Growth Factor-Stimulated Phosphorylation Cascades: Activation of Growth Factor-Stimulated Map Kinase

Abstract Background Heat shock protein 22 (HSP22) belongs to class I of the small HSP family that displays ubiquitous expression in osteoblasts. We previously demonstrated that prostaglandin F2α (PGF2α), a potent bone remodeling factor, induces the synthesis of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether HSP22 is implicated in the PGF2α-induced synthesis of IL-6 and VEGF and the mechanism of MC3T3-E1 cells. Methods MC3T3-E1 cells were transfected with HSP22-siRNA. IL-6 and VEGF release was assessed by ELISA. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was detected by Western blotting. Results The PGF2α-induced release of IL-6 in HSP22 knockdown cells was significantly suppressed compared with that in the control cells. HSP22 knockdown also reduced the VEGF release by PGF2α. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was attenuated by HSP22 downregulation. Conclusions Our results strongly suggest that HSP22 acts as a positive regulator in the PGF2α-induced synthesis of IL-6 and VEGF in osteoblasts.

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Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors.

The Journal of Cell Biology ◽

10.1083/jcb.135.6.1633 ◽

1996 ◽

Vol 135 (6) ◽

pp. 1633-1642 ◽

Cited By ~ 536

Author(s):

S Miyamoto ◽

H Teramoto ◽

J S Gutkind ◽

K M Yamada

Keyword(s):

Signal Transduction ◽

Growth Factors ◽

Growth Factor ◽

Map Kinase ◽

Tyrosine Kinases ◽

Map Kinases ◽

Egf Receptor ◽

Growth Factor Receptor ◽

Kinase Activation ◽

Transient Activation

Integrins mediate cell adhesion, migration, and a variety of signal transduction events. These integrin actions can overlap or even synergize with those of growth factors. We examined for mechanisms of collaboration or synergy between integrins and growth factors involving MAP kinases, which regulate many cellular functions. In cooperation with integrins, the growth factors EGF, PDGF-BB, and basic FGF each produced a marked, transient activation of the ERK (extracellular signal-regulated kinase) class of MAP kinase, but only if the integrins were both aggregated and occupied by ligand. Transmembrane accumulation of total tyrosine-phosphorylated proteins, as well as nonsynergistic MAP kinase activation, could be induced by simple integrin aggregation, whereas enhanced transient accumulation of the EGF-receptor substrate eps8 required integrin aggregation and occupancy, as well as EGF treatment. Each type of growth factor receptor was itself induced to aggregate transiently by integrin ligand-coated beads in a process requiring both aggregation and occupancy of integrin receptors, but not the presence of growth factor ligand. Synergism was also observed between integrins and growth factors for triggering tyrosine phosphorylation of EGF, PDGF, and FGF receptors. This collaborative response also required both integrin aggregation and occupancy. These studies identify mechanisms in the signal transduction response to integrins and growth factors that require various combinations of integrin aggregation and ligands for integrin or growth factor receptors, providing opportunities for collaboration between these major regulatory systems.

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Insulin Induces Heterologous Desensitization of G Protein-Coupled Receptor and Insulin-Like Growth Factor I Signaling by Downregulating β-Arrestin-1

Molecular and Cellular Biology ◽

10.1128/mcb.22.17.6272-6285.2002 ◽

2002 ◽

Vol 22 (17) ◽

pp. 6272-6285 ◽

Cited By ~ 60

Author(s):

Stéphane Dalle ◽

Takeshi Imamura ◽

David W. Rose ◽

Dorothy Sears Worrall ◽

Satoshi Ugi ◽

...

Keyword(s):

Growth Factor ◽

Map Kinase ◽

Insulin Treatment ◽

Ectopic Expression ◽

Wild Type ◽

Factor I ◽

Igf I ◽

Heterologous Desensitization ◽

Kinase Phosphorylation ◽

G Protein Coupled

ABSTRACT β-Arrestin-1 mediates agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCRs) and is also essential for GPCR mitogenic signaