Protein phosphatase 5 as a negative key regulator of Raf-1 activation

Growth factors such as the epidermal growth factor cause sequential activation of receptor tyrosine kinases, adaptor molecules and the Raf-MEK-ERK pathway. The kinetics and intensity of these signals are dependent on the balance between phosphorylation and dephosphorylation of these molecules by numerous kinases and phosphatases, respectively. Recently, protein phosphatase 5 has been characterized as a key dephosphorylation regulator of Raf-1 activation in growth factor-mediated signaling, leading to attenuation of the MEK-ERK cascade.     

Protein kinase Czeta is required for oleic acid-induced secretion of glucagon-like peptide-1 by intestinal endocrine L cells

Long-chain, monounsaturated fatty acids (FAs) stimulate secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1) from the intestinal L cell. Because the atypical protein kinase C (PKC), PKCzeta, is involved in FA signaling in many cells, the role of PKCzeta in FA-induced GLP-1 secretion was investigated, using the murine GLUTag L cell line and primary rat intestinal L cells. GLUTag cells expressed mRNA for several PKC isoforms, including PKCzeta, and PKCzeta protein was localized throughout the cytoplasm in GLUTag and primary L cells as well as normal mouse and rat L cells. Treatment with oleic acid (150-1000 microm) for 2 h increased GLP-1 secretion (P < 0.001), and this was abrogated by the PKCzeta inhibitor ZI (P < 0.05) and PKCzeta small interfering RNA transfection (P < 0.05) but not inhibition of classical/novel PKC isoforms. Although most PKCzeta was localized in the particulate compartment of GLUTag cells, oleate treatment did not alter PKCzeta levels or activity in this cell fraction. GLUTag cells expressed mRNA for the Gq-coupled FA receptor GPR120; however, oleic acid did not induce any changes in Akt, MAPK, or calcium, and pretreatment with LY294002 and PD98059 to inhibit phosphatidylinositol 3-kinase and MAPK, respectively, did not prevent the effects of oleic acid. Finally, GLUTag cells also released GLP-1 in response to arachidonic acid (P < 0.001) but were not affected by other long-chain FAs. These findings demonstrate that PKCzeta is required for oleic acid-induced GLP-1 secretion. This enzyme may therefore serve as a therapeutic target to enhance GLP-1 release in type 2 diabetes.     

Raf-1 and p21v-ras cooperate in the activation of mitogen-activated protein kinase.

Mitogen-activated protein (MAP) kinases Raf-1, pp60src, and p21ras all play important roles in the transfer of signals from the cell surface to the nucleus. We have used the baculovirus/Sf9 insect cell system to elucidate the regulatory relationships between pp60v-src, p21v-ras, MAP kinase (p44erk1/mapk), and Raf-1. In Sf9 cells, p44erk1/mapk is activated by coexpression with either v-Raf or a constitutively activated form of Raf-1 (Raf22W). In contrast, p44erk1/mapk is activated to only a limited extent by coexpression with either Raf-1 or p21v-ras alone. This activation of p44erk1/mapk is greatly enhanced by coexpression with both p21v-ras and Raf-1. Since we have previously shown that p21v-ras stimulates Raf-1 activity, the activation of p44erk1/mapk by p21v-ras may occur exclusively via a Raf-1-dependent pathway. However, a dominant-inhibitory mutant of Raf-1 (Raf301) does not block the activation of p44erk1/mapk by p21-v-ras. Further, pp60v-src, which activates Raf-1 at least as effectively as p21v-ras, fails to enhance p44erk1/mapk activity greatly when coexpressed with Raf-1. These data suggest that activation of p44erk1/mapk by p21v-ras may occur via both Raf-1-dependent and Raf-1-independent pathways.     

Protein kinase activation and myocardial ischemia/reperfusion injury

Myocardial ischemia and ischemia/reperfusion activate several protein kinase pathways. Protein kinase activation potentially regulates the onset of myocardial cell injury and the reduction of this injury by ischemic and pharmacologic preconditioning. The primary protein kinase pathways that are potentially activated by myocardial ischemia/reperfusion include: the MAP kinases, ERK 1/2, JNK 1/2, p38 MAPKalpha/beta; the cell survival kinase, Akt; and the sodium-hydrogen exchanger (NHE) kinase, p90RSK. The literature does not support a role for ischemia/reperfusion in the activation of the tyrosine kinases, Src and Lck, or the translocation and activation of PKC. This review will detail the role of these protein kinases in the onset of myocardial cell death by necrosis and apoptosis and the reduction of this injury by preconditioning.     

