Somatostatin receptor 1 (SSTR1)-mediated inhibition of cell proliferation correlates with the activation of the MAP kinase cascade: role of the phosphotyrosine phosphatase SHP-2.

The mitogen activated protein (MAP) kinase cascade represents one of the major regulator of cell growth by hormones and growth factors. However, although the activation of this intracellular pathway has been often regarded as mediator of cell proliferation, in many cell types the increase in MAP kinase (also called extra-cellular signal regulated kinase: ERK) activity may result in cell growth arrest, depending on the length or the intensity of the stimulation. In this review we examine recent data concerning the effects of somatostatin on the MAP kinase cascade through one of its major receptor subtype, the somatostatin receptor 1 (SSTR1), stably expressed in CHO-K1 cells. Somatostatin inhibits the proliferative effects of basic FGF (bFGF) in CHO-SSTR1 cell line. However, in these cells, somatostatin robustly activates the MAP kinase and augments bFGF-induced stimulation of ERK. We show that the activation of ERK via SSTR1 is mediated by the betagamma subunit of a pertussis toxin-sensitive G-protein and requires both the small G protein Ras and the serine/threonine kinase Raf-1. Moreover the phosphatidyl inositol-3kinase and the cytosolic tyrosine kinase c-src participate in the signal transduction regulated by SSTRI to activate ERK, as well as it is involved the protein tyrosine phosphatase (PTP) SHP-2. Previous studies have suggested that somatostatin-stimulated PTP activity mediates the growth inhibitory actions of somatostatin, in CHO-SSTR1 cells. Thus, the activation of SHP-2 by SSTR1 may mediate the antiproliferative activity of somatostatin. SHP-2 may. in turn, regulate the activity of kinases upstream of ERK that require tyrosine dephosphorylation to be activated, such as c-src. Finally, the synergism between somatostatin and bFGF in the activation of ERK results in an increased expression of the cyclin-dependent kinase inhibitor p21cip/WAF1 as molecular effector of the antiproliferative activity of somatostatin.     

GAP-43 Augmentation of G Protein-Mediated Signal Transduction Is Regulated by Both Phosphorylation and Palmitoylation

Abstract: The neuronal protein GAP-43 is concentrated at the growth cone membrane, where it is thought to amplify the signal transduction process. As a model for its neuronal effects, GAP-43 protein injection into Xenopus laevis oocytes strongly augments the calcium-sensitive chloride current evoked by the G protein-coupled receptor stimulation. We have now examined a series of GAP-43 mutants in this system and determined those regions of GAP-43 required for this increase in current flux. As expected, palmitoylation inhibits signal amplification in oocytes by blocking G protein activation. Unexpectedly, a second domain of GAP-43 (residues 35–50) containing a protein kinase C phosphorylation site at residue 41 is also necessary for augmentation of G protein-coupled signals in oocytes. This region is not required for activation of isolated G_o but is necessary for GAP-43 binding to isolated calmodulin and to isolated protein kinase C. Substitution of Asp for Ser⁴¹ inactivates GAP-43 as a signal facilitator in oocytes. This mutation blocks GAP-43 binding to both protein kinase C and calmodulin. Thus, GAP-43 regulates an oocyte signaling cascade via coordinated, simultaneous G protein activation and interaction with either calmodulin or protein kinase C.     

Extracellular ATP stimulates an inhibitory pathway towards growth factor-induced cRaf-1 and MEKK activation in astrocyte cultures

ATP, acting via P2Y, G protein-coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor-2 (FGF-2)-induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF-2 cotreatment on the main components of the extracellular-signal regulated protein kinase (ERK) cascade, cRaf-1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf-1 by FGF-2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf-1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf-1 by other growth factors, epidermal growth factor and platelet-derived growth factor, as well as other MEK1 activators stimulated by FGF-2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF-2-induced cRaf-1 activation, thereby indicating specificity in the ATP-induced inhibitory cross-talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor-induced activation of cRaf-1 and MEKKS: Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y-GPCRS:     

Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3

The c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) is activated in response to the treatment of cells with inflammatory cytokines and by exposure to environmental stress. JNK activation is mediated by a protein kinase cascade composed of a MAPK kinase and a MAPK kinase kinase. Here we describe the molecular cloning of a putative molecular scaffold protein, JIP3, that binds the protein kinase components of a JNK signaling module and facilitates JNK activation in cultured cells. JIP3 is expressed in the brain and at lower levels in the heart and other tissues. Immunofluorescence analysis demonstrated that JIP3 was present in the cytoplasm and accumulated in the growth cones of developing neurites. JIP3 is a member of a novel class of putative MAPK scaffold proteins that may regulate signal transduction by the JNK pathway.     

