G protein-coupled receptor-mediated ERK1/2 phosphorylation: towards a generic sensor of GPCR activation

The development of new analytical methods, aimed at profiling G protein-coupled receptor (GPCR) ligands, regardless of the G protein-coupling pattern of their respective receptor, remains a key goal in drug discovery. Considerable evidence has recently revived the central role that could be played by extracellular-signal-regulated kinase (ERK), the cornerstone protein kinase of the first tyrosine kinase receptor-mediated pathway identified, in response to the activation of various types of GPCRs. Here we reveal a conceptual study in which the potential of ERK phosphorylation is evaluated as a generic readout in response to three different receptors activating three main classes of G proteins: Galphas, Galphai and Galphaq. GPCR-mediated ERK phosphorylation was compared with different readouts such as GTPgammaS, CAMP, or Ca2 +. We propose the measurement of GPCR-activated ERK phosphorylation as an alternative assay to better understand the molecular pharmacology of ligands of promiscuous GPCRs.     

Agrin-induced activation of acetylcholine receptor-bound Src family kinases requires Rapsyn and correlates with acetylcholine receptor clustering

During neuromuscular synaptogenesis, neurally released agrin induces aggregation and tyrosine phosphorylation of acetylcholine receptors (AChRs) by acting through both the receptor tyrosine kinase MuSK (muscle-specific kinase) and the AChR-associated protein, rapsyn. To elucidate this signaling mechanism, we examined tyrosine phosphorylation of AChR-associated proteins, particularly addressing whether agrin activates Src family kinases bound to the AChR. In C2 myotubes, agrin induced tyrosine phosphorylation of these kinases, of AChR-bound MuSK, and of the AChR beta and delta subunits, as observed in phosphotyrosine immunoblotting experiments. Kinase assays revealed that the activity of AChR-associated Src kinases was increased by agrin, whereas phosphorylation of the total cellular kinase pool was unaffected. In both rapsyn-deficient myotubes and staurosporine-treated C2 myotubes, where AChRs are not clustered, agrin activated MuSK but did not cause either Src family or AChR phosphorylation. In S27 mutant myotubes, which fail to aggregate AChRs, no agrin-induced phosphorylation of AChR-bound Src kinases, MuSK, or AChRs was observed. These results demonstrate first that agrin leads to phosphorylation and activation of AChR-associated Src-related kinases, which requires rapsyn, occurs downstream of MuSK, and causes AChR phosphorylation. Second, this activation intimately correlates with AChR clustering, suggesting that these kinases may play a role in agrin-induced AChR aggregation by forming an AChR-bound signaling cascade.     

Rac and Cdc42-dependent regulation of c-Jun N-terminal kinases by the delta-opioid receptor

Heptahelical opioid receptors utilize Gi proteins to regulate a multitude of effectors including the classical adenylyl cyclases and the more recently discovered mitogen-activated protein kinases (MAPKs). The c-Jun NH2-terminal kinases (JNKs) belong to one of three subgroups of MAPKs. In NG108-15 neuroblastoma x glioma hybrid cells that endogenously express delta-opioid receptors, delta-agonist dose-dependently stimulated JNK activity in a pertussis toxin-sensitive manner. By using COS-7 cells transiently transfected with the cDNAs of delta-opioid receptor and hemagglutinin (HA)-tagged JNK, we delineated the signaling components involved in this pathway. Sequestration of Gbetagamma subunits by transducin suppressed the opioid-induced JNK activity. The possible involvement of the small GTPases was also examined. Expression of dominant negative mutants of Rac and Cdc42 blocked the opioid-induced JNK activation, and a partial inhibition was observed in the presence of the dominant negative mutant of Ras. In contrast, the dominant negative mutant of Rho did not affect the opioid-induced JNK activation. In addition, the receptor-mediated JNK activation was dependent on Src family tyrosine kinases, but independent of phosphatidylinositol-3 kinase and EGF receptor tyrosine kinases. Collectively, these results demonstrate functional regulation of JNK by the delta-opioid receptor, and this pathway requires Gbetagamma, Src kinases and the small GTPases Rac and Cdc42.     