Insulin stimulates primary beta-cell proliferation via Raf-1 kinase

A relative decrease in beta-cell mass is key in the pathogenesis of type 1 diabetes, type 2 diabetes, and in the failure of transplanted islet grafts. It is now clear that beta-cell duplication plays a dominant role in the regulation of adult beta-cell mass. Therefore, knowledge of the endogenous regulators of beta-cell replication is critical for understanding the physiological control of beta-cell mass and for harnessing this process therapeutically. We have shown that concentrations of insulin known to exist in vivo act directly on beta-cells to promote survival. Whether insulin stimulates adult beta-cell proliferation remains unclear. We tested this hypothesis using dispersed primary mouse islet cells double labeled with 5-bromo-2-deoxyuridine and insulin antisera. Treating cells with 200-pm insulin significantly increased proliferation from a baseline rate of 0.15% per day. Elevating glucose from 5-15 mm did not significantly increase beta-cell replication. beta-Cell proliferation was inhibited by somatostatin as well as inhibitors of insulin signaling. Interestingly, inhibiting Raf-1 kinase blocked proliferation stimulated by low, but not high (superphysiological), insulin doses. Insulin-stimulated mouse insulinoma cell proliferation was dependent on both phosphatidylinositol 3-kinase/Akt and Raf-1/MAPK kinase pathways. Overexpression of Raf-1 was sufficient to increase proliferation in the absence of insulin, whereas a dominant-negative Raf-1 reduced proliferation in the presence of 200-pm insulin. Together, these results demonstrate for the first time that insulin, at levels that have been measured in vivo, can directly stimulate beta-cell proliferation and that Raf-1 kinase is involved in this process. These findings have significant implications for the understanding of the regulation of beta-cell mass in both the hyperinsulinemic and insulin-deficient states that occur in the various forms of diabetes.     

Angiotensin II-induced extracellular signal-regulated kinase 1/2 activation is mediated by protein kinase Cdelta and intracellular calcium in adult rat cardiac fibroblasts

Angiotensin II (Ang II)-induced proliferation of cardiac fibroblasts is a major contributing factor to the pathogenesis of cardiac fibrosis. Ang II activates extracellular signal-regulated kinase (ERK) 1/2 to induce cardiac fibroblast proliferation, but the signaling pathways leading to ERK 1/2 activation have not been elucidated in these cells. The goal of the current study was to identify the intracellular mediators of Ang II-induced ERK 1/2 activation in adult rat cardiac fibroblasts. We determined that 100 nmol/L of Ang II-induced ERK 1/2 phosphorylation is inhibited by simultaneous chelation of cytosolic calcium and downregulation of protein kinase C (PKC) by phorbol ester or by the specific PKCdelta inhibitor rottlerin, as well as PKCdelta small interfering RNA, but not by inhibition of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate, phorbol ester, rottlerin, or PKCdelta small interfering RNA alone. We also found that Ang II does not transactivate the epidermal growth factor receptor in adult cardiac fibroblasts, because pretreatment with 1 mumol/L of AG 1478 did not significantly inhibit [(3)H]-thymidine incorporation or ERK 1/2 activation. In addition, immunoprecipitation of the epidermal growth factor receptor demonstrated no significant Ang II-induced phosphorylation of tyrosine residues. Inhibition of phosphatidylinositide 3-kinase, PKCzeta, and src tyrosine kinase had no effect on Ang II-induced ERK 1/2 activation. Collectively, these data demonstrate that Ang II does not transactivate the epidermal growth factor receptor in adult rat cardiac fibroblasts to activate ERK 1/2, a common pathway described in vascular smooth muscle and other cell types, but rather occurs via activation of distinct parallel signaling pathways mechanistically controlled by intracellular Ca(2+) and PKCdelta.     

Interleukin-4 synergizes with Raf-1 to promote long-term proliferation and activation of c-jun N-terminal kinase.

This report shows that interleukin-4 (IL-4), which plays a key role in regulating immune responses, fails to support cellular growth. We investigated whether this failure of IL-4 to promote growth was because of its unique inability to activate the Ras/Raf/Erk pathway. Consistent with other reports, expression in Ba/F3, a factor-dependent hematopoietic cell line, of either activated Q61KN-Ras or a hormone-inducible activated Raf-1, resulted in suppression of apoptosis but not in long-term growth. However, in the presence of IL-4, Ba/F3 cells that expressed either Q61KN-Ras or activated Raf-1 grew continuously at a rate comparable with that stimulated by IL-3. Investigation of the biochemical events associated with the stimulation of long-term growth showed that, as expected, the presence of activated Raf-1 resulted in an increased activity of extracellular signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) but not of c-jun N-terminal kinase/stress-activated protein kinase (JNK). However, surprisingly, if IL-4 was present, cells expressing active Raf-1 exhibited increases in JNK activity. These observations point to a novel mechanism for JNK activation involving synergy between Raf-1 and pathways activated by IL-4 and suggest that in hematopoietic cells proliferation is correlated not only with "mitogen activated" ERK activity, but also with JNK activity.     

