Galardin (GM 6001), a broad-spectrum matrix metalloproteinase inhibitor, blocks bombesin- and LPA-induced EGF receptor transactivation and DNA synthesis in rat-1 cells

Matrix metalloproteinases (MMPs) have been implicated in the transactivation of the epidermal growth factor receptor (EGFR) induced by G-protein coupled receptor (GPCR) agonists. Although EGFR phosphorylation and downstream signaling have been shown to be dependent on MMP activity in many systems, a role for MMPs in GPCR-induced DNA synthesis has not been studied in any detail. In this study we utilized the broad-spectrum matrix metalloproteinase inhibitor, galardin (Ilomastat, GM 6001), to study the mechanism of bombesin- or LPA-induced EGFR transactivation and the role of MMPs in early and late response mitogenic signaling in Rat-1 cells stably transfected with the bombesin/GRP receptor (BoR-15 cells). Addition of galardin to cells stimulated with bombesin or LPA specifically inhibited total EGFR phosphorylation, as well as site-specific phosphorylation of tyrosine 845, a putative Src phosphorylation site, and tyrosine 1068, a typical autophosphorylation site. Galardin treatment also inhibited extracellular signal-regulated kinase (ERK) activation induced by bombesin or LPA, but not by EGF. In addition, galardin inhibited bombesin- or LPA-induced DNA synthesis in a dose dependent manner, when stimulated by increasing concentrations of bombesin, and when added after bombesin stimulation. Furthermore, addition of galardin post-bombesin stimulation indicated that by 3 h sufficient accumulation of EGFR ligands had occurred to continue to induce transactivation despite an inhibition of MMP activity. Taken together, our results suggest that MMPs act as early as 5 min, and up to around 3 h, to mediate GPCR-induced EGFR transactivation, ERK activation, and stimulation of DNA synthesis.     

Src-dependent phosphorylation of the epidermal growth factor receptor on tyrosine 845 is required for zinc-induced Ras activation

Previous studies have shown that exposure of cells to Zn2+ ions induces Ras and MAPK activation through the EGF receptor (EGFR). To further determine the role of EGFR in Zn2+-induced signaling, mouse B82L fibroblasts expressing no detectable EGFR protein (B82L-par), wild type EGFR (B82L-wt), kinase-deficient EGFR (B82L-K721M), or COOH-truncated EGFR (B82L-c'958) were tested. Exposure to Zn2+ induced Ras activity in B82L-wt, B82L-K721M, and B82L-c'958 but not in B82L-par cells, indicating that the tyrosine kinase domain and the auto-phosphorylation sites of the EGFR were not required for Zn2+-induced Ras activation. Zn2+ induced Src activation in all B82L cell lines, including B82L-par, indicating that Src activation is independent of the presence of the EGFR. A Src kinase inhibitor blocked Zn2+-induced Ras activation in all the B82L cell lines capable of this response, suggesting the involvement of Src kinase in Zn2+-induced Ras activation via the EGFR. Zn2+ induced the association of the EGFR with Src and specifically increased the phosphorylation of EGFR at tyrosine 845 (Tyr-845), a known Src phosphorylation site. Stably transfected B82L cells with a point mutation of the EGFR at Tyr-845 (B82L-Y845F) exhibited only basal Ras activity following exposure to Zn2+. These data demonstrate that Src-dependent phosphorylation of the EGFR at Tyr-845 is required for EGFR transactivation and Zn2+-induced Ras activation.     

EGFR plays a pivotal role in the regulation of polyamine-dependent apoptosis in intestinal epithelial cells

Intracellular polyamine synthesis is regulated by the enzyme ornithine decarboxylase (ODC), and its inhibition by -difluromethylornithine (DFMO), confers resistance to apoptosis. We have previously shown that DFMO leads to the inhibition of de novo polyamine synthesis, which in turn rapidly activates Src, STAT3 and NF-κB via integrin β3 in intestinal epithelial cells. One mechanism to explain these effects involves the activation of upstream growth factor receptors, such as the epidermal growth factor receptor (EGFR). We therefore hypothesized that EGFR phosphorylation regulates the early response to polyamine depletion. DFMO increased EGFR phosphorylation on tyrosine residues 1173 (pY1173) and 845 (pY845) within 5 min. Phosphorylation declined after 10 min and was prevented by the addition of exogenous putrescine to DFMO containing medium. Phosphorylation of EGFR was concomitant with the activation of ERK1/2. Pretreatment with either DFMO or EGF for 1 h protected cells from TNF-/CHX-induced apoptosis. Exogenous addition of polyamines prevented the protective effect of DFMO. In addition, inhibition of integrin β3 activity (with RGDS), Src activity (with PP2), or EGFR kinase activity (with AG1478), increased basal apoptosis and prevented protection conferred by either DFMO or EGF. Polyamine depletion failed to protect B82L fibroblasts lacking the EGFR (PRN) and PRN cells expressing either a kinase dead EGFR (K721A) or an EGFR (Y845F) mutant lacking the Src phosphorylation site. Conversely, expression of WT-EGFR (WT) restored the protective effect of polyamine depletion. Fibronectin activated the EGFR, Src, ERKs and protected cells from apoptosis. Taken together, our data indicate an essential role of EGFR kinase activity in MEK/ERK-mediated protection, which synergizes with integrin β3 leading to Src-mediated protective responses in polyamine depleted cells.     

EGFR Tyrosine 845 Phosphorylation-Dependent Proliferation and Transformation of Breast Cancer Cells Require Activation of p38 MAPK

Phosphorylation of epidermal growth factor receptor (EGFR) on tyrosine 845 by c-Src has been shown to be important for cell proliferation and migration in several model systems. This cross talk between EGFR and Src family kinases (SFKs) is one mechanism for resistance to EGFR inhibitors both in cell models and in the clinic. Here, we show that phosphorylation of tyrosine 845 on EGFR is required for proliferation and transformation using several cell models of breast cancer. Overexpression of EGFR-Y845F or treating cells with the SFK inhibitor dasatinib abrogated tyrosine 845 phosphorylation, yet had little to no effect on other EGFR phosphorylation sites or EGFR kinase activity. Abrogation of Y845 phosphorylation inhibited cell proliferation and transformation, even though extracellular signal-regulated kinase (ERK) and Akt remained active under these conditions. Importantly, cotransfection of mitogen-activated protein kinase (MAPK) kinase 3 and p38 MAPK restored cell proliferation in the absence of EGFR tyrosine 845 phosphorylation. Taken together, these data demonstrate a novel role for p38 MAPK signaling downstream of EGFR tyrosine 845 phosphorylation in the regulation of breast cancer cell proliferation and transformation and implicate SFK inhibitors as a potential therapeutic mechanism for overcoming EGFR tyrosine kinase inhibitor resistance in breast cancer.     

Regulation of lysophosphatidic acid-induced epidermal growth factor receptor transactivation and interleukin-8 secretion in human bronchial epithelial cells by protein kinase Cdelta, Lyn kinase, and matrix metalloproteinases

We have demonstrated earlier that lysophosphatidic acid (LPA)-induced interleukin-8 (IL-8) secretion is regulated by protein kinase Cdelta (PKCdelta)-dependent NF-kappaB activation in human bronchial epithelial cells (HBEpCs). Here we provide evidence for signaling pathways that regulate LPA-mediated transactivation of epidermal growth factor receptor (EGFR) and the role of cross-talk between G-protein-coupled receptors and receptor-tyrosine kinases in IL-8 secretion in HBEpCs. Treatment of HBEpCs with LPA stimulated tyrosine phosphorylation of EGFR, which was attenuated by matrix metalloproteinase (MMP) inhibitor (GM6001), heparin binding (HB)-EGF inhibitor (CRM 197), and HB-EGF neutralizing antibody. Overexpression of dominant negative PKCdelta or pretreatment with a PKCdelta inhibitor (rottlerin) or Src kinase family inhibitor (PP2) partially blocked LPA-induced MMP activation, proHB-EGF shedding, and EGFR tyrosine phosphorylation. Down-regulation of Lyn kinase, but not Src kinase, by specific small interfering RNA mitigated LPA-induced MMP activation, proHB-EGF shedding, and EGFR phosphorylation. In addition, overexpression of dominant negative PKCdelta blocked LPA-induced phosphorylation and translocation of Lyn kinase to the plasma membrane. Furthermore, down-regulation of EGFR by EGFR small interfering RNA or pretreatment of cells with EGFR inhibitors AG1478 and PD158780 almost completely blocked LPA-dependent EGFR phosphorylation and partially attenuated IL-8 secretion, respectively. These results demonstrate that LPA-induced IL-8 secretion is partly dependent on EGFR transactivation regulated by PKCdelta-dependent activation of Lyn kinase and MMPs and proHB-EGF shedding, suggesting a novel mechanism of cross-talk and interaction between G-protein-coupled receptors and receptor-tyrosine kinases in HBEpCs.     

