A Novel Cross-talk in Diacylglycerol Signaling: THE RAC-GAP β2-CHIMAERIN IS NEGATIVELY REGULATED BY PROTEIN KINASE Cδ-MEDIATED PHOSPHORYLATION*

Although the family of chimaerin Rac-GAPs has recently gained significant attention for their involvement in development, cancer, and neuritogenesis, little is known about their molecular regulation. Chimaerins are activated by the lipid second messenger diacylglycerol via their C1 domain upon activation of tyrosine kinase receptors, thereby restricting the magnitude of Rac signaling in a receptor-regulated manner. Here we identified a novel regulatory mechanism for β2-chimaerin via phosphorylation. Epidermal growth factor or the phorbol ester phorbol 12-myristate 13-acetate caused rapid phosphorylation of β2-chimaerin on Ser¹⁶⁹ located in the SH2-C1 domain linker region via protein kinase Cδ, which retained β2-chimaerin in the cytosol and prevented its C1 domain-mediated translocation to membranes. Furthermore, despite the fact that Ser¹⁶⁹ phosphorylation did not alter intrinsic Rac-GAP activity in vitro, a non-phosphorylatable β2-chimaerin mutant was highly sensitive to translocation, and displayed enhanced association with activated Rac, enhanced Rac-GAP activity, and anti-migratory properties when expressed in cells. Our results not only revealed a novel regulatory mechanism that facilitates Rac activation, but also identified a novel mechanism of cross-talk between diacylglycerol receptors that restricts β2-chimaerin relocalization and activation.     

Phospholipase Cgamma/diacylglycerol-dependent activation of beta2-chimaerin restricts EGF-induced Rac signaling

Although receptor-mediated regulation of small G-proteins and the cytoskeleton is intensively studied, the mechanisms for attenuation of these signals are poorly understood. In this study, we have identified the Rac-GAP beta2-chimaerin as an effector of the epidermal growth factor receptor (EGFR) via coupling to phospholipase Cgamma (PLCgamma) and generation of the lipid second messenger diacylglycerol (DAG). EGF redistributes beta2-chimaerin to promote its association with the small GTPase Rac1 at the plasma membrane, as determined by FRET. This relocalization and association with Rac1 were impaired by disruption of the beta2-chimaerin C1 domain as well as by PLCgamma1 RNAi, thus defining beta2-chimaerin as a novel DAG effector. On the other hand, GAP-deficient beta2-chimaerin mutants show enhanced translocation and sustained Rac1 association in the FRET assays. Remarkably, RNAi depletion of beta2-chimaerin significantly extended the duration of Rac activation by EGF, suggesting that beta2-chimaerin serves as a mechanism that self-limits Rac activity in response to EGFR activation. Our results represent the first direct evidence of divergence in DAG signaling downstream of a tyrosine-kinase receptor via a PKC-independent mechanism.     

T Cell Receptor-dependent Tyrosine Phosphorylation of ��2-Chimaerin Modulates Its Rac-GAP Function in T Cells