Role of protein kinase Czeta in thrombin-induced endothelial permeability changes: inhibition by angiopoietin-1

Endothelial cell leakiness is regulated by mediators such as thrombin, which promotes endothelial permeability, and anti-inflammatory agents, such as angiopoietin-1. Here we define a new pathway involved in thrombin-induced permeability that involves the atypical protein kinase C isoform, PKCzeta. Chemical inhibitor studies implicated the involvement of an atypical PKC isoform in thrombin-induced permeability changes in human umbilical vein endothelial cells. Thrombin stimulation resulted in PKCzeta, but not the other atypical PKC isoform, PKClambda, translocating to the membrane, an event known to be critical to enzyme activation. The involvement of PKCzeta was confirmed by overexpression of constitutively active PKCzeta, resulting in enhanced basal permeability. Dominant-negative PKCzeta prevented the thrombin-mediated effects on endothelial cell permeability and inhibited thrombin-induced activation of PKCzeta. Rho activation does not appear to play a role, either upstream or downstream of PKCzeta, as C3 transferase does not block thrombin-induced PKCzeta activation and dominant-negative PKCzeta does not block thrombin-induced Rho activation. Finally, we show that angiopoietin-1 inhibits thrombin-induced PKCzeta activation, Rho activation, and Ca(++) flux, thus demonstrating that the powerful antipermeability action of angiopoietin-1 is mediated by its action on a number of signaling pathways induced by thrombin and implicated in permeability changes.     

Protein kinase A mediated modulation of acto-myosin kinetics

The effects of protein kinase A (PKA) mediated phosphorylation on thin filament and cross-bridge function is not fully understood. To delineate the effects of troponin I (TnI) phosphorylation by PKA on contractile protein performance, reconstituted thin filaments were treated with PKA. With the use of the in vitro motility assay, PKA treated thin filament function was assessed relative to non-phosphorylated thin filaments in a calcium-regulated system. At maximal calcium activation, unloaded shortening velocity and force did not differ between the groups. However, at submaximal activation, an increase in calcium sensitivity of the thin filament was observed for velocity but a decrease in calcium sensitivity was observed for force. Activation of the thin filament by myosin strong-binding did not elicit a calcium-independent effect. The rightward shift in calcium sensitivity for force and the leftward shift in calcium sensitivity for velocity indicate that PKA phosphorylation of TnI directly modulates the kinetics of the myosin cross-bridge. In addition, the altered velocity dependence on thin filament length implicates reduced myosin cross-bridge binding with PKA treatment. These data highlight the importance of TnI serine 23 and 24 phosphorylation in the modulation of cardiac function.     

Protein kinase C mediated inhibition of endothelial l-arginine transport is mediated by MARCKS protein

The endothelium plays a vital role in the maintenance of vascular tone and structural vascular integrity, principally mediated via the actions of nitric oxide (NO). l-arginine is the immediate substrate for NO synthesis, and the availability of extracellular l-arginine is critical for the production of NO. Activation of protein kinase C (PKC) dependent signalling pathways are a feature of a number of cardiovascular disease states, and in this study we aimed to systematically evaluate the mechanism by which PKC regulates l-arginine transport in endothelial cells. In response to PKC activation (PMA 100 nM, 30 min), [³H]l-arginine uptake by bovine aortic endothelial cells (BAEC) was reduced to 45 + 4% of control (p < 0.05). This resulted from a 53% reduction in the Vmax (p < 0.05), with no change in the K_m for l-arginine. Western blot analysis and confocal microscopy revealed no change in the expression or membrane distribution of CAT-1, the principal BAEC l-arginine transporter. Moreover in ³²P-labeling studies, PMA exposure did not result in CAT-1 phosphorylation. We therefore explored the possibility that PKC altered and interaction with MARCKS protein, a candidate membrane associated protein. By co-immunoprecipitation we show that CAT-1 interacts with, a membrane associated protein, that was significantly inhibited by PKC activation (p < 0.05). Moreover antisense inhibition of MARCKS abolished the PMA effect on l-arginine transport. PKC dependent mechanisms regulate the transport of l-arginine, mediated via process involving MARCKS.     