Enhancement of lysophosphatidic acid-induced ERK phosphorylation by phospholipase D1 via the formation of phosphatidic acid

We made stable cell lines overexpressing PLD1 (GP-PLD1) from GP+envAm12 cell, a derivative of NIH 3T3 cell. PLD1 activity and extracellular signal-regulated kinase (ERK) phosphorylation were enhanced in GP-PLD1 cells by the treatment of lysophosphatidic acid (LPA). In contrast, these LPA-induced effects were attenuated with the pretreatment of pertussis toxin (PTX) or protein kinase C (PKC) inhibitor. Moreover, accumulation of phosphatidic acid (PA), a product of PLD action, potentiated the LPA-induced ERK activation in GP-PLD1 cells while blocking of PA production with the treatment of 1-butanol attenuated LPA-induced ERK phosphorylation. From these results, we suggest that LPA activate PLD1 through pertussis toxin-sensitive G protein and PKC-dependent pathways, then PA produced from PLD1 activation facilitate ERK phosphorylation.     

Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma.

Two novel sites of autophosphorylation were localized to the C-terminal tail of the PDGF beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) compared to the wild type PDGF beta-receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC-gamma could be detected. These data show that tyrosine phosphorylation of PLC-gamma is dependent on autophosphorylation of the PDGF beta-receptor at Tyr1009 and Tyr1021.     

c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function

Accumulating evidence indicates that interactions between the epidermal growth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may contribute to an aggressive phenotype in multiple human tumors. Previous work from our laboratory demonstrated that murine fibroblasts which overexpress both these tyrosine kinases display synergistic increases in DNA synthesis, soft agar growth, and tumor formation in nude mice, and increased phosphorylation of the receptor substrates Shc and phospholipase gamma as compared with single overexpressors. These parameters correlated with the ability of c-Src and EGFR to form an EGF-dependent heterocomplex in vivo. Here we provide evidence that association between c-Src and EGFR can occur directly, as shown by receptor overlay experiments, and that it results in the appearance of two novel tyrosine phosphorylations on the receptor that are seen both in vitro and in vivo following EGF stimulation. Edman degradation analyses and co-migration of synthetic peptides with EGFR-derived tryptic phosphopeptides identify these sites as Tyr845 and Tyr1101. Tyr1101 lies within the carboxyl-terminal region of the EGFR among sites of receptor autophosphorylation, while Tyr845 resides in the catalytic domain, in a position analogous to Tyr416 of c-Src. Phosphorylation of Tyr416 and homologous residues in other tyrosine kinase receptors has been shown to be required for or to increase catalytic activity, suggesting that c-Src can influence EGFR activity by mediating phosphorylation of Tyr845. Indeed, EGF-induced phosphorylation of Tyr845 was increased in MDA468 human breast cancer cells engineered to overexpress c-Src as compared with parental MDA 468 cells. Furthermore, transient expression of a Y845F variant EGFR in murine fibroblasts resulted in an ablation of EGF-induced DNA synthesis to nonstimulated levels. Together, these data support the hypothesis that c-Src-mediated phosphorylation of EGFR Tyr845 is involved in regulation of receptor function, as well as in tumor progression.     

Budding yeast Cdc5 phosphorylates Net1 and assists Cdc14 release from the nucleolus