Regulation of MAP kinase-dependent apoptotic pathway: implication of reactive oxygen and nitrogen species

Mitogen-activated protein (MAP) kinase signaling cascades are multi-functional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase that triggers apoptogenic kinase cascade leading to the phosphorylation/activation of c-Jun N-terminal kinases and p38-MAP kinase, which are responsible for inducing apoptotic cell death. This pathway plays a pivotal role in transduction of signals from different apoptotic stimuli. In the present review, we summarized the recent evidence concerning MAP kinase-dependent apoptotic pathway and its regulation in the mammalian cells and organism in vivo. We have shown that the key messengers of regulation of this pathway are the reactive oxygen and nitrogen species. The role of protein oxidation and S-nitrosation in induction of apoptotic cell death via ASK1 is discussed. Also we have outlined other recently discovered signal transduction processes involved in the regulation of ASK1 activity and downstream pathway.     

Na+/K+/Cl- cotransporter activates MAP-kinase cascade downstream to protein kinase C, and upstream to MEK

In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl- cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl- cotransporter, is downstream of tyrosine kinase receptors, as well as of the G-protein-coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl- cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.     

Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice

The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery.     

Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.     

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.     

p73 cooperates with Ras in the activation of MAP kinase signaling cascade

The p73 gene is capable of inducing cell cycle arrest, apoptosis, senescence, differentiation and to cooperate with oncogenic Ras in cellular transformation. Ras can be considered as a branch point in signal transduction, where diverse extracellular stimuli converge. The intensity of the mitogen-activated protein kinase (MAPK) cascade activation influences the cellular response to Ras. Despite the fundamental role of p53 in Ras-induced growth arrest and senescence, it remains unclear how the Ras/MEK/ERK pathway induces growth arrest in the absence of p53. We report here that oncogenic Ras stabilizes p73 resulting in p73 accumulation and enhancement of its activity. p73, in turn, induces a sustained activation of the MAP kinase cascade synergizing with oncogenic Ras. We also found that inhibition of p73 function modifies the cellular outcome to Ras activation inhibiting Ras-dependent differentiation. Here, we show for the first time that there is a signaling loop between Ras-dependent MAPK cascade activation and p73 function.     

Hematopoietic progenitor kinase 1 is a component of transforming growth factor beta-induced c-Jun N-terminal kinase signaling cascade

The c-Jun N-terminal kinase (JNK) signaling pathway is involved in transforming growth factor beta (TGF-beta) signaling in a variety of cell systems. We report here that hematopoietic progenitor kinase 1 (HPK1), a novel Ste20-like protein serine/threonine kinase, serves as an upstream mediator for the TGF-beta-activated JNK1 cascade in 293T cells. TGF-beta treatment resulted in a time-dependent activation of HPK1, which was accompanied by similar kinetics of JNK1 activation. The activation of JNK1 by TGF-beta was abrogated by a kinase-defective HPK1 mutant but not by a kinase-defective mutant of kinase homologous to Ste20/Sps1. This result indicates that HPK1 is specifically required for TGF-beta-induced activation of JNK1. We also found that TGF-beta-induced JNK1 activation was blocked by a kinase-defective mutant of TGF-beta-activated kinase 1 (TAK1). In addition, interaction between HPK1 and TAK1 was observed in transient transfection assays, and this interaction was enhanced by TGF-beta treatment. Both stress-activated protein kinase/extracellular signal-regulated kinase kinase (SEK) and mitogen-activated protein kinase kinase 7 (MKK7) are immediate upstream activators of JNK1. Although SEK and MKK7 acted downstream of TAK1, only a kinase-defective SEK mutant blocked TGF-beta-induced activation of JNK1, indicating that the TGF-beta signal is relayed solely through SEK, but not MKK7, in vivo. Furthermore, TGF-beta-induced activating protein 1 activation was blocked by a HPK1 mutant, as well as by TAK1 and SEK mutants. Taken together, these studies establish a potential cascade of TGF-beta-activated interacting kinases beginning with HPK1, a Ste20 homolog, and ending in JNK1 activation: HPK1 --> TAK1 --> SEK --> JNK1.     

