ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKCzeta-controlled ERK1/2 activity within the nucleus

High-affinity binding of angiotensin II (ANG II) to the ANG II type 1 receptor (AT₁R) results in the activation of ERK1/2 mitogen-activated protein kinases (MAPK). However, the precise mechanism of ANG II-induced ERK1/2 activation has not been fully characterized. Here, we investigated the signaling events leading to ANG II-induced ERK1/2 activation using a c-Src/Yes/Fyn tyrosine kinase-deficient mouse embryonic fibroblast (MEF) cell line stably transfected with the AT₁R (SYF/AT₁). ERK1/2 activation was reduced by 50% within these cells compared with wild-type controls (WT/AT₁). The remaining 50% of intracellular ERK1/2 activation was dependent upon heterotrimeric G protein and protein kinase C zeta (PKC) activation. Therefore, ANG II-induced ERK1/2 activation occurs via two independent mechanisms. We next investigated whether a loss of either c-Src/Yes/Fyn or PKC signaling affected ERK1/2 nuclear translocation and cell proliferation in response to ANG II. ANG II-induced cell proliferation was markedly reduced in SYF/AT₁ cells compared with WT/AT₁ cells (P < 0.01), but interestingly, ERK2 nuclear translocation was normal. ANG II-induced nuclear translocation of ERK2 was blocked via pretreatment of WT/AT₁ cells with a PKC pseudosubstrate. ANG II-induced cell proliferation was significantly reduced in PKC pseudosubstrate-treated WT/AT₁ cells (P < 0.01) and was completely blocked in SYF/AT₁ cells treated with this same compound. Thus ANG II-induced cell proliferation appears to be regulated by both ERK1/2-driven nuclear and cytoplasmic events. In response to ANG II, the ability of ERK1/2 to remain within the cytoplasm or translocate into the nucleus is controlled by c-Src/Yes/Fyn or heterotrimeric G protein/PKC signaling, respectively. Src family tyrosine kinases; angiotensin II     

Signalling Pathways for Cardiac Hypertrophy

Mechanical stretch is an initial factor for cardiac hypertrophy in response to haemodynamic overload (high blood pressure). Stretch of cardiomyocytes activates second messengers such as phosphatidylinositol, protein kinase C, Raf-1 kinase and extracellular signal-regulated protein kinases (ERKs), which are involved in increased protein synthesis. The cardiac renin–angiotensin system is linked to the formation of pressure-overload hypertrophy. Angiotensin II increases the growth of cardiomyocytes by an autocrine mechanism. Angiotensin II-evoked signal transduction pathways differ among cell types. In cardiac fibroblasts, angiotensin II activates ERKs through a pathway including the Gβγ subunit of Gi protein, Src family tyrosine kinases, Shc, Grb2 and Ras, whereas Gq and protein kinase C are important in cardiac myocytes. In addition, mechanical stretch enhances the endothelin-1 release from the cardiomyocytes. Further, the Na⁺–H⁺ exchanger mediates mechanical stretch-induced Raf-1 kinase and ERK activation followed by increased protein synthesis in cardiomyocytes. Not only mechanical stress, but also neurohumoral factors induce cardiac hypertrophy. The activation of protein kinase cascades by norepinephrine is induced by protein kinase A through β-adrenoceptors as well as by protein kinase C through -adrenoceptors.     

Selective down-regulation of angiotensin II receptor type 1A signaling by protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells

The heptahelical AT(1) G-protein-coupled receptor lacks inherent tyrosine kinase activity. Angiotensin II binding to AT(1) nevertheless activates several tyrosine kinases and stimulates both tyrosine phosphorylation and phosphatase activity of the SHP-2 tyrosine phosphatase in vascular smooth muscle cells. Since a balance between tyrosine kinase and tyrosine phosphatase activities is essential in angiotensin II signaling, we investigated the role of SHP-2 in modulating tyrosine kinase signaling pathways by stably transfecting vascular smooth muscle cells with expression vectors encoding wild-type SHP-2 protein or a catalytically inactive SHP-2 mutant. Our data indicate that SHP-2 is an efficient negative regulator of angiotensin II signaling. SHP-2 inhibited c-Src catalytic activity by dephosphorylating a positive regulatory tyrosine 418 within the Src kinase domain. Importantly, SHP-2 expression also abrogated angiotensin II-induced activation of ERK, whereas expression of catalytically inactive SHP-2 caused sustained ERK activation. Thus, SHP-2 likely regulates angiotensin II-induced MAP kinase signaling by inactivating c-Src. These SHP-2 effects were specific for a subset of angiotensin II signaling pathways, since SHP-2 overexpression failed to influence Jak2 tyrosine phosphorylation or Fyn catalytic activity. These data show SHP-2 represents a critical negative regulator of angiotensin II signaling, and further demonstrate a new function for this phosphatase in vascular smooth muscle cells.     

Protein tyrosine phosphatase epsilon activates Yes and Fyn in Neu-induced mammary tumor cells

The receptor-type form of protein tyrosine phosphatase epsilon (RPTP) is among the few tyrosine phosphatases that can support the transformed phenotype of tumor cells. Accordingly, cells from mammary epithelial tumors induced by activated Neu in mice genetically lacking RPTP appear morphologically less transformed and exhibit reduced proliferation. The effect of RPTP in these cells is mediated at least in part by its ability to activate Src, the prototypic member of a family of related kinases. We show here that RPTP is a physiological activator of two additional Src family kinases, Yes and Fyn. Activities of both kinases are inhibited in mammary tumor cells lacking RPTP, and phosphorylation at their C-terminal inhibitory tyrosines is increased. In agreement, opposite effects on activities and phosphorylation of Yes and Fyn are observed following increased expression of PTP. RPTP also forms stable complexes with either kinase, providing physical opportunity for their activation by RPTP. Surprisingly, expression of Yes or of Fyn does not rescue the morphological phenotype of RPTP-deficient tumor cells in contrast with the strong ability of Src to do so. We conclude that RPTP activates Src, Yes, and Fyn, but that these related kinases play distinct roles in Neu-induced mammary tumor cells.     

Co-operative effects of angiotensin II and caerulein in NFκB activation in pancreatic acinar cells in vitro

Angiotensin II is a vasoactive peptide that controls blood pressure and homeostasis. Emerging evidence shows that locally generated angiotensin II plays a crucial role in normal physiology, as well as pathophysiological conditions such as pancreatitis. We recently reported that angiotensin II activates pancreatic NFκB in obstructive pancreatitis. However, the specific cell type responsible for this activation remains unclear. In this study, we investigated whether pancreatic acinar cells respond to angiotensin II. These cells are the most abundant pancreatic cells and the most vulnerable to pancreatitis. Pancreatic acinar AR42J cells were used as an in vitro model of pancreatic inflammation. Our results demonstrated that treatment with caerulein, a cholecystokinin receptor agonist, induced hypersecretion and NFκB activation, as demonstrated by elevated amylase secretion and degradation of inhibitor of NFκB (IκBβ). Angiotensin II, either alone or in combination with caerulein, augmented IκBβ degradation. Pre-treatment with losartan, an antagonist of the angiotensin type I (AT₁) receptor, abolished NFκB activation by angiotensin II and caerulein in a dose-dependent manner. Treatment with PD123319, a blocker of the angiotensin type II (AT₂) receptor, enhanced the activation of NFκB by angiotensin II and caerulein. Preliminary data further demonstrated that angiotensin II could extend caerulein-induced ERK1/2 activation in acinar cells. These results indicated that inflammation triggered by hyperstimulation of pancreatic acinar cells is enhanced by angiotensin II, via the AT₁ receptor. In contrast, stimulation of the AT₂ receptor protects against caerulein-induced NFκB activation. The differential roles of the AT₁ and AT₂ receptors might be useful in developing potential therapies for pancreatic inflammation.     

Activation of p42/p44 mitogen-activated protein kinase by angiotensin II,vasopressin, norepinephrine,and prostaglandin F2α in hepatocytes is sustained,and like the effect of epidermal growth factor,mediated through pertussis toxin-sensitive mechanisms

Several agents that act through G-protein-coupled receptors and also stimulate phosphoinositide-specific phospholipase C (PI-PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F_2α, activated the ERK1 (p44^mapk) and ERK2 (p42^mapk) members of the mitogen-activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in-gel assays. All these agonists induced a peak activation (two to threefold increase in MBP-phosphorylation) at 3–5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI-PLC, implicating a G_q-dependent pathway, the elevations of the concentration of inositol (1,4,5)-trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PGF_2α. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G-protein-coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that G_i-dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G-protein-coupled receptors as well as the EGF receptor. J. Cell. Physiol. 175:348–358, 1998. © 1998 Wiley-Liss, Inc.     

Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

Engagement of the B-cell antigen receptor (BCR) leads to activation of the Raf-MEK-ERK pathway and Raf kinases play an important role in the modulation of ERK activity. B lymphocytes express two Raf isoforms, Raf-1 and B-Raf. Using an inducible deletion system in DT40 cells, the contribution of Raf-1 and B-Raf to BCR signalling was dissected. Loss of Raf-1 has no effect on BCR-mediated ERK activation, whereas B-Raf-deficient DT40 cells display a reduced basal ERK activity as well as a shortened BCR-mediated ERK activation. The Raf-1/B-Raf double deficient DT40 cells show an almost complete block both in ERK activation and in the induction of the immediate early gene products c-Fos and Egr-1. In contrast, BCR-mediated activation of nuclear factor of activated T cells (NFAT) relies predominantly on B-Raf. Furthermore, complementation of Raf-1/B-Raf double deficient cells with various Raf mutants demonstrates a requirement for Ras-GTP binding in BCR-mediated activation of both Raf isoforms and also reveals the important role of the S259 residue for the regulation of Raf-1. Our study shows that BCR-mediated ERK activation involves a cooperation of both B-Raf and Raf-1, which are activated specifically in a temporally distinct manner.     

The protein-tyrosine phosphatase SHP-2 is required during angiotensin II-mediated activation of cyclin D1 promoter in CHO-AT1A cells

Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary (CHO) cells stably expressing a rat vascular angiotensin II type 1A receptor (CHO-AT(1A)). Cyclin D1 protein expression is regulated by mitogens, and its assembly with the cyclin-dependent kinases induces phosphorylation of the retinoblastoma protein pRb, a critical step in G(1) to S phase cell cycle progression contributing to the proliferative responses. In the present study, we found that in CHO-AT(1A) cells, Ang II induced a rapid and reversible tyrosine phosphorylation of various intracellular proteins including the protein-tyrosine phosphatase SHP-2. Ang II also induced cyclin D1 protein expression in a phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner. Using a pharmacological and a co-transfection approach, we found that p21(ras), Raf-1, phosphatidylinositol 3-kinase and also the catalytic activity of SHP-2 and its Src homology 2 domains are required for cyclin D1 promoter/reporter gene activation by Ang II through the regulation of MAPK/ERK activity. Our findings suggest for the first time that SHP-2 could play an important role in the regulation of a gene involved in the control of cell cycle progression resulting from stimulation of a G protein-coupled receptor independently of epidermal growth factor receptor transactivation.     

Receptor-specific regulation of ERK1/2 activation by members of the “free fatty acid receptor” family

Context: The “free fatty acid receptors” (FFARs) GPR40, GPR41, and GPR43 regulate various physiological homeostases, and are all linked to activation of extracellular signal-regulated kinases (ERK)1/2.

Objective: Investigation of coupling of FFARs to two other mitogen-activated protein kinases (MAPKs) sometimes regulated by G protein-coupled receptors (GPCRs), c-Jun N-terminal kinase (JNK) and p38MAPK, and characterization of signaling proteins involved in the regulation of FFAR-mediated ERK1/2 activation.

Methods: FFARs were recombinantly expressed, cells challenged with the respective agonist, and MAPK activation quantitatively determined using an AlphaScreen SureFire assay. Inhibitors for signaling proteins were utilized to characterize ERK1/2 pathways.

Results: Propionate-stimulated GPR41 strongly coupled to ERK1/2 activation, while the coupling of linoleic acid-activated GPR40 and acetate-activated GPR43 was weaker. JNK and p38MAPK were weakly activated by FFARs. All three receptors activated ERK1/2 fully or partially via G_i/o and Rac. PI3K was relevant for GPR40- and GPR41-mediated ERK1/2 activation, and Src was essential for GPR40- and GPR43-induced activation. Raf-1 was not involved in the GPR43-triggered activation.