Lysophosphatidic acid (LPA) has been shown to be a potent mitogen for vascular smooth muscle cells. Src-dependent transactivation of receptor tyrosine kinases has been previously demonstrated to mediate LPA-induced activation of MAP kinase ERK1/2. Furthermore, generation of reactive oxygen species (ROS) by LPA is also known to contribute to MAP kinase activation. Rho family small G-proteins Rac and Cdc42, and their immediate downstream effector p21-activated kinase (PAK), have been demonstrated to mediate important effects on the cytoskeleton that are relevant for cell migration and proliferation. In the present report we evaluated stimulation of PAK by LPA in rat aortic vascular smooth muscle cells (VSMC) by PAK immunocomplex MBP in-gel kinase assay. LPA increased PAK activity 3-fold, peaking at 5 min and showing sustained activation up to 45 min. Inhibition of tyrosine kinases by pretreatment of VSMC with genistein or specific inhibition of Src by PP1 greatly diminished LPA-induced PAK activation, whereas specific inhibition of PDFG- and EGF receptor kinase by tyrphostin AG1296 and AG1478 had no effect. Furthermore, inhibition of Galpha(i) by pertussis toxin and inhibition of NADH/NADPH oxidase by diphenylene iodonium also diminished LPA-induced stimulation of PAK. This is the first study to demonstrate that LPA activates PAK. In VSMC, PAK activation by LPA is mediated by Galpha(i) and is dependent on Src, whereas EGF- or PDGF receptor transactivation are not involved. Furthermore, generation of ROS is required for LPA-induced activation of PAK.     

Tyrosyl phosphorylation of Shp2 is required for normal ERK activation in response to some,but not all,growth factors

The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542F- or Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the "Grb2-adapter" model of Shp2 action.     

Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts.

The receptor for the myeloid cell growth factor colony stimulating factor 1 (CSF-1) is a protein tyrosine kinase that is closely related to the PDGF receptor. Ligand binding results in kinase activation and autophosphorylation. Three autophosphorylation sites, Tyr697, Tyr706 and Tyr721, have been mapped to the kinase insert domain. Deletion of the entire kinase insert domain completely abrogates signal transduction by the CSF-1 receptor expressed in Rat-2 fibroblasts. To investigate the function of individual phosphorylation sites present in the CSF-1 receptor kinase insert domain, a number of phosphorylation site mutants were expressed in Rat-2 fibroblasts. Mutation of either Tyr697 or Tyr721 compromised signal transduction by the CSF-1 receptor. A mutant receptor, in which both Tyr697 and Tyr721 were replaced by phenylalanine, has lost all ability to induce changes in morphology or to increase cell growth rate in response to CSF-1. Tyr721 has been identified recently as the binding site for PI 3-kinase. Here we report that GRB2 associates with the CSF-1 receptor upon ligand binding. The phosphorylation on tyrosine of SHC and several other GRB2-associated proteins increased upon stimulation with CSF-1. Tyr697 was identified as a binding site for GRB2. We suggest that PI 3-kinase, GRB2 and some of the GRB2-associated proteins could play an important role in signal transduction by the CSF-1 receptor.     

Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation

Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.     

Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Delta1022-1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRDelta1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Delta1022-1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro and in vivo.     

Mechanisms of extracellularly regulated kinases 1/2 activation in adrenal glomerulosa cells by lysophosphatidic acid and epidermal growth factor

The regulation of adrenal function, including aldosterone production from adrenal glomerulosa cells, is dependent on a variety of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In many cell types, GPCR-mediated MAPK activation is mediated through transactivation of RTKs, in particular the epidermal growth factor (EGF) receptor (EGF-R). However, the extent to which this cross-communication between GPCRs and RTKs is operative in the adrenal glomerulosa has not been defined. Bovine adrenal glomerulosa cells express receptors for lysophosphatidic acid (LPA) and EGF. In cultured bovine adrenal glomerulosa cells, LPA, which is predominantly coupled to Gi and partially to Gq/protein kinase C alpha and epsilon, caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), EGF-R, protein kinase B/Akt, extracellularly regulated signal kinases 1/2, and their dependent protein, p90 ribosomal S6 kinase. Overexpression of dominant negative mutants of Ras or EGF-R, and selective inhibition of EGF-R kinase with AG1478, significantly reduced LPA-induced ERK1/2 phosphorylation. However, this was not impaired by inhibition of matrix metalloproteinase (MMP) and heparin-binding EGF. LPA-induced ERK1/2 activation occurs predominantly through EGF-R transactivation by Gi/Src and partly through activation of protein kinase C, which acts downstream of EGF-R and Ras. In contrast, LPA-induced phosphorylation of Shc and ERK1/2 in clonal hepatocytes (C9 cells) was primarily mediated through MMP-dependent transactivation of the EGF-R. These observations in adrenal glomerulosa and hepatic cells demonstrate that LPA phosphorylates ERK1/2 through EGF-R transactivation in a MMP-dependent or -independent manner in individual target cells. This reflects the ability of GPCRs expressed in cell lines and neoplastic cells to utilize distinct signaling pathways that can elicit altered responses compared with those of native tissues.     