A Role for the Protein Tyrosine Phosphatase CD45 in Macrophage Adhesion through the Regulation of Paxillin Degradation

CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility. These defects are accompanied by substantially decreased levels of the cytoskeletal-associated protein paxillin, without affecting the levels of other proteins. Degradation of paxillin in CD45-deficient macrophages is calpain-mediated, as treatment with a calpain inhibitor restores paxillin levels in these cells and enhances cell spreading. Inhibition of the tyrosine kinases proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK), kinases that are capable of mediating tyrosine phosphorylation of paxillin, also restored paxillin levels, indicating a role for these kinases in the CD45-dependent regulation of paxillin. These data demonstrate that CD45 functions to regulate Pyk2/FAK activity, likely through the activity of Src family kinases, which in turn regulates the levels of paxillin to modulate macrophage adhesion and migration.     

Signal-transduction pathways that regulate visceral smooth muscle function. III. Coupling of muscarinic receptors to signaling kinases and effector proteins in gastrointestinal smooth muscles

Stimulation of muscarinic M3 and M2 receptors on gastrointestinal smooth muscle elicits contraction via activation of G proteins that are coupled to a diverse set of downstream signaling pathways and effector proteins. Many studies suggest a canonical excitation-contraction coupling pathway that includes activation of phospholipases, production of inositol 1,4,5-trisphosphate and diacylglycerol, release of calcium from the sarcoplasmic reticulum, activation of L-type calcium channels, and activation of nonselective cation channels. These events lead to elevated intracellular calcium concentration, which activates myosin light chain kinase to phosphorylate and activate myosin II thus causing contraction. In addition, muscarinic receptors are coupled to signaling pathways that modulate the effect of activator calcium. The Rho/Rho kinase pathway inhibits myosin light chain phosphatase, one of the key steps in sensitization of the contractile proteins to calcium. Phosphatidylinositol 3-kinases and Src family tyrosine kinases are also activated by muscarinic agonists. Src family tyrosine kinases regulate L-type calcium and nonselective cation channels. Src activation also leads to activation of ERK and p38 MAPKs. ERK MAPKs phosphorylate caldesmon, an actin filament binding protein. P38 MAPKs activate phospholipases and MAPKAP kinase 2/3, which phosphorylate HSP27. HSP27 may regulate cross-bridge function, actin filament formation, and actin filament attachment to the cell membrane. In addition to the well-known role of M3 muscarinic receptors to regulate myoplasmic calcium levels, the integrated effect of muscarinic activation probably also includes signaling pathways that modulate phospholipases, cyclic nucleotides, contractile protein function, and cytoskeletal protein function.     

Dual role of Fyn in the regulation of FAK+6,7 by cannabinoids in hippocampus

In hippocampus endocannabinoids modulate synaptic function and plasticity and increase tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK). Autophosphorylation of FAK on Tyr-397 is generally a critical step for its activation, allowing the recruitment of Src family kinases, and phosphorylation of FAK and associated proteins. We have examined the mechanisms of the regulation of FAK by cannabinoids in rat and mouse hippocampal slices. Anandamide and 2-arachidonoylglycerol, two endocannabinoids, and Delta9-tetrahydrocannabinol, stimulated tyrosine phosphorylation of FAK+6,7, a neuronal splice isoform of FAK, on several residues including Tyr-397. Cannabinoids increased phosphorylation of p130-Cas, a protein associated with FAK, but had no effect on PYK2, a tyrosine kinase related to FAK and enriched in hippocampus. Pharmacological experiments and the use of knockout mice demonstrated that the effects of cannabinoids were mediated through CB1 receptors. These effects were sensitive to manipulation of cAMP-dependent protein kinase, suggesting that they were mediated by inhibition of a cAMP pathway. PP2, an Src family kinase inhibitor, prevented the effects of cannabinoids on p130-Cas and on FAK+6,7 tyrosines 577 and 925, but not 397, indicating that FAK autophosphorylation was upstream of Src family kinases in response to CB1-R stimulation. Endocannabinoids increased the association of Fyn, but not Src, with FAK+6,7. In hippocampal slices from Fyn -/- mice, the levels of p130-Cas were increased, and the effects of endocannabinoids on tyrosine phosphorylation, including of Tyr-397, were completely abolished. These results demonstrate the specific functional association of Fyn with FAK+6,7 in a pathway regulated by endocannabinoids, in which Fyn may play roles dependent and independent of its catalytic activity.     