The actin cytoskeleton has an important role in the organization and function of the immune synapse during antigen recognition. Dynamic rearrangement of the actin cytoskeleton in response to T cell receptor (TCR) triggering requires the coordinated activation of Rho family GTPases that cycle between active and inactive conformations. This is controlled by GTPase-activating proteins (GAP), which regulate inactivation of Rho GTPases, and guanine exchange factors, which mediate their activation. Whereas much attention has centered on guanine exchange factors for Rho GTPases in T cell activation, the identity and functional roles of the GAP in this process are largely unknown. We previously reported β2-chimaerin as a diacylglycerol-regulated Rac-GAP that is expressed in T cells. We now demonstrate Lck-dependent phosphorylation of β2-chimaerin in response to TCR triggering. We identify Tyr-153 as the Lck-dependent phosphorylation residue and show that its phosphorylation negatively regulates membrane stabilization of β2-chimaerin, decreasing its GAP activity to Rac. This study establishes the existence of TCR-dependent regulation of β2-chimaerin and identifies a novel mechanism for its inactivation.T cell activation requires presentation of an antigen by antigen-presenting cells (APC)² to the T cell receptor (TCR); this event involves the reorganization of several scaffolds and signaling proteins, leading to formation of the immunological synapse (IS) (1). Correct protein redistribution during synapse formation is critical for an efficient T cell response, and it is largely regulated by actin polymerization at the T cell/APC contact site as a result of TCR-regulated Rac-dependent signals (2, 3). Like other Rho GTPases, Rac cycles between a GTP-bound active state and a GDP-bound inactive state. This continuous recycling is regulated by the concerted action of two proteins as follows: GEF, which activates Rac by mediating GDP/GTP exchange (4), and GAP, which induces Rac inactivation by accelerating intrinsic Rac GTPase activity, converting GTP to GDP (5).Vav-1 is the best studied GEF for Rac, and it has critical roles in T cell-dependent functions (6). In naive, unstimulated T cells, Vav-1 is in an inactive state through autoinhibition, as the GEF domain is blocked by the N-terminal region (7). This autoinhibition is relieved by TCR-mediated tyrosine phosphorylation (8, 9). Thymocytes from Vav-1-deficient mice have a developmental block, and their mature T cells show severe defects in IS formation, as well as reduced Ca²⁺ influx, IL-2 production, T cell proliferation, and cytotoxic activity (10–13). Although several studies have shown a key role for Vav-1, the mechanisms that govern Rac inactivation downstream of the TCR remain elusive.The chimaerins are a family of Rho-GAP, with specific activity for Rac. In addition to their catalytic domain, they have an N-terminal SH2 domain and a C1 domain required for interaction with the lipid messenger diacylglycerol (DAG) and with phorbol esters (14). There are two mammalian chimaerin genes (CHN1 and CHN2), which encode the full-lengthα2-(ARHGAP2) and β2-chimaerins (ARHGAP3), and at least one splice variant each (α1 and β1) that lack the SH2 domain. The α-chimaerins are expressed abundantly in brain and are linked to neuritogenesis and axon guidance (15–20). β2-Chimaerin expression is ubiquitous (21) and is involved in EGF-dependent Rac regulation (22, 23). Experiments in T cells showed that β2-chimaerin participates in chemokine-dependent regulation of T cell migration and adhesion (24). A very recent study implicates chimaerins in the modulation of Rac activity during T cell synapse formation, suggesting that this protein family contributes to DAG-mediated regulation of cytoskeletal remodeling during T cell activation (25).Determination of the β2-chimaerin crystal structure provided important clues regarding its mechanism of action. In the absence of stimulation, the protein is in an inactive state in which the N-terminal domain maintains a “closed” conformation, blocking Rac binding and concealing the C1 domain (26). These structural data were fully supported by experiments in live T lymphocytes showing that phorbol myristate acetate (PMA)-dependent translocation of β2-chimaerin was less effective than that of its isolated C1 domain (24). These data not only confirmed the lack of accessibility of the β2-chimaerin C1 domain but also suggested that there are negative regulatory mechanisms that promote β2-chimaerin release from the membrane.DAG-dependent signaling is critical for the modulation of T cell functions, by virtue of its ability to bind and regulate C1 domain-containing proteins such as protein kinase Cθ, protein kinase D, and RasGRP1 (27). An important issue is to determine how the different DAG-binding proteins discriminate between distinct DAG pools, and how DAG activates certain C1-containing proteins and not others. Some mechanisms that allow discrimination between DAG receptors include the distinct affinity of C1 domains for different DAG pools, association of C1 domain-containing proteins to specific scaffolds, and/or structural determinants in these proteins that limit C1 domain accessibility to membrane DAG (28–30).To explore the events that contribute to the specific regulation of β2-chimaerin, we studied β2-chimaerin phosphorylation in the context of TCR stimulation. We show that β2-chimaerin is phosphorylated in tyrosine residues after TCR stimulation, and we identify Lck as the Tyr kinase responsible for this phosphorylation. Generation of point mutants identified Tyr-153, at the hinge of the SH2 and C1 domains, as the main tyrosine residue phosphorylated in response to TCR stimulation. Cells expressing a β2-chimaerin mutant defective for Tyr-153 phosphorylation show anomalies in TCR clustering, conjugate formation, NF-AT activation, and IL-2 production that correlate with elevated Rac-GAP activity in this mutant. Subcellular localization analysis of the β2-chimaerin mutants reveals that impairment of β2-chimaerin phosphorylation at Tyr-153 promotes C1-mediated β2-chimaerin stabilization at the plasma membrane, providing a mechanistic explanation for its higher Rac-GAP activity. In summary, our results demonstrate for the first time that tyrosine kinase-mediated negative regulation of β2-chimaerin is elicited by physiological stimulation in T lymphocytes, and suggest that TCR stimulation provides both positive and negative signals for β2-chimaerin activation.     