Requirement of SHP2 binding to Grb2-associated binder-1 for mitogen-activated protein kinase activation in response to lysophosphatidic acid and epidermal growth factor

Grb2-associated binder-1 (Gab1) is a multisite docking protein containing a pleckstrin homology (PH) domain, multiple potential tyrosine phosphorylation sites, and several proline-rich sequences. Gab1 becomes tyrosine-phosphorylated in cells stimulated with growth factors, cytokines, and ligands for G protein-coupled receptors. A major Gab1-binding protein detected in cells treated with extracellular stimuli is the tyrosine phosphatase, SHP2. Although the role of SHP2-Gab1 interaction in cell signaling has not yet been characterized, SHP2 is known to mediate mitogen-activated protein (MAP) kinase activation induced by the epidermal growth factor (EGF). However, the mechanism by which the SHP2 phosphatase exerts a positive signaling role remains obscure. In this study, we prepared Gab1 mutants lacking the SHP2 binding site (Gab1Y627F), the phosphatidylinositol 3-kinase (PI3K) binding sites (Gab1DeltaPI3K), and the PH domain (Gab1DeltaPH). Expression of Gab1Y627F blocked the extracellular signal-regulated kinase-2 (ERK2) activation by lysophosphatidic acid (LPA) and EGF. Conversely, expression of the wild-type Gab1 in HEK293 cells augmented the LPA receptor Edg2-mediated ERK2 activation. Whereas the PH domain was required for Gab1 mediation of ERK2 activation by LPA, it was not essential for EGF-induced ERK2 activation. Expression of Gab1DeltaPI3K had no apparent effect on ERK2 activation by LPA and EGF in the cells that we have examined. These results establish a role for Gab1 in the LPA-induced MAP kinase pathway and clearly demonstrate that Gab1-SHP2 interaction is essential for ERK2 activation by LPA and EGF. These findings also suggest that the positive role of SHP2 in the MAP kinase pathway depends on its interaction with Gab1.     

RAFTK/Pyk2 mediates LPA-induced PC12 cell migration

The phospholipid lysophosphatidic acid (LPA) is a normal constituent of serum that functions as a lipid growth factor and intracellular signaling molecule. In this report, we have investigated the signaling mechanism and function of the tyrosine kinase RAFTK/Pyk2 in LPA-induced cell migration. Analysis of tyrosine phosphorylation upon LPA stimulation in neuroendocrine PC12 cells revealed 6 major tyrosine-phosphorylated proteins with estimated sizes of 180, 120, 115, 68, 44, and 42 kDa. These proteins were identified as epidermal growth factor receptor (EGFR), focal adhesion kinase, RAFTK/Pyk2, paxillin, Erk 1, and Erk 2, respectively. Using specific pharmacological inhibitors, we found that the tyrosine phosphorylation of RAFTK/Pyk2 was intracellular Ca2+-dependent, but not EGFR-dependent, during LPA stimulation of these cells. Moreover, the cytoskeletal and signal scaffolding protein, paxillin, associated with and was regulated by RAFTK/Pyk2 in a Ca2+-dependent manner. Characterization of LPA receptors showed that LPA1 (Edg2) and LPA2 (Edg4) are major receptors for LPA, while LPA3 receptor (Edg7) expression was limited. Upon using the LPA1/LPA3 receptor-specific antagonist VPC 32179, we observed that inhibition of the LPA1/LPA3 receptors had no effect on the LPA-induced phosphorylation of RAFTK, strongly suggesting that the LPA2 receptor is a key mediator of RAFTK phosphorylation. Furthermore, LPA induced PC12 cell migration, which was subsequently blocked by the dominant-negative form of FAK, FRNK. Expression of a dominant-negative form of the small GTPase Ras also blocked LPA-induced cell migration and RAFTK phosphorylation. Taken together, these results indicate that RAFTK is a key signaling molecule that mediates LPA-induced PC12 cell migration in a Ras-dependent manner.     

Convergence of multiple signaling cascades at glycogen synthase kinase 3: Edg receptor-mediated phosphorylation and inactivation by lysophosphatidic acid through a protein kinase C-dependent intracellular pathway

Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor beta subunit (PDGFRbeta) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRbeta mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cgamma (PLCgamma) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRbeta. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCgamma-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.