Ephrin-A1 induces c-Cbl phosphorylation and EphA receptor down-regulation in T cells

Eph receptor tyrosine kinases are expressed by T lineage cells, and stimulation with their ligands, the ephrins, has recently been shown to modulate T cell behavior. We show that ephrin-A1 stimulation of Jurkat T cells induces tyrosine phosphorylation of EphA3 receptors and cytoplasmic proteins, including the c-cbl proto-oncogene. Cbl phosphorylation was also observed in peripheral blood T cells. In contrast, stimulation of Jurkat cells with the EphB receptor ligand ephrin-B1 does not cause Cbl phosphorylation. EphA activation also induced Cbl association with Crk-L and Crk-II adapters, but not the related Grb2 protein. Induction of Cbl phosphorylation upon EphA activation appeared to be dependent upon Src family kinase activity, as Cbl phosphorylation was selectively abrogated by the Src family inhibitor 4-amino-5(4-chlorophenyl-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine, while EphA phosphorylation was unimpaired. Ephrin-A1 stimulation of Jurkat cells was also found to cause down-regulation of endogenous EphA3 receptors from the cell surface and their degradation. In accordance with the role of Cbl as a negative regulator of receptor tyrosine kinases, overexpression of wild-type Cbl, but not its 70-Z mutant, was found to down-regulate EphA receptor expression. Receptor down-regulation could also be inhibited by blockage of Src family kinase activity. Our findings show that EphA receptors can actively signal in T cells, and that Cbl performs multiple roles in this signaling pathway, functioning to transduce signals from the receptors as well as regulating activated EphA receptor expression.     

Cholecystokinin-stimulated protein kinase C-delta kinase activation,tyrosine phosphorylation,and translocation are mediated by Src tyrosine kinases in pancreatic acinar cells

Protein kinase C-delta (PKC-delta) is involved in growth, differentiation, tumor suppression, and regulation of other cellular processes. PKC-delta activation causes translocation, tyrosine phosphorylation, and serine-threonine kinase activity. However, little is known about the ability of G protein-coupled receptors to activate these processes or the mediators involved. In the present study, we explored the ability of the neurotransmitter/hormone, CCK, to stimulate these changes in PKC-delta and explored the mechanisms. In rat pancreatic acini under basal conditions, PKC-delta is almost exclusively located in cytosol. CCK and TPA stimulated a rapid PKC-delta translocation to membrane and nuclear fractions, which was transient with CCK. CCK stimulated rapid tyrosine phosphorylation of PKC-delta and increased kinase activity. Using tyrosine kinase (B44) and a tyrosine phosphatase inhibitor (orthovanadate), changes in both CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation were shown to correlate with changes in its kinase activity but not translocation. Both PKC-delta tyrosine phosphorylation and activation occur exclusively in particulate fractions. The Src kinase inhibitors, SU6656 and PP2, but not the inactive related compound, PP3, inhibited CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation and activation. In contrast, PP2 also had a lesser effect on CCK- but not TPA-stimulated PKC-delta translocation. CCK stimulated the association of Src kinases with PKC-delta, demonstrated by co-immunoprecipitation. These results demonstrate that CCKA receptor activation results in rapid translocation, tyrosine phosphorylation, and activation of PKC-delta. Stimulation of PKC-delta translocation precedes tyrosine phosphorylation, which is essential for activation to occur. Activation of Src kinases is essential for the tyrosine phosphorylation and kinase activation to occur and plays a partial role in translocation.     

An intracellular multi-effector complex mediates somatostatin receptor 1 activation of phospho-tyrosine phosphatase eta

The receptor-like phosphotyrosine phosphatase eta (PTPeta) is an important intracellular effector of the cytostatic action of SST. Here we characterize, in Chinese hamster ovary-k1 cells, the intracellular pathway that from somatostatin receptor 1 (SSTR1), leads to the activation of PTPeta and that involves, in a multimeric complex and sequential activation, the tyrosine kinases Janus kinase (JAK) 2 and Src, and the cytosolic phosphotyrosine phosphatase SHP-2. We show that inhibitors of JAK2 and Src and dominant-negative mutants of SHP-2 and Src abolished the SSTR1-mediated PTPeta activation, suggesting that all these effectors participate in the activation of PTPeta. In basal conditions, JAK2 forms a multimeric complex with SHP-2, Src and PTPeta. In response to SST, JAK2 is activated in a G protein-dependent manner, dissociates from and phosphorylates SHP-2, increasing its activity. Subsequently, SHP-2 dissociates from Src, dephosphorylates the Src inhibitory tyrosine-529, and causes an autocatalytical increase of the phosphorylation of Src tyrosine 418, located inside its kinase activation loop. Active Src, in turn, controls the activity of PTPeta, via a direct interaction and phosphorylation of the phosphatase. These data for the first time depict an intracellular pathway involving a precise sequence of interactions and cross-activation among tyrosine phosphatases and kinases acting upstream of PTPeta. In particular the sequential activation of JAK2, SHP-2, and Src conveys the molecular signaling from SSTR1 to the activation of this phosphatase that is responsible for the final biological effects of SST.     