ERK mediates effects of glycated albumin in mesangial cells

The alterations in glomerular cell biology induced by glycated albumin resemble those caused by high ambient glucose, but are operative in physiologic (5.5 mM) glucose concentration. Recently, high glucose has been shown to activate extracellular signal-related kinase (ERK) in mesangial cells, but whether the mitogen-activated protein kinase (MAPK) cascade participates in signal transduction triggered by glycated albumin is unknown. Using a specific inhibitor of MAPK/ERK kinase, we demonstrate for the first time that activation of ERK is required for the inhibition of cell growth and enhanced elaboration of extracellular matrix protein provoked by glycated albumin. These findings indicate that the MAPK/ERK pathway mediates biologic activities of this glycated protein.     

A Highlights from MBoC Selection: Combined computational and experimental analysis reveals mitogen-activated protein kinase–mediated feedback phosphorylation as a mechanism for signaling specificity

Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity.     

Activation of extracellular signal-regulated kinases (ERK 44/42) by chlorpyrifos oxon in Chinese hamster ovary cells

Acetylcholinesterase inhibition explains most but not all of the toxicological manifestations of exposure to the major organophosphorus insecticide chlorpyrifos (CP) and its metabolically activated form chlorpyrifos oxon (CPO); CPO is also reported to interact with muscarinic acetylcholine receptors and alter secondary messenger status. We find that CP and CPO activate extracellular signal-regulated kinases (ERK 44/42) in both wild-type (CHOK1) and human muscarinic receptor-expressing Chinese hamster ovary cells (CHO-M2). The degree of ERK 44/42 activation on treatment with 50 microM CPO for 40 minutes is 2- to 3-fold compared with control cells and is both concentration- and time-dependent. CP is at least 2-fold less potent than CPO as an activator of ERK 44/42 and the hydrolysis products 3,5,6-trichloropyridinol and diethyl phosphate are not activators. ERK 44/42 activation by CPO is insensitive to the protein kinase A inhibitor H-89, but is completely abolished by the phosphatidylinositol 3-kinase (P13-K) inhibitor wortmannin, the protein kinase C (PKC) inhibitor GF-109203X, and the mitogen-activated extracellular signal-regulated protein kinase kinase (MEK) inhibitor PD 098059. Therefore, CPO activates the ERK 44/42 signaling cascade in CHOK1 cells via a pathway dependent on P13-K, PKC, and MEK but not requiring PKA or the human M2 muscarinic receptor. In summary we find that CPO activates a mammalian signal transduction cascade involved in cell growth and differentiation. This occurs through a pathway common to growth factors and mitogens, consistent with a receptor-mediated event. However, CPO may also inhibit an enzyme involved in signal transduction. The specific target of CPO leading to the activation of ERK 44/42 and the potential effects of this activation on cell function remain to be determined.     

Activation and targeting of mitogen-activated protein kinases by G-protein-coupled receptors

Over the past decade, it has become apparent that many G-protein-coupled receptors (GPCRs) generate signals that control cellular differentiation and growth, including stimulation of Ras family GTPases and activation of mitogen-activated protein (MAP) kinase pathways. The mechanisms that GPCRs use to control the activity of MAP kinases vary between receptor and cell type but fall broadly into one of three categories: signals initiated by classical G protein effectors, e.g., protein kinase (PK)A and PKC, signals initiated by cross-talk between GPCRs and classical receptor tyrosine kinases, e.g., "transactivation" of epidermal growth factor (EGF) receptors, and signals initiated by direct interaction between beta-arrestins and components of the MAP kinase cascade, e.g., beta-arrestin "scaffolds". While each of these pathways results in increased cellular MAP kinase activity, emerging data suggest that they are not functionally redundant. MAP kinase activation occurring via PKC-dependent pathways and EGF receptor transactivation leads to nuclear translocation of the kinase and stimulates cell proliferation, while MAP kinase activation via beta-arrestin scaffolds primarily increases cytosolic kinase activity. By controlling the spatial and temporal distribution of MAP kinase activity within the cell, the consequences of GPCR-stimulated MAP kinase activation may be determined by the mechanism by which they are activated.     

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.