Phorbol esters and related analogs regulate the subcellular localization of beta 2-chimaerin, a non-protein kinase C phorbol ester receptor

The novel phorbol ester receptor beta2-chimaerin is a Rac-GAP protein possessing a single copy of the C1 domain, a 50-amino acid motif initially identified in protein kinase C (PKC) isozymes that is involved in phorbol ester and diacylglycerol binding. We have previously shown that, like PKCs, beta2-chimaerin binds phorbol esters with high affinity in a phospholipid-dependent manner (Caloca, M. J., Fernandez, M. N., Lewin, N. E., Ching, D., Modali, R., Blumberg, P. M., and Kazanietz, M. G. (1997) J. Biol. Chem. 272, 26488-26496). In this paper we report that like PKC isozymes, beta2-chimaerin is translocated by phorbol esters from the cytosolic to particulate fraction. Phorbol esters also induce translocation of alpha1 (n)- and beta1-chimaerins, suggesting common regulatory mechanisms for all chimaerin isoforms. The subcellular redistribution of beta2-chimaerin by phorbol esters is entirely dependent on the C1 domain, as revealed by deletional analysis and site-directed mutagenesis. Interestingly, beta2-chimaerin translocates to the Golgi apparatus after phorbol ester treatment, as revealed by co-staining with the Golgi marker BODIPY-TR-ceramide. Structure relationship analysis of translocation using a series of PKC ligands revealed substantial differences between translocation of beta2-chimaerin and PKCalpha. Strikingly, the mezerein analog thymeleatoxin is not able to translocate beta2-chimaerin, although it very efficiently translocates PKCalpha. Phorbol esters also promote the association of beta2-chimaerin with Rac in cells. These data suggest that chimaerins can be positionally regulated by phorbol esters and that each phorbol ester receptor class has distinct pharmacological properties and targeting mechanisms. The identification of selective ligands for each phorbol ester receptor class represents an important step in dissecting their specific cellular functions.     

Identification of an autoinhibitory mechanism that restricts C1 domain-mediated activation of the Rac-GAP alpha2-chimaerin

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the alpha2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of alpha2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.     

PYK2 autophosphorylation,but not kinase activity,is necessary for adhesion-induced association with c-Src,osteoclast spreading,and bone resorption