Conclusion: The results demonstrate a novel role of Rac in GPCR-mediated ERK1/2 signaling, and that GPCRs belonging to the same family can regulate ERK1/2 activation by different receptor-specific mechanisms.     

p120 Catenin-associated Fer and Fyn tyrosine kinases regulate beta-catenin Tyr-142 phosphorylation and beta-catenin-alpha-catenin Interaction

beta-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and alpha-catenin. We show here that interaction of beta-catenin with alpha-catenin is regulated by the phosphorylation of beta-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupted the association between both catenins. We have also examined whether these kinases are involved in the regulation of this interaction by K-ras. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little alpha-catenin-beta-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of beta-catenin-alpha-catenin interaction.     

Activation of ERK by Ca2+ store depletion in rat liver epithelial cells

In rat liver epithelial (WB) cells,Ca²⁺ pool depletion induced by twoindependent methods resulted in activation of extracellular signal-regulated protein kinase (ERK). In the first method,Ca²⁺ pool depletion bythapsigargin increased the activity of ERK, even when rise in cytosolicCa²⁺ was blocked with theCa²⁺ chelator BAPTA-AM. For thesecond method, addition of extracellular EGTA at a concentration shownto deplete intracellular Ca²⁺pools also increased ERK activity. In each instance, ERK activation, asmeasured by an immunocomplex kinase assay, was greatly reduced by thetyrosine kinase inhibitor genistein, suggesting thatCa²⁺ store depletion increased ERKactivity through a tyrosine kinase pathway. The intracellularCa²⁺-releasing agent thapsigarginincreased Fyn activity, which was unaffected by BAPTA-AM pretreatment,suggesting that Fyn activity was unaffected by increased cytosolic freeCa²⁺. Furthermore, depletion ofintracellular Ca²⁺ with EGTAcaused inactivation of protein phosphatase 2A and protein tyrosinephosphatases. ANG II-induced activations of Fyn, Raf-1, and ERK wereaugmented in cells pretreated with BAPTA-AM, but ANG II-inducedexpression of the dual-specificity phosphatase mitogen-activatedprotein kinase phosphatase-1 was blocked by BAPTA-AM pretreatment.Together these results indicate that ERK activity is regulated by thebalance of phosphorylation vs. dephosphorylation reactions in intactcells and that the amount of Ca²⁺stored in intracellular pools plays an important role in this regulation.

     

The mixed-lineage kinase DLK undergoes Src-dependent tyrosine phosphorylation and activation in cells exposed to vanadate or platelet-derived growth factor (PDGF)

Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling.     

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

The calcium/calmodulin-dependent kinase II (CaMKII) participates with Ras to Raf-1 activation, and it is necessary for activation of the extracellular signal-regulated kinase (ERK) by different factors in epithelial and mesenchimal cells. Raf-1 activation is a complex multistep process, and its maximal activation is achieved by phosphorylation at Y341 by Src and at S338 by other kinase/s. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 remains to be definitively identified. In this study, we verified the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To do so, we determined the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors. Serum, fibronectin, Src^Y527 and Ras^V12 activated CaMKII and ERK, at different extents. The inhibition of CaMKII attenuated Raf-1 and ERK activation by all these factors. CaMKII was also necessary for the phosphorylation of Raf-1 at S338 by serum, fibronectin and Ras. Conversely, inhibition of Pak3 activation by blocking phosphatidylinositol 3-kinase was ineffective. The direct phosphorylation of S338 Raf-1 by CaMKII was demonstrated in vitro by interaction of purified kinases. These results demonstrate that Ras activates CaMKII, which, in turn, phosphorylates Raf-1 at S338 and participates in ERK activation upon different stimuli.     

Vascular Smooth Muscle Cell Motility Is Mediated by a Physical and Functional Interaction of Ca2+/Calmodulin-dependent Protein Kinase IIδ2 and Fyn

In vascular smooth muscle (VSM) cells, Ca²⁺/calmodulin-dependent protein kinase IIδ₂ (CaMKIIδ₂) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ₂ was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ₂ and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ₂ and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ₂ and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ₂ and Fyn, implicating CaMKIIδ₂ as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ₂ and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ₂ and Fyn may coordinately regulate VSM cell motility.     

