PYK2/CAKbeta represents a redox-sensitive tyrosine kinase in vascular smooth muscle cells

In vascular smooth muscle cells (VSMCs), the focal adhesion kinase-related tyrosine kinase PYK2/CAKbeta is activated by vascular mitogens. Because reactive oxygen species (ROS) are assumed to mediate mitogenic signals by these agonists, we examined the possible link between ROS and PYK2 in cultured rat VSMCs. Here we present several lines of evidence showing that PYK2 is activated by ROS in VSMCs. The inhibitory effect of an antioxidant, N-acetyl-cysteine, on PYK2 activation by its specific agonists further suggests the pivotal role of PYK2 in vascular remodeling associated with enhanced ROS production.     

Oxytocin activates calcium signaling in rat sensory neurons through a protein kinase C-dependent mechanism

In addition to its well-known effects on parturition and lactation, oxytocin (OT) plays an important role in modulation of pain and nociceptive transmission. But, the mechanism of this effect is unclear. To address the possible role of OT on pain modulation at the peripheral level, the effects of OT on intracellular calcium levels ([Ca²⁺]_i) in rat dorsal root ganglion (DRG) neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1- or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of OT on [Ca²⁺]_i and role of the protein kinase C (PKC)-mediated pathway in OT effect were assessed. OT caused a significant increase in basal levels of [Ca²⁺]_i after application at the doses of 30 nM (n?=?34, p?<?0.01), 100 nM (n?=?41, p?<?0.001) and 300 nM (n?=?46, p?<?0.001). The stimulatory effect of OT (300 nM) on [Ca²⁺]_i was persistent in Ca²⁺-free conditions (n?=?56, p?<?0.01). Chelerythrine chloride, a PKC inhibitor, significantly reduced the OT-induced increase in [Ca²⁺]_i (n?=?28, p?<?0.001). We demonstrated that OT activates intracellular calcium signaling in cultured rat primary sensory neurons in a dose- and PKC-dependent mechanism. The finding of the role of OT in peripheral pain modification may serve as a novel target for the development of new pharmacological strategies for the management of pain.     

Inhibition of a signaling pathway in cardiac muscle cells by active mitogen-activated protein kinase kinase.

Signaling via the Ras pathway involves sequential activation of Ras, Raf-1, mitogen-activated protein kinase kinase (MKK), and the extracellular signal-regulated (ERK) group of mitogen-activated protein (MAP) kinases. Expression from the c-Fos, atrial natriuretic factor (ANF), and myosin light chain-2 (MLC-2) promoters during phenylephrine-induced cardiac muscle cell hypertrophy requires activation of this pathway. Furthermore, constitutively active Ras or Raf-1 can mimic the action of phenylephrine in inducing expression from these promoters. In this study, we tested whether constitutively active MKK, the molecule immediately downstream of Raf, was sufficient to induce expression. Expression of constitutively active MKK induce ERK2 kinase activity and caused expression from the c-Fos promoter, but did not significantly activate expression of reporter genes under the control of either the ANF or MLC-2 promoters. Expression of CL100, a phosphatase that inactivates ERKs, prevented expression from all of the promoters. Taken together, these data suggest that ERK activation is required for expression from the Fos, ANF, and MLC-2 promoters but MKK and ERK activation is sufficient for expression only from the Fos promoter. Constitutively active MKK synergized with phenylephrine to increase expression from a c-Fos- or an AP1-driven reporter. However, active MKK inhibited phenylephrine- and Raf-1-induced expression from the ANF and MLC-2 promoters. A DNA sequence in the MLC-2 promoter that is a target for inhibition by active MKK, but not CL100, was mapped to a previously characterized DNA element (HF1) that is responsible for cardiac specificity. Thus, activation of cardiac gene expression during phenylephrine-induced hypertrophy requires ERK activation but constitutive activation by MKK can inhibit expression by targeting a DNA element that controls the cardiac specificity of gene expression.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism

The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38 MAPK signaling pathway are currently unknown. Here we demonstrate that pp125 focal adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. We observe that the dominant-negative mutant of MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, a membrane-permeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the p38 MAPK signaling pathway in response to certain cytotoxic agents.     

Macrophage colony-stimulating factor induces the expression of mitogen-activated protein kinase phosphatase-1 through a protein kinase C-dependent pathway.

M-CSF triggers the activation of extracellular signal-regulated protein kinases (ERK)-1/2. We show that inhibition of this pathway leads to the arrest of bone marrow macrophages at the G0/G1 phase of the cell cycle without inducing apoptosis. M-CSF induces the transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), which correlates with the inactivation of ERK-1/2. Because the time course of ERK activation must be finely controlled to induce cell proliferation, we studied the mechanisms involved in the induction of MKP-1 by M-CSF. Activation of ERK-1/2 is not required for this event. Therefore, M-CSF activates ERK-1/2 and induces MKP-1 expression through different pathways. The use of two protein kinase C (PKC) inhibitors (GF109203X and calphostin C) revealed that M-CSF induces MKP-1 expression through a PKC-dependent pathway. We analyzed the expression of different PKC isoforms in bone marrow macrophages, and we only detected PKCbetaI, PKCepsilon, and PKCzeta. PKCzeta is not inhibited by GF109203X/calphostin C. Of the other two isoforms, PKCepsilon is the best candidate to mediate MKP-1 induction. Prolonged exposure to PMA slightly inhibits MKP-1 expression in response to M-CSF. In bone marrow macrophages, this treatment leads to a complete depletion of PKCbetaI, but only a partial down-regulation of PKCepsilon. Moreover, no translocation of PKCbetaI or PKCzeta from the cytosol to particulate fractions was detected in response to M-CSF, whereas PKCepsilon was constitutively present at the membrane and underwent significant activation in M-CSF-stimulated macrophages. In conclusion, we remark the role of PKC, probably isoform epsilon, in the negative control of ERK-1/2 through the induction of their specific phosphatase.     

E-selectin-dependent signaling via the mitogen-activated protein kinase pathway in vascular endothelial cells

E-selectin, a cytokine-inducible adhesion molecule, supports rolling and stable arrest of leukocytes on activated vascular endothelium. Previous studies have suggested that this transmembrane protein can also transduce signals into the endothelial cell. We now demonstrate activation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured HUVEC in response to E-selectin-dependent leukocyte adhesion and Ab-mediated cross-linking of cell surface E-selectin. Adhesion of increasing numbers of HL60 cells to IL-1beta-activated HUVEC stimulated robust increases in MAPK activity that were abrogated by an E-selectin blocking Ab. Cross-linking of cell surface E-selectin with Abs, as a mimic of multivalent ligand engagement, strongly stimulated MAPK/extracellular signal-related kinase (ERK) kinase (MEK)-dependent MAPK activation and concomitant up-regulation of mRNA for c-fos, an immediate early response gene, whereas Ab cross-linking of HLA class I molecules (present at comparable density) failed to do so. Coimmunoprecipitation documented Ras, Raf-1 and, phospho-MEK complex formation. Unactivated HUVEC transduced with a full-length adenoviral E-selectin construct also exhibited cross-link-induced MAPK activation, macromolecular complex formation, and c-fos up-regulation, whereas HUVEC transduced with a cytoplasmic domain deletion mutant failed to respond. These observations indicate that E-selectin can transduce an activating stimulus via the MAPK cascade into the endothelial cell during leukocyte adhesion.     

Fibronectin fragment activation of proline-rich tyrosine kinase PYK2 mediates integrin signals regulating collagenase-3 expression by human chondrocytes through a protein kinase C-dependent pathway

Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the alpha5beta1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the alpha5beta1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCdelta inhibitor rottlerin. Furthermore, PKCdelta translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after cotransfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves FN-f stimulation of the alpha5beta1 integrin and activation of the nonreceptor tyrosine kinase PYK2 by PKC, most likely PKCdelta     

Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C.

In this study we show that phosphorylation of extracellular signal-regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO(-)) exposure occurs via a MAPK kinase (MEK)-independent but PKC-dependent pathway in rat-1 fibroblasts. ONOO(-)-mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126. Furthermore, no increase in MEK phosphorylation was detected upon ONOO(-) treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down-regulation of PKC by phorbol-12,13-dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO(-), implying that activation of ERK by ONOO(-) depends on activation of PKC. Indeed, PKCalpha and epsilon were activated upon ONOO(-) exposure. When cells were treated with ONOO(-) in a calcium-free buffer, no activation of PKCalpha was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCalpha and ERK phosphorylation by ONOO(-). Indeed, ONOO(-) exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using G?6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO(-)-mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC.     

Muscarinic receptor stimulation increases regulators of G-protein signaling 2 mRNA levels through a protein kinase C-dependent mechanism.