Proline-rich tyrosine kinase 2 (PYK2) is the main adhesion-induced kinase in bone-resorbing osteoclasts. Previous studies have shown that ligation of alpha(v)beta(3) integrin in osteoclasts induces c-Src-dependent tyrosine phosphorylation and PYK2 activation, leading to cytoskeletal rearrangement, migration, and polarization of these cells. In this study, we examined the role of PYK2 kinase activity and its major autophosphorylation site in adhesion-dependent signaling and cytoskeletal organization during osteoclast spreading and migration. By infecting pre-fusion osteoclasts using recombinant adenovirus expressing PYK2 and its mutants, we demonstrated that mutation at the autophosphorylation site (Y402F) abolishes PYK2 association with c-Src and reduces significantly phosphorylation at tyrosines 579/580 and 881 resulting in inhibition of osteoclast spreading and bone resorption. Overexpression of the kinase-dead PYK2(K475A) mutant had no effect on cell spreading, interaction with c-Src, or the phosphorylation level of Tyr-402, Tyr-579/580, and Tyr-881 relative to PYK2(wt)-expressing cells. Taken together these findings suggest that Tyr-402 is the major docking site for c-Src and can be phosphorylated by another tyrosine kinase in osteoclasts but not in HEK293 cells. Interestingly, both PYK2(Y402F) and PYK2(K457A) translocate normally to podosomes and have no effect on macrophage colony-stimulating factor-induced osteoclast migration. Whereas PYK2(Y402F) dominant negatively blocks osteoclast spreading and bone resorption, PYK2(K457A) may function in part as an adaptor by initially recruiting c-Src to the adhesion complex, which appears to activate PYK2 by phosphorylating additional tyrosines in its regulatory and C-terminal domains. We thus concluded that phosphorylation at Tyr-402 in PYK2 is essential in the regulation of adhesion-dependent cytoskeletal organization in osteoclasts.     

Diacylglycerol kinase gamma interacts with and activates beta2-chimaerin, a Rac-specific GAP, in response to epidermal growth factor

Diacylglycerol kinase (DGK)gamma was shown to act as an upstream suppressor of Rac1. Here we report that, in COS7 cells stimulated with epidermal growth factor (EGF), DGKgamma specifically interacts and co-localizes at the plasma membrane with beta2-chimaerin, a GTPase-activating protein (GAP) for Rac. Moreover, DGKgamma enhanced EGF-dependent translocation of beta2-chimaerin to the plasma membrane. Interestingly, DGKgamma markedly augmented EGF-dependent GAP activity of beta2-chimaerin through its catalytic action. These results indicate that DGKgamma is a novel regulator of beta2-chimaerin, and thus suggest that beta2-chimaerin is an effector molecule, linking DGKgamma functionally with Rac1.     

Rac-GAP-dependent inhibition of breast cancer cell proliferation by {beta}2-chimerin

beta2-Chimerin is a member of the "non-protein kinase C" intracellular receptors for the second messenger diacylglycerol and the phorbol esters that is yet poorly characterized, particularly in the context of signaling pathways involved in proliferation and cancer progression. beta2-Chimerin possesses a C-terminal Rac-GAP (GTPase-activating protein) domain that accelerates the hydrolysis of GTP from the Rac GTPase, leading to its inactivation. We found that beta2-chimerin messenger levels are significantly down-regulated in human breast cancer cell lines as well as in breast tumors. Adenoviral delivery of beta2-chimerin into MCF-7 breast cancer cells leads to inhibition of proliferation and G(1) cell cycle arrest. Mechanistic studies show that the effect involves the reduction in Rac-GTP levels, cyclin D1 expression, and retinoblastoma dephosphorylation. Studies using the mutated forms of beta2-chimerin revealed that these effects were entirely dependent on its C-terminal GAP domain and Rac-GAP activity. Moreover, MCF-7 cells stably expressing active Rac (V12Rac1) but not RhoA (V14RhoA) were insensitive to beta2-chimerin-induced inhibition of proliferation and cell cycle progression. The modulation of G(1)/S progression by beta2-chimerin not only implies an essential role for Rac in breast cancer cell proliferation but also raises the intriguing possibility that diacylglycerol-regulated non-protein kinase C pathways can negatively impact proliferation mechanisms controlled by Rho GTPases.     