NF-kappa B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-theta

Protein kinase C-theta (PKCtheta) is a Ca(2+)-independent member of the PKC family that is selectively expressed in skeletal muscle and T lymphocytes and plays an important role in T cell activation. However, the molecular basis for the important functions of PKCtheta in T cells and the manner in which it becomes coupled to the T cell receptor-signaling machinery are unknown. We addressed the functional relationship between PKCtheta and CD28 costimulation, which plays an essential role in T cell receptor-mediated IL-2 production. Here, we provide evidence that PKCtheta is functionally coupled to CD28 costimulation by virtue of its selective ability to activate the CD28RE/activator protein-1 (AP-1) element in the IL-2 gene promoter. First, CD28 costimulation enhanced the membrane translocation and catalytic activation of PKCtheta. Second, among several PKC isoforms, PKCtheta was the only one capable of activating NF-kappaB or CD28RE/AP-1 reporters in T cells (but not in 293T cells). Third, wild-type PKCtheta synergized with CD28/CD3 signals to activate CD28RE/AP-1. In addition, PKCtheta selectively synergized with Tat to activate a CD28RE/AP-1 reporter. Fourth, CD3/CD28-induced CD28RE/AP-1 activation and NF-kappaB nuclear translocation were blocked by a selective PKCtheta inhibitor. Last, PKCtheta-mediated activation of the same reporter was inhibited by the proteasome inhibitor MG132 (which blocks IkappaB degradation) and was found to involve IkappaB-kinase beta. These findings identify a unique PKCtheta-mediated pathway for the costimulatory action of CD28, which involves activation of the IkappaB-kinase beta/IkappaB/NF-kappaB-signaling cascade.     

Fibronectin-induced proliferation in thyroid cells is mediated by alphavbeta3 integrin through Ras/Raf-1/MEK/ERK and calcium/CaMKII signals

We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.     

Interleukin 2 induces tyrosine phosphorylation and activation of p72-74 Raf-1 kinase in a T-cell line.

Interleukin 2 (IL-2) is a lymphokine, produced by T cells upon antigenic or mitogenic stimulation, that is a critical regulator of T-cell proliferation. Although the binding of IL-2 to its receptor has been well characterized, the molecular mechanisms by which IL-2 transmits its signal from the membrane to the interior of the cell are poorly understood. Like most other growth factors, IL-2 causes rapid phosphorylation of proteins within its target cells. Unlike many other growth factors, however, the known subunits of the IL-2 receptor lack tyrosine-specific kinase activity, and little is known about the kinases whose activities are regulated by IL-2. Here we show that IL-2 (but not IL-4) induces rapid phosphorylation of the p72-74 serine/threonine-specific kinase encoded by the c-Raf-1 protooncogene in an IL-2-dependent murine T-cell line, CTLL-2, and that this phosphorylation is associated with increased kinase activity in p72-74 Raf-1-containing immune complexes. The concentration dependence of IL-2-mediated elevations in Raf-1 kinase activity correlated well with IL-2-stimulated proliferation of CTLL-2 cells. Furthermore, much of the IL-2-stimulated phosphorylation of p72-74 Raf-1 occurred on tyrosines. To our knowledge, the Raf-1 kinase represents the first endogenous substrate of an IL-2-regulated tyrosine kinase to be identified.     

Protein kinase C mediated phosphorylation blocks juvenile hormone action

Juvenile hormones (JH) regulate a wide variety of developmental and physiological processes in insects. Although the biological actions of JH are well documented, the molecular mechanisms underlying JH action are poorly understood. We studied the molecular basis of JH action using a JH response element (JHRE) identified in the promoter region of JH esterase gene cloned from Choristoneura fumiferana, which is responsive to JH and 20-hydroxyecdysone (20E). In Drosophila melanogaster L57 cells, the JHRE-regulated reporter gene was induced by JH I, JH III, methoprene, and hydroprene. Nuclear proteins isolated from L57 cells bound to the JHRE and exposure of these proteins to ATP resulted in a reduction in their DNA binding. Either JH III or calf intestinal alkaline phosphatase (CIAP) was able to restore the binding of nuclear proteins to the DNA. In addition, protein kinase C inhibitors increased and protein kinase C activators reduced the binding of nuclear proteins to the JHRE. In transactivation assays, protein kinase C inhibitors induced the luciferase gene placed under the control of a minimal promoter and the JHRE. These data suggest that protein kinase C mediated phosphorylation prevents binding of nuclear proteins to juvenile hormone responsive promoters resulting in suppression of JH action.     