Distinct roles for Src tyrosine kinase in beta2-adrenergic receptor signaling to MAPK and in receptor internalization

G protein-coupled receptors form the largest family of membrane receptors and transmit diverse ligand signals to modulate various cellular responses. After activation by their ligands, some of these G protein-coupled receptors are desensitized, internalized (endocytosed), and down-regulated (degraded). In HEK 293 cells, the G(s)-coupled beta2-adrenergic receptor was postulated to initiate a second wave of signaling, such as the activation of the mitogen-activated protein kinase (MAPK) pathway after the receptor is internalized. The tyrosine kinase c-Src plays a critical role in these events. Here we used mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases to examine the role of Src in beta2-adrenergic receptor signaling to the MAPK pathway and in receptor internalization. We found that in Src-deficient cells the beta2-adrenergic receptor could activate the MAPK pathway. However, the internalization of beta2-adrenergic receptors was blocked in Src-deficient MEF cells. Furthermore, we observed that in MEF cells deficient in beta-arrestin 2 the internalization of the beta2-adrenergic receptor was impaired, whereas the activation of the MAPK pathway by the beta2-adrenergic receptor was normal. Our data demonstrate that although Src and beta-arrestin 2 play essential roles in beta2-adrenergic receptor internalization, they are not required for the activation of the MAPK pathway by the beta2-adrenergic receptor. In other words, our finding suggests that receptor internalization is not required for beta2-adrenergic receptor signaling to the MAPK pathway in MEF cells.     

Brain-specific Gαz interacts with Src tyrosine kinase to regulate Mu-opioid receptor-NMDAR signaling pathway

There is a certain cross-talk in the nervous system between N-methyl-D-aspartate receptors (NMDARs) and Mu-opioid receptors (MORs). While NMDARs participate in the desensitization of MORs, these in turn modulate NMDAR-mediated glutamate responses. The G protein coupled receptors (GPCRs) activate NMDARs via Src although the role of Gα subunits in this process is not well defined. We have found that in the absence of MOR activation, the brain specific Gαz subunit binds to and stabilizes Src in its inactive form. The administration of morphine provokes the phosphorylation of specific cytosolic tyrosine residues in NMDAR2A subunits. This was achieved by PKCγ disrupting this Gαz–Src complex, enabling Src to be activated (pTyr416) by binding to GαiGTP proteins. These changes increased the activation of the calcium/calmodulin-dependent protein kinase II (CaMKII), thereby promoting MOR desensitization. This regulatory pathway is disrupted by inhibiting PKC, preventing MOR-activated Gαi2 subunits from gaining control over Src. Thus, in neural cells the Gαz subunits exert a negative control on Src function reducing the activating influence of MORs on this tyrosine kinase. This MOR-triggered signaling pathway recruits PKCγ and Gαi subunits to activate Src tyrosine kinase, resulting in the potentiation of NMDAR function. Most relevant, this mechanism which operates in neural cells is essential for the development of tolerance to the analgesic effects of morphine.     

Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signaling

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.     

Regulation of microtubule formation in activated mast cells by complexes of gamma-tubulin with Fyn and Syk kinases

Aggregation of the high-affinity IgE receptors (FcepsilonRIs) on the surface of granulated mast cells initiates a chain of signaling events culminating in the release of allergy mediators. Although microtubules are involved in mast cell degranulation, the molecular mechanism that controls microtubule rearrangement after FcepsilonRI triggering is poorly understood. In this study, we show that the activation of bone marrow-derived mast cells (BMMCs) induced by FcepsilonRI aggregation or treatment with pervanadate leads to a rapid polymerization of microtubules. This polymerization was not dependent on the presence of Lyn kinase as determined by experiments with BMMCs isolated from Lyn-negative mice. One of the key regulators of microtubule polymerization is gamma-tubulin. Immunoprecipitation experiments revealed that gamma-tubulin from activated cells formed complexes with Fyn and Syk protein tyrosine kinases and several tyrosine phosphorylated proteins from both wild-type and Lyn(-/-) BMMCs. Pretreatment of the cells with Src-family or Syk-family selective tyrosine kinase inhibitors, PP2 or piceatannol, respectively, inhibited the formation of microtubules and reduced the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, suggesting that Src and Syk family kinases are involved in the initial stages of microtubule formation. This notion was corroborated by pull-down experiments in which gamma-tubulin complex bounds to the recombinant Src homology 2 and Src homology 3 domains of Fyn kinase. We propose that Fyn and Syk kinases are involved in the regulation of binding properties of gamma-tubulin and/or its associated proteins, and thus modulate the microtubule nucleation in activated mast cells.     