RGS2, a member of the Regulators of G-protein Signaling family, modulates the activity of G-proteins coupled to the phosphoinositide signal transduction system, but little is known about what regulates RGS2. In human neuroblastoma SH-SY5Y cells stimulation of muscarinic receptors by carbachol activates phosphoinositide signaling and also caused a rapid, large, and long lasting increase in RGS2 mRNA levels. Direct activation of protein kinase C also rapidly increased RGS2 mRNA levels. Inhibition of protein kinase C with Ro31-8220, GF109203x, or Go6976 or down-regulation of protein kinase C inhibited increases in RGS2 mRNA levels induced by carbachol or by the activation of protein kinase C. Blockade of calcium signaling did not alter carbachol-induced increases in RGS2 mRNA levels. Neither activation of epidermal growth factor receptors nor stimulation of cyclic AMP production with forskolin increased RGS2 mRNA levels. Pretreatment with actinomycin D blocked increases in RGS2 mRNA levels but caused a surprisingly small, although statistically significant, partial blockade of protein kinase C-mediated feedback inhibition of carbachol-induced phosphoinositide hydrolysis. Thus, RGS2 mRNA levels are increased by activation of muscarinic receptors coupled to the phosphoinositide signal transduction system through a protein kinase C-dependent mechanism. This action may contribute to negative feedback control of this signaling cascade, but because the small contribution to negative feedback contrasts with the large and prolonged elevations in RGS2 mRNA levels, we speculate that its primary role may be in modulating other signaling components.     

Dehydroepiandrosterone negatively regulates the p38 mitogen-activated protein kinase pathway by a novel mitogen-activated protein kinase phosphatase

Dehydroepiandrosterone-sulfate, the sulfated form of dehydroepiandrosterone, is the most abundant steroid in young adults, but gradually declines with aging. In humans, the clinical application of dehydroepiandrosterone targeting some collagen diseases, such as systemic lupus erythematosus, as an adjunctive treatment has been applied in clinical trial. Here, we report that dehydroepiandrosterone may negatively regulate the mitogen-activated protein kinase pathway in humans via a novel dual specificity protein phosphatase, DDSP (dehydroepiandrosterone-enhanced dual specificity protein phosphatase). DDSP is highly homologous to LCPTP/HePTP, a tissue-specific protein tyrosine phosphatase (PTP) which negatively regulates both ERK and p38-mitogen-activated protein kinase, and is transcribed from the PTPN7 locus by alternative splicing. Although previous reports have shown that the mRNA expression of the LCPTP/HePTP gene was inducible by extracellular signals such as T-cell antigen receptor stimulation, reverse transcribed (RT)-PCR experiments using specific sets of primers suggested that the expression of LCPTP/HePTP was constitutive while the actual inducible sequence was that of DDSP. Furthermore DDSP was widely distributed among different types of human tissues and specifically interacted with p38-mitogen-activated protein kinase. This inducible negative regulation of the p38-mitogen-activated protein kinase-dependent pathway may help to clarify the broad range of dehydroepiandrosterone actions, thereby aiding the development of new preventive or adjunctive applications for human diseases.     

Cholecystokinin activates Gi1-, Gi2-, Gi3- and several Gs-proteins in rat pancreatic acinar cells.

On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.     

The Src kinase Yes is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters, but not pancreatic growth factors, which stimulate its association with numerous other signaling molecules

For growth factors, cytokines, G-protein-coupled receptors and numerous other stimuli, the Src Family of kinases (SFK) play a central signaling role. SFKs also play an important role in pancreatic acinar cell function including metabolism, secretion, endocytosis, growth and cytoskeletal integrity, although the specific SFKs involved are not fully known. In the present study we used specific antibodies for the SFK, Yes, to determine its presence, activation by pancreatic secretagogues or growth factors, and interaction with cellular signaling cascades mediated by CCK in which Yes participates in to cause acinar cell responses. Yes was identified in acini and secretagogues known to activate phospholipase C (PLC) [CCK, carbachol, bombesin] as well as post-receptor stimulants activating PKC [TPA] or mobilizing cellular calcium [thapsigargin/calcium ionophore (A23187)] each activated Yes. Secretin, which activates adenylate cyclase did not stimulate Yes, nor did pancreatic growth factors. CCK activation of Yes required both high- and low-affinity CCK(1)-receptor states. TPA-/CCK-stimulated Yes activation was completely inhibited by thapsigargin and the PKC inhibitor, GF109203X. CCK/TPA stimulated the association of Yes with focal adhesion kinases (Pyk2, FAK) and its autophosphorylated forms (pY397FAK, pY402Pyk2). Moreover, CCK/TPA stimulated Yes interacted with a number of other signaling proteins, including Shc, PKD, p130(Cas), PI3K and PTEN. This study demonstrates that in rat pancreatic acini, the SFK member Yes is expressed and activated by CCK and other gastrointestinal hormones/neurotransmitters. Because its activation results in the direct activation of many cellular signaling cascades that have been shown to mediate CCK's effect in acinar cell function our results suggest that it is one of the important pancreatic SFKs mediating these effects.