Resistance of bcr-abl-positive acute lymphoblastic leukemia to daunorubicin is not mediated by mdr1 gene expression

In vitro resistance to anthracyclines is thought to be a poor prognosis in achieving long-term remission in patients with acute lymphoblastic leukemia (ALL). Expression of a multidrug resistance gene (mdr1) that codes for 170 Kd transmembrane glycoprotein is responsible for conferring resistance to malignant cells to anthracyclines. The t(9:22) translocation, resulting in bcr-abl fusion gene, is commonly found in B-lineage ALL and is known to be a poor prognostic factor for long-term remission. To investigate whether resistance to anthracyclines contributes to poor prognosis in bcr-abl-positive ALL, we studied daunorubicin sensitivity by an in vitro colorimetric methyl tetrazolium (MTT) assay in B-lineage ALL patients who were bcr-abl-positive and compared them with the B-lineage, age-matched bcr-abl-negative group. We also looked for and compared the presence of mdr1 gene expression in these two groups of patients by RT-PCR. Of the 46 patients included in the study, 16 (34.7%) were positive for the bcr-abl fusion gene. mdr1 gene expression was seen in 14 of these 46 patients (30.4%). However, the expression of the mdr1 gene was relatively lower in the bcr-abl-positive group (3 out of 16, 18.7%) compared to the bcr-abl-negative group (11 out of 30, 36.6%). The median LD(50) of daunorubicin (concentration lethal to 50% of the leukemic blasts) differed significantly between bcr-abl-positive and -negative patients (P = 0.018). This in vitro study suggests that bcr-abl-positive ALL is relatively resistant to daunorubicin, but this resistance is not mediated through mdr1 gene expression.     

B-lymphocyte activation mediated by anti-immunoglobulin antibody in the absence of protein kinase C

B-cell activation induced by crosslinking of surface immunoglobulin is known to stimulate hydrolysis of phosphatidylinositol to diacylglycerol and inositol trisphosphate. We now provide evidence that alternative pathways of activation may also be recruited during such activation. We utilized depletion of protein kinase C activity to determine whether this enzyme is required under all conditions for anti-immunoglobulin-stimulated B-cell activation. Although anti-immunoglobulin does not induce B-cell proliferation in protein kinase C-depleted cells, it stimulates an earlier event in B-cell activation as reflected by its ability to enhance the expression of major histocompatibility complex-encoded class II molecules. Furthermore, the ribonucleoside 8-mercaptoguanosine restores the ability of anti-immunoglobulin to induce B-cell proliferation in protein kinase C-depleted cells. This restoration is also demonstrated by an enhancement of synthesis of a nuclear protein that we find is increased during B-cell mitogenesis. These results indicate that B-cell activation stimulated by anti-immunoglobulin may recruit pathways in addition to the one dependent on protein kinase C.     

Raf-1 kinase activation by angiotensin II in adrenal glomerulosa cells: roles of Gi, phosphatidylinositol 3-kinase, and Ca2+ influx

Little is known of the mechanisms leading to mitogen-activated protein kinase (MAPK) activation via Gq-coupled receptors. We therefore examined the pathways by which angiotensin II (Ang II) activates Raf-1 kinase, an upstream intermediate in the pathway to MAPK, via the Gq-coupled AT1 angiotensin receptor in bovine adrenal glomerulosa (BAG) cells. Ang II caused a rapid and transient activation of Raf-1 that reached a peak at 5-10 min. Ang II was a potent stimulus of Raf-1 activation with an ED50 of 10 pM and a maximal response at 1 nM, although higher Ang II concentrations elicited a submaximal response. Ang II-stimulated Raf-1 activity was unaffected by down-regulation of protein kinase C and intracellular Ca2+ chelation (using BAPTA) but was partially inhibited by pertussis toxin, and was abolished by manumycin A. Removal of extracellular Ca2+ (by EGTA) or blockade of L type Ca2+ channels (by nifedipine), as well as inhibition of MEK-1 kinase (by PD98059), enhanced Raf-1 activity, whereas wortmannin (100 nM) inhibited approximately one half of Ang II-stimulated Raf-1 activity. Hence, Raf-1 kinase activation by Ang II in BAG cells is dependent on Ras, is mediated in part via Gi and phosphatidylinositol 3-kinase, and is negatively regulated via Ca2+ influx and a downstream signaling element(s).