Phorbol ester and hydrogen peroxide synergistically induce the interaction of diacylglycerol kinase gamma with the Src homology 2 and C1 domains of beta2-chimaerin

DGKgamma (diacylglycerol kinase gamma) was reported to interact with beta2-chimaerin, a GAP (GTPase-activating protein) for Rac, in response to epidermal growth factor. Here we found that PMA and H2O2 also induced the interaction of DGKgamma with beta2-chimaerin. It is noteworthy that simultaneous addition of PMA and H2O2 synergistically enhanced the interaction. In this case, PMA was replaceable by DAG (diacylglycerol). The beta2-chimaerin translocation from the cytoplasm to the plasma membrane caused by PMA plus H2O2 was further enhanced by the expression of DGKgamma. Moreover, DGKgamma apparently enhanced the beta2-chimaerin GAP activity upon cell stimulation with PMA. PMA was found to be mainly required for a conversion of beta2-chimaerin into an active form. On the other hand, H2O2 was suggested to induce a release of Zn2+ from the C1 domain of beta2-chimaerin. By stepwise deletion analysis, we demonstrated that the SH2 (Src homology 2) and C1 domains of beta2-chimaerin interacted with the N-terminal half of catalytic region of DGKgamma. Unexpectedly, the SH2 domain of beta2-chimaerin contributes to the interaction independently of phosphotyrosine. Taken together, these results suggest that the functional link between DGKgamma and beta2-chimaerin has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein-protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in co-operation with the C1 domain.     

Rac GTPase-activating protein (Rac GAP) α1-Chimaerin undergoes proteasomal degradation and is stabilized by diacylglycerol signaling in neurons

α1-Chimaerin is a neuron-specific member of the Rho GTPase-activating protein family that selectively inactivates the small GTPase Rac. It is known to regulate the structure of dendrites and dendritic spines. We describe here that under basal conditions α1-chimaerin becomes polyubiquitinated and undergoes rapid proteasomal degradation. This degradation is partly dependent on the N-terminal region that is unique to this isoform. Mimicking diacylglycerol (DAG) signaling with a phorbol ester stabilizes endogenous α1-chimaerin against degradation and causes accumulation of the protein. The stabilization requires phorbol ester binding via the C1 domain of the protein and is independent of PKC activity. In addition, overexpression of a constitutively active Rac1 mutant is sufficient to cause an accumulation of α1-chimaerin through a phospholipase C-dependent mechanism, showing that endogenous DAG signaling can also stabilize the protein. These results suggest that signaling via DAG may regulate the abundance of α1-chimaerin under physiological conditions, providing a new model for understanding how its activity could be controlled.     

Heregulin beta1 promotes breast cancer cell proliferation through Rac/ERK-dependent induction of cyclin D1 and p21Cip1

Accumulating evidence indicates that heregulins, EGF (epidermal growth factor)-like ligands, promote breast cancer cell proliferation and are involved in the progression of breast cancer towards an aggressive and invasive phenotype. However, there is limited information regarding the molecular mechanisms that mediate these effects. We have recently established that HRG (heregulin beta1) promotes breast cancer cell proliferation and migration via cross-talk with EGFR (EGF receptor) that involves the activation of the small GTPase Rac1. In the present paper we report that Rac1 is an essential player for mediating the induction of cyclin D1 and p21(Cip1) by HRG in breast cancer cells. Inhibition of Rac function by expressing either the Rac-GAP (GTPase-activating protein) beta2-chimaerin or the dominant-negative Rac mutant N17Rac1, or Rac1 depletion using RNAi (RNA interference), abolished the cyclin D1 and p21(Cip1) induction by HRG. Interestingly, the proliferative effect of HRG was impaired not only when the expression of Rac1 or cyclin D1 was inhibited, but also when cells were depleted of p21(Cip1) using RNAi. Inhibition of EGFR, PI3K (phosphoinositide 3-kinase; kinases required for Rac activation by HRG) or MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] also blocked the up-regulation of cyclin D1 and p21(Cip1) by HRG. In addition, we found that HRG activates NF-kappaB (nuclear factor kappaB) in a Rac1- and MEK-dependent fashion, and inhibition of NF-kappaB abrogates cyclin D1/p21(Cip1) induction and proliferation by HRG. Taken together, these findings establish a central role for Rac1 in the control of HRG-induced breast cancer cell-cycle progression and proliferation through up-regulating the expression of cyclin D1 and p21(Cip1).     

