specificity. Finally. Ras inhibitors and expression of a dominant negative form of Ras (N17Ras) while affecting initial cell attachment, did not prevent cell migration, and instead favored the dissociated state on collagen. The specific signaling pathways identified for adhesion and motility should help to understand the sequential processes associated with cell migration.     

Transformation of chicken embryo fibroblasts by v-src uncouples beta1 integrin-mediated outside-in but not inside-out signaling

Adhesion of cells to extracellular matrix is mediated by integrin family receptors. The process of receptor-ligand binding is dependent on metabolic energy and is regulated by intracellular signals, termed inside-out signals. The strength of the initial alpha5beta1-mediated adhesion of v-src-transformed chicken embryo fibroblasts (v-srcCEF) was similar to that of normal CEF. A chemically cross-linked fibronectin substrate was able to restore cell spreading and the ability of v-srcCEF to assemble a fibronectin matrix. Over time, v-srcCEF showed decreased adhesion due to the reduction of alpha5beta1-fibronectin bonds consequent on the reduction of substrate-bound fibronectin due to the secretion of proteases by v-srcCEF. Excess synthesis of hyaluronic acid by v-srcCEF also reduced the alpha5beta1-fibronectin bonds and contributed to cell detachment at later times in culture. Thus, the adhesion defects were not due to a failure of alpha5beta1 function and adhesion of the v-srcCEF was alpha5beta1 dependent. Integrin-mediated adhesion also produces signals that affect cell proliferation and cell differentiation. An early consequence of these "outside-in" signals was the phosphorylation of FAK Y397 in direct proportion to the number of alpha5beta1-fibronectin bonds formed. In contrast, v-srcCEF had an increased level of phosphorylation on five different tyrosines in FAK, and none of these phosphorylation levels were sensitive to the number of alpha5beta1-fibronectin bonds. In the absence of serum, CEF proliferation was sensitive to changes in alpha5beta1-mediated adhesion levels. Transformation by v-src increased the serum-free proliferation rate and made it insensitive to alpha5beta1-mediated adhesion. Thus, the v-srcCEF were insensitive to the normal outside-in signals from alpha5beta1 integrin.     

The Shc family protein adaptor, Rai, negatively regulates T cell antigen receptor signaling by inhibiting ZAP-70 recruitment and activation

Rai/ShcC is a member of the Shc family of protein adaptors expressed with the highest abundance in the central nervous system, where it exerts a protective function by coupling neurotrophic receptors to the PI3K/Akt survival pathway. Rai is also expressed, albeit at lower levels, in other cell types, including T and B lymphocytes. We have previously reported that in these cells Rai attenuates antigen receptor signaling, thereby impairing not only cell proliferation but also, opposite to neurons, cell survival. Here we have addressed the mechanism underlying the inhibitory activity of Rai on TCR signaling. We show that Rai interferes with the TCR signaling cascade one of the earliest steps--recruitment of the initiating kinase ZAP-70 to the phosphorylated subunit of the TCR/CD3 complex, which results in a generalized dampening of the downstream signaling events. The inhibitory activity of Rai is associated to its inducible recruitment to phosphorylated CD3, which occurs in the physiological signaling context of the immune synapse. Rai is moreover found as a pre-assembled complex with ZAP-70 and also constitutively interacts with the regulatory p85 subunit of PI3K, similar to neuronal cells, notwithstanding the opposite biological outcome, i.e. impairment of PI-3K/Akt activation. The data highlight the ability of Rai to establish interactions with the TCR and key signaling mediators which, either directly (e.g. by inhibiting ZAP-70 recruitment to the TCR or sequestering ZAP-70/PI3K in the cytosol) or indirectly (e.g. by promoting the recruitment of effectors responsible for signal extinction) prevent full triggering of the TCR signaling cascade.     

Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor

cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1-GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the G alpha s-coupled beta 2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.     

Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. alpha-Casein and beta-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.     

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.     

Inhibition of beta(2) integrin-mediated leukocyte cell adhesion by leucine-leucine-glycine motif-containing peptides

Many integrins mediate cell attachment to the extracellular matrix by recognizing short tripeptide sequences such as arginine-glycine-aspartic acid and leucine-aspartate-valine. Using phage display, we have now found that the leukocyte-specific beta(2) integrins bind sequences containing a leucine-leucine-glycine (LLG) tripeptide motif. An LLG motif is present on intercellular adhesion molecule (ICAM)-1, the major beta(2) integrin ligand, but also on several matrix proteins, including von Willebrand factor. We developed a novel beta(2) integrin antagonist peptide CPCFLLGCC (called LLG-C4), the structure of which was determined by nuclear magnetic resonance. The LLG-C4 peptide inhibited leukocyte adhesion to ICAM-1, and, interestingly, also to von Willebrand factor. When immobilized on plastic, the LLG-C4 sequence supported the beta(2) integrin-mediated leukocyte adhesion, but not beta(1) or beta(3) integrin-mediated cell adhesion. These results suggest that LLG sequences exposed on ICAM-1 and on von Willebrand factor at sites of vascular injury play a role in the binding of leukocytes, and LLG-C4 and peptidomimetics derived from it could provide a therapeutic approach to inflammatory reactions.     

(-)-Epigallocatechin gallate regulates CD3-mediated T cell receptor signaling in leukemia through the inhibition of ZAP-70 kinase

The zeta chain-associated 70-kDa protein (ZAP-70) of tyrosine kinase plays a critical role in T cell receptor-mediated signal transduction and the immune response. A high level of ZAP-70 expression is observed in leukemia, which suggests ZAP-70 as a logical target for immunomodulatory therapies. (-)-Epigallocatechin gallate (EGCG) is one of the major green tea catechins that is suggested to have a role as a preventive agent in cancer, obesity, diabetes, and cardiovascular disease. Here we identified ZAP-70 as an important and novel molecular target of EGCG in leukemia cells. ZAP-70 and EGCG displayed high binding affinity (Kd = 0.6207 micromol/liter), and additional results revealed that EGCG effectively suppressed ZAP-70, linker for the activation of T cells, phospholipase Cgamma1, extracellular signaling-regulated kinase, and MAPK kinase activities in CD3-activated T cell leukemia. Furthermore, the activation of activator protein-1 and interleukin-2 induced by CD3 was dose-dependently inhibited by EGCG treatment. Notably, EGCG dose-dependently induced caspase-mediated apoptosis in P116.cl39 ZAP-70-expressing leukemia cells, whereas P116 ZAP-70-deficient cells were resistant to EGCG treatment. Molecular docking studies, supported by site-directed mutagenesis experiments, showed that EGCG could form a series of intermolecular hydrogen bonds and hydrophobic interactions within the ATP binding domain, which may contribute to the stability of the ZAP-70-EGCG complex. Overall, these results strongly indicated that ZAP-70 activity was inhibited specifically by EGCG, which contributed to suppressing the CD3-mediated T cell-induced pathways in leukemia cells.     

Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling

Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivo capacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT. Collectively, our data reveal the pivotal role of Tyr319 phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling.     

Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin alpha(v)beta(3) induces the [Ca(2+)](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. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alpha(v)beta(3) 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.