Mitogen-activated Protein Kinase In Response Research Articles

A Synthetic Peptide Derived from a COOH-terminal Domain of the Insulin Receptor Specifically Enhances Insulin Receptor Signaling

The role of the insulin receptor COOH-terminal domain in the regulation of insulin signal transduction was explored with a variety of synthetic peptides. One of the peptides, termed peptide HC, whose structure corresponds to residues 1293-1307 of the insulin proreceptor sequence, enhanced insulin-stimulated autophosphorylation of the insulin receptor in cell-free systems and in semipermeabilized Chinese hamster ovary (CHO) cells that had been transfected with an expression plasmid encoding the human insulin receptor (CHO/HIRc) at concentrations where there was no detectable effect on basal autophosphorylation levels or on receptor dephosphorylation. A lipophilic analogue of peptide HC, stearyl peptide HC, added to intact CHO/HIRc cells enhanced significantly insulin-stimulated insulin receptor autophosphorylation while having no effect on ligand-stimulated receptor phosphorylation in CHO cells overexpressing either the IGF-1 receptor or epidermal growth factor receptor. Addition of stearyl peptide HC to CHO/HIRc cells resulted in a 2.4 +/- 0.3-fold increase in the amount of insulin-stimulated phosphatidylinositol 3-kinase detected in anti-IRS-1 immunoprecipitates and a 2.1 +/- 0.6-fold increase in the levels of tyrosine phosphorylation of mitogen-activated protein kinase in response to insulin. Finally, a derivative of peptide HC coupled to a biotin moiety was prepared and showed to bind with the beta-subunit of the wild-type insulin receptor and a truncated receptor that lacks 43 amino acids from its carboxyl terminus. However, there was little binding, if any, of the peptide with the IGF-1 receptors or the epidermal growth factor receptors. Taken together, our data demonstrate that a pentadecapeptide related to the carboxyl terminus of the insulin receptor binds to the insulin receptor beta-subunit and that this interaction may contribute to the increased receptor's intrinsic activity and signal transduction.

Involvement of stress-activated protein kinase and p38 mitogen-activated protein kinase in mIgM-induced apoptosis of human B lymphocytes.

Despite intensive efforts, the intracellular signaling pathways that mediate apoptosis remain unclear. The human B lymphoma cell line, B104, possesses characteristics that make it an attractive model for analysis of receptor-mediated apoptosis. Although these cells express both membrane IgM (mIgM) and membrane IgD (mIgD) crosslinking mIgM results in significant apoptosis while crosslinking mIgD does not. Our results show that crosslinking mIgM but not mIgD induced a delayed and sustained activation of the mitogen-activated protein kinase (MAPK) family members stress-activated protein kinase (SAPK) and p38 MAPK. The calcium ionophore ionomycin, which also induces apoptosis in B104 cells, stimulated a similar SAPK and p38 MAPK response. Cyclosporin A, a potent inhibitor of apoptosis induced by either mIgM or ionomycin, inhibited activation of both SAPK and p38 MAPK, suggesting that stimulation of these kinases may be required for induction of apoptosis. Collectively, our results indicate that SAPK and p38 MAPK may be downstream targets during mIgM-induced, calcium-mediated, apoptosis in human B lymphocytes.

5-Oxo-eicosanoids and Hematopoietic Cytokines Cooperate in Stimulating Neutrophil Function and the Mitogen-activated Protein Kinase Pathway

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B4 (LTB4). All three eicosanoids stimulate Ca2+ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB4, 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB4, GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA2, as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [14C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca2+ depletion, inhibited MAPK responses to 5-oxoETE and LTB4, indicating that responses to both agents are coupled through G proteins but not dependent upon Ca2+ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3- and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB4 antagonist, blocked the responses to LTB4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB4. The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation.