alpha2-Chimaerin is an essential EphA4 effector in the assembly of neuronal locomotor circuits

The assembly of neuronal networks during development requires tightly controlled cell-cell interactions. Multiple cell surface receptors that control axon guidance and synapse maturation have been identified. However, the signaling mechanisms downstream of these receptors have remained unclear. Receptor signals might be transmitted through dedicated signaling lines defined by specific effector proteins. Alternatively, a single cell surface receptor might couple to multiple effectors with overlapping functions. We identified the neuronal RacGAP alpha2-chimaerin as an effector for the receptor tyrosine kinase EphA4. alpha2-Chimaerin interacts with activated EphA4 and is required for ephrin-induced growth cone collapse in cortical neurons. alpha2-Chimaerin mutant mice exhibit a rabbit-like hopping gait with synchronous hindlimb movements that phenocopies mice lacking EphA4 kinase activity. Anatomical and functional analyses of corticospinal and spinal interneuron projections reveal that loss of alpha2-chimaerin results in impairment of EphA4 signaling in vivo. These findings identify alpha2-chimaerin as an indispensable effector for EphA4 in cortical and spinal motor circuits.     

Differential Phosphorylation of RhoGDI Mediates the Distinct Cycling of Cdc42 and Rac1 to Regulate Second-phase Insulin Secretion

Cdc42 cycling through GTP/GDP states is critical for its function in the second/granule mobilization phase of insulin granule exocytosis in pancreatic islet beta cells, although the identities of the Cdc42 cycling proteins involved remain incomplete. Using a tandem affinity purification-based mass spectrometry screen for Cdc42 cycling factors in beta cells, RhoGDI was identified. RNA interference-mediated depletion of RhoGDI from isolated islets selectively amplified the second phase of insulin release, consistent with the role of RhoGDI as a Cdc42 cycling factor. Replenishment of RhoGDI to RNA interference-depleted cells normalized secretion, confirming the action of RhoGDI to be that of a negative regulator of Cdc42 activation. Given that RhoGDI also regulates Rac1 activation in beta cells, and that Rac1 activation occurs in a Cdc42-dependent manner, the question as to how the beta cell utilized RhoGDI for differential Cdc42 and Rac1 cycling was explored. Co-immunoprecipitation was used to determine that RhoGDI-Cdc42 complexes dissociated upon stimulation of beta cells with glucose for 3 min, correlating with the timing of glucose-induced Cdc42 activation and the onset of RhoGDI tyrosine phosphorylation. Glucose-induced disruption of RhoGDI-Rac1 complexes occurred subsequent to this, coincident with Rac1 activation, which followed the onset of RhoGDI serine phosphorylation. RhoGDI-Cdc42 complex dissociation was blocked by mutation of RhoGDI residue Tyr-156, whereas RhoGDI-Rac1 dissociation was blocked by RhoGDI mutations Y156F and S101A/S174A. Finally, expression of a triple Y156F/S101A/S174A-RhoGDI mutant specifically inhibited only the second/granule mobilization phase of glucose-stimulated insulin secretion, overall supporting the integration of RhoGDI into the activation cycling mechanism of glucose-responsive small GTPases.     

Beta3 tyrosine phosphorylation and alphavbeta3-mediated adhesion are required for Vav1 association and Rho activation in leukocytes

Integrin alpha(v)beta(3)-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of beta(3) tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in alpha(v)beta(3)-mediated adhesion. We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with alpha(v)beta(3) upon cell adhesion to vitronectin and that this association requires beta(3) tyrosine phosphorylation. Expression of exogenous Vav1 demonstrates that Y160F, but not wild type or the Vav1Y174F mutant, inhibits Rac and Rho activation during alpha(v)beta(3)-mediated cell adhesion to vitronectin. Cells expressing Vav1Y160F exhibit a sustained Cdc42 activation similar to nonphosphorylatable beta(3) mutants. In addition, cytoskeletal reorganization and cell adhesion are severely suppressed in Vav1Y160F-transfected cells, and Vav1Y160F fails to associate with beta(3) integrins. Furthermore, Vav1 itself is selectively phosphorylated upon tyrosine 160 after alpha(v)beta(3)-mediated adhesion, and the association between Vav1 and beta(3) occurs in specific response to adhesion to substrate. These studies describe a phosphorylation-dependent association between beta(3) integrin and Vav1 which is essential for cell progression to a Rho-dominant phenotype during cell adhesion.     

Epidermal growth factor induces tyrosine phosphorylation, membrane insertion, and activation of transient receptor potential channel 4

Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca(2+) store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na(+)/H(+) exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca(2+) influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function.     

Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins.

Cyclin-dependent kinases (Cdks) are key regulators of the eukaryotic cell division cycle. Cdk1 (Cdc2) and Cdk2 should be bound to regulatory subunits named cyclins as well as phosphorylated on a conserved Thr located in the T-loop for full enzymatic activity. Cdc2- and Cdk2-cyclin complexes can be inactivated by phosphorylation on the catalytic cleft-located Thr-14 and Tyr-15 residues or by association with inhibitory subunits such as p21(Cip1). We have recently identified a novel Cdc2 regulator named RINGO that plays an important role in the meiotic cell cycle of Xenopus oocytes. RINGO can bind and activate Cdc2 but has no sequence homology to cyclins. Here we report that, in contrast with Cdc2- cyclin complexes, the phosphorylation of Thr-161 is not required for full activation of Cdc2 by RINGO. We also show that RINGO can directly stimulate the kinase activity of Cdk2 independently of Thr-160 phosphorylation. Moreover, RINGO-bound Cdc2 and Cdk2 are both less susceptible to inhibition by p21(Cip1), whereas the Thr-14/Tyr-15 kinase Myt1 can negatively regulate the activity of Cdc2-RINGO with reduced efficiency. Our results indicate that Cdk-RINGO complexes may be active under conditions in which cyclin-bound Cdks are inhibited and can therefore play different regulatory roles.     

Phosphorylation of Tyr-176 of the yeast MAPK Hog1/p38 is not vital for Hog1 biological activity

Mitogen-activated protein kinases are crucial components in the life of eukaryotic cells. The current dogma for MAPK activation is that dual phosphorylation of neighboring Thr and Tyr residues at the phosphorylation lip is an absolute requirement for their catalytic and biological activity. In this study we addressed the role of Tyr and Thr phosphorylation in the yeast MAPK Hog1/p38. Taking advantage of the recently isolated hyperactive mutants, whose intrinsic basal activity is independent of upstream regulation, we demonstrate that Tyr-176 is not required for basal catalytic and biological activity but is essential for the salt-induced amplification of Hog1 catalysis. We show that intact Thr-174 is absolutely essential for biology and catalysis of the mutants but is mainly required for structural reasons and not as a phosphoacceptor. The roles of Thr-174 and Tyr-176 in wild type Hog1 molecules were also tested. Unexpectedly we found that Hog1(Y176F) is biologically active, capable of induction of Hog1 target genes and of rescuing hog1Delta cells from osmotic stress. Hog1(Y176F) was not able, however, to mediate growth arrest induced by constitutively active MAPK kinase/Pbs2. We propose that Thr-174 is essential for stabilizing the basal active conformation, whereas Tyr-176 is not. Tyr-176 serves as a regulatory element required for stimuli-induced amplification of kinase activity.