Attenuated 5-HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase,phosphatidylinositol 3-kinase,Akt and mitogen-activated protein kinase

Positron emission tomography studies in major depression show reduced serotonin (5-HT)1A receptor antagonist-binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5-HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Measurements of [35S]GTPgammaS binding to Galphai/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5-HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Galphai/o, Galphaq/11 or Galphas proteins, or in the activity of cAMP-dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5-HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3-kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate. Finally, the activation of extracellular signal-regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist-stimulated 5-HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5-HT1A receptor transduction pathways that may have implications for cell survival.     

HD-PTP inhibits endothelial migration through its interaction with Src

Endothelial migration, early step in angiogenesis, is tightly regulated by the coordinated action of tyrosine kinases and tyrosine phosphatases. HD-PTP contributes to endothelial motility, since endothelial cells silencing HD-PTP after transfection with iRNA acquire a scattered and spindle-shaped phenotype and migrate faster than controls. Since (i) the proto-oncogene Src contributes to the regulation of cell motility and (ii) HD-PTP has a potential binding site for Src, we investigated whether an interplay exists between these two proteins. We found that Src binds HD-PTP and this interaction is enhanced after exposure to basic fibroblast growth factor. While HD-PTP does not modulate the levels of Src phosphorylation both in vitro and in vivo, we found that Src phosphorylates HD-PTP on tyrosine residues. Here we show for the first time that (i) HD-PTP has a tyrosine phosphatase activity; (ii) HD-PTP phosphorylation by Src inhibits its enzymatic activity. Interestingly, pharmacological and genetic inhibition of Src abrogates the migratory phenotype of endothelial cells silencing HD-PTP. On these bases, and because we have previously demonstrated that HD-PTP binds and dephosphorylates focal adhesion kinase (FAK), another crucial regulator of cell migration, we hypothesize that HD-PTP participates to the regulation of endothelial motility through its interactions with Src and FAK.     

Src kinases Fyn and Lck facilitate the accumulation of phosphorylated CTLA-4 and its association with PI-3 kinase in intracellular compartments of T-cells.

Src kinases bind to surface receptors and mediate signaling events at the surface of cells. Little is known regarding whether these kinases can mediate events within intracellular compartments. The T-cell antigen CTLA-4 resides primarily in the trans-Golgi network (TGN), and as such could serve as a model to study the intracellular function of src kinases in their ability to phosphorylate the receptor. In this study, we show that tyrosine kinases p56lck and p59fyn phosphorylate the cytoplasmic domain of CTLA-4 in T-cells. Most interestingly, these kinases are also found in the Golgi apparatus, the intracellular compartment where most of CTLA-4 is localized. Transfection of Lck or Fyn resulted in increased phosphorylation of intracellular CTLA-4 and recruitment of PI-3 kinase. By contrast, phosphorylation did not influence the presence of the receptor in the TGN. These data demonstrate that src kinases operate to modulate receptor binding to intracellular signaling proteins introducing the possibility that intracellular forms of receptors may generate growth signals.     

Coordinate expression of the alpha and beta subunits of heterotrimeric G proteins involves regulation of protein degradation in CHO cells

Individual cell types express a characteristic balance between heterotrimeric G protein alpha and betagamma subunits, but little is known about the regulatory mechanism. We systemically examined the regulatory mechanism in CHO cells. We found that expression of Galphas, Galphai2, and Galphaq proteins increased in direct proportion to the increase of Gbeta1gamma2 overexpressed transiently. Expression of Gbeta protein also increased following overexpression of Galphas, Galphai2, and Galphaq. The Gbetagamma overexpression stimulated degradation of Gbeta in contrast to reduction of Galphas degradation. We conclude that coordinate expression of the G protein subunits involves regulation of protein degradation via proteasome in CHO cells.