The Src family kinase Yes triggers hyperosmotic activation of the epidermal growth factor receptor and CD95

Hyperosmotic exposure of rat hepatocytes triggers epidermal growth factor receptor (EGFR) activation, which results in an activation of the CD95 system and sensitizes the cells toward apoptosis (Reinehr, R., Schliess, F., and Haüssinger, D. (2003) FASEB J. 17, 731-733). The mechanisms underlying the hyperosmotic EGFR activation were studied. Hyperosmotic exposure (405 mosm) resulted in a rapid activation of the Src kinase family members Yes, Fyn, and Lck. Hyperosmotic Yes, but not Fyn activation, was antioxidant-sensitive and was followed by a rapid Yes/EGFR association. PP-2 abolished the hyperosmotic activation of Fyn and Lck but not activation of Yes and EGFR and their association. However, these latter processes were prevented in the presence of SU6656. SU6656 and antioxidants, but not PP-2 and AG1478, also inhibited the hyperosmotic JNK activation. Cyclic AMP had no effect on hyperosmotic Yes and JNK activation but prevented EGFR/Yes association and EGFR activation in an H89-sensitive way. When the hyperosmolarity-induced Yes-EGFR protein complex started to disappear after 30 min, an association between EGFR and CD95 became apparent, which was followed by CD95 tyrosine phosphorylation and activation. SU6656 but not PP-2 also inhibited EGFR/CD95 association, CD95 tyrosine phosphorylation, CD95 membrane trafficking, and death-inducing signaling complex (DISC) formation. EGFR knockdown had no effect on hyperosmotic Yes activation but prevented CD95 tyrosine phosphorylation, membrane targeting, and DISC formation. Hyperosmotic EGFR and CD95 activation was also largely blunted following Yes knockdown. The data suggest that hyperosmotic signaling triggers an oxidative stress-dependent Yes activation, which is followed by JNK and EGFR activation and subsequent activation of the CD95 system. However, the functional relevance of hyperosmolarity-induced Fyn and Lck activation remains to be elucidated.     

Functional activation of Src family kinase yes protein is essential for the enhanced malignant properties of human melanoma cells expressing ganglioside GD3

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.     

Molecular aspects of mechanical stress-induced cardiac hypertrophy

To elucidate the signal transduction pathway from external stimuli to nuclear gene expression in mechanical stress-induced cardiac hypertrophy, we examined the time course of activation of protein kinases such as Raf-1 kinase (Raf-1), mitogen-activated protein kinase kinase (MAPKK), MAP kinases (MAPKs) and 90-kDa ribosomal S6 kinase (p90^rsk) in neonatal rat cardiomyocytes. Mechanical stretch rapidly activated Raf-1 and its maximal activation was observed at 1–2 min after stretch. The activity of MAPKK was also increased by stretch, with a peak at 5 min after stretch. In addition, MAPKs and p90^rsk were maximally activated at 8 min and at 10–30 min after stretch, respectively. Next, the relationship between mechanical stress-induced hypertrophy and the cardiac renin-angiotensin system was investigated. When the stretch-conditioned culture medium was transferred to the culture dish of non-stretched cardiac myocytes, the medium activated MAPK activity slightly but significantly, and the activation was completely blocked by the type 1 angiotensin II receptor antagonist, CV-11974. However, activation of Raf-1 and MAPKs provoked by stretching cardiomyocytes was only partially suppressed by pretreatment with CV-11974. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1, MAPKK, MAPKs and p90^rsk, and that angiotensin II, which is secreted from stretched myocytes, activates a part of these protein kinases.Abbreviations MAPK mitogen-activated protein kinase - MAPKK MAP kinase kinase - Raf-1 - Raf- 1 kinase p90^rsk, 90 kDa ribosomal S6 kinase; AngII - angiotensin II - MAPKKK MAP kinase kinase kinase - rMAPK recombinant MAPKK fused to gluthathione S transferase - MMAKK recombinant MAPK fused to maltose binding protein - MBP myelin basic protein - ACE angiotensin-converting enzyme     

Cholecystokinin stimulates extracellular signal-regulated kinase through activation of the epidermal growth factor receptor,Yes, and protein kinase C. Signal amplification at the level of Raf by activation of protein kinase Cepsilon

Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through G(q)-coupled receptors (CCK(A) and CCK(B)) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCK(A) and CCK(B). CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cepsilon (PKCepsilon), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCepsilon, but not PKCalpha or PKCdelta, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCK(B) receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCepsilon, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level.