CAMP-mediated Growth Inhibition in Fibroblasts Is Not Mediated via Mitogen-activated Protein (MAP) Kinase (ERK) Inhibition: cAMP-DEPENDENT PROTEIN KINASE INDUCES A TEMPORAL SHIFT IN GROWTH FACTOR-STIMULATED MAP KINASES

Growth factors stimulate fibroblast cell division by activating the recently identified mitogen-activated protein kinase (MAP kinase) signaling cascade. In contrast to our previous work (Kahan, K., Seuwen, K., Meloche, S. and Pouysségur, J. (1992) J. Biol. Chem. 267, 13369-13375), several reports have suggested that an elevation in intracellular cAMP blocks cell proliferation by attenuating MAP kinase activation. Hence we re-examined the effect of a long term increase in intracellular cAMP and therefore cAMP-dependent protein kinase (PKA) activation on the MAP kinase cascade in CCL39 fibroblasts. The concomitant addition of cAMP-elevating agents prostaglandin E, (PGE1) and IBMX did not inhibit the mitogen-mediated activation of p44 MAP kinase. However, a 5-min PGE1/IBMX pretreatment abolished the MAP kinase response, in a manner correlating with the extent of PKA activity. This inhibition was temporal in nature, and while modifying the time course of growth factor-mediated p44 MAP kinase, activation did not diminish the magnitude of the response. Thus the major peak of MAP kinase activity normally present 5 min after alpha-thrombin addition was now evident at 10 min in the presence of PGE1/IBMX. CCL39 cell proliferation is inhibited by elevated cAMP levels. Such an inhibition could reflect either a reduction in the number of cells entering the cell cycle or a delay in the time required to go through the cycle. Bromodeoxyuridine labeling experiments revealed that the cAMP-mediated inhibition of DNA synthesis in CCL39 cells was not due to a delay in S phase entry, but was due to a reduction in the number of cells entering S phase. Thus we conclude that although PKA activation may slightly modify the time course of MAP kinase activation in response to mitogens in CCL39 cells, the PKA-mediated inhibition of cell division occurs through modulation of an intracellular target, distinct from the p42/p44 MAP kinase cascade.

Dual Effect of β-Adrenergic Receptors on Mitogen-activated Protein Kinase: EVIDENCE FOR A βγ-DEPENDENT ACTIVATION AND A Gαs-cAMP-MEDIATED INHIBITION

The enzymatic activity of mitogen-activated protein kinases (MAP kinases) increases in response to agents acting on a variety of cell surface receptors, including receptors linked to heterotrimeric G proteins of the Gi and Gq family. Recently, it has been shown that stimulation of beta-adrenergic receptors, which are typical of those that act through Gs to activate adenylyl cyclases, potently activates MAP kinases in the heart, resulting in the hypertrophy of the cardiac muscle (Lazou, A., Bogoyevitch, M.A., Clerk, A., Fuller, S.J., Marshall, C.J., and Sudgen, P.H. (1994) Circ. Res. 75, 938-941). We have observed that exposure of COS-7 cells to a beta-adrenergic agonist, isoproterenol, raises intracellular levels of cAMP and effectively activates protein kinase A (PKA) and an epitope-tagged MAP kinase. However, MAP kinase stimulation by isoproterenol was neither mimicked by expression of an activated mutant of G alpha s, nor by treatment with PKA-stimulating agents. Moreover, pretreatment of COS-7 with a permeable cAMP analog, 8-Br-cAMP, markedly decreased MAP kinase activation by either isoproterenol or epidermal growth factor. Thus, in COS-7 cells cAMP and PKA do not appear to mediate MAP kinase activation by beta-adrenergic receptors. Signaling from beta-adrenergic receptors to MAP kinase was inhibited by transfection of a chimeric molecule consisting of the CD8 receptor and the carboxyl terminus of the beta-adrenergic receptor kinase, which includes the beta gamma-binding domain. MAP kinase activation by isoproterenol was not affected by depletion of protein kinase C, but it was completely abolished by expression of Ras-inhibiting molecules. We conclude that signaling from beta-adrenergic receptors to MAP kinase involves an activating signal mediated by beta gamma subunits acting on a Ras-dependent pathway and a G alpha s-induced inhibitory signal mediated by cAMP and PKA. The balance between these two opposing mechanisms of regulation would be expected to control the MAP kinase response to beta-adrenergic agonists as well as to other biologically active agents known to act on Gs coupled receptors, including a number of hormones, neurotransmitters, and lipid mediators.

Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line.

Differential activation of mitogen-activated protein kinase in response to basic fibroblast growth factor in skeletal muscle cells.

In the MM14 mouse myoblast cell line, fibroblast growth factor (FGF) stimulates proliferation and represses differentiation. However, the intracellular signaling pathways used by FGF to affect these cellular processes are unknown. The predominant FGF receptor present on MM14 cells, FGFR1, is a receptor tyrosine kinase capable of activating the mitogen-activated protein kinase (MAPK) cascade in fibroblast and neuronal cell lines. To determine whether the FGF signal is mediated via the MAPK cascade in myoblasts, MM14 cells were stimulated with basic FGF (bFGF) and activities of the various kinases were measured. After withdrawal from serum and bFGF for 3 hr, bFGF stimulated MAPK kinase (MAPKK) activity, but MAPK and S6 peptide kinase activities were not detected. In contrast, when serum and bFGF were withdrawn for 10 hr, the activities of MAPKK, MAPK, and S6 peptide kinase were all stimulated by bFGF treatment. The inability of bFGF to stimulate MAPK after 3 hr of withdrawal may be due, in part, to the presence of a MAPK phosphatase activity that was detected in MM14 cell extracts. This dephosphorylating activity diminishes during commitment to terminal differentiation and is inhibited by sodium orthovanadate. Thus, the ability of bFGF to stimulate MAPK in MM14 cells is correlated with the loss of a MAPK phosphatase activity. These results show that although bFGF activates MAPKK in proliferating myoblasts, the mitogenic signal does not progress to the downstream kinases, providing a physiological example of an uncoupling of the MAPK cascade.

Regulation of endothelin-1- and lysophosphatidic acid-stimulated tyrosine phosphorylation of focal adhesion kinase (pp125fak) in Rat-1 fibroblasts.

The characteristics of protein tyrosine phosphorylation were examined in Rat-1 fibroblasts in response to endothelin-1 (ET-1) and 1-oleoyl-lysophosphatidic acid (LPA). Both agonists stimulated the biphasic tyrosine phosphorylation of at least three major proteins of approx. 120 kDa (pp116, pp120 and pp130) and two of 80 kDa (pp80 and pp70). Immunoprecipitation experiments indicated that the pp120 protein corresponded to the recently described focal adhesion protein kinase pp125fak. Phorbol 12-myristate 13-acetate, alone or in combination with the calcium ionophore A23187, also stimulated the phosphorylation of pp125fak but to a smaller extent than LPA or ET-1. Removal of both extracellular and intracellular Ca2+ did not significantly reduce LPA- and ET-1-stimulated tyrosine phosphorylation of pp125fak. In cells where protein kinase C activity was down-regulated or inhibited, ET-1-stimulated tyrosine phosphorylation of pp125fak was reduced to a greater extent than phosphorylation in response to LPA. In addition, ET-1-stimulated tyrosine phosphorylation of pp80 was decreased by 50-70% in response to protein kinase C inhibition at both 2 and 60 min whereas LPA-stimulated tyrosine phosphorylation of this protein was only reduced at 2 min. Pretreatment with pertussis toxin reduced the tyrosine phosphorylation of pp42 and pp44 forms of mitogen-activated protein kinase in response to both ET-1 and LPA but reduced the tyrosine phosphorylation of pp125fak only in response to LPA. These results indicate agonist-specific differences in the regulation of pathways mediating the tyrosine phosphorylation of pp125fak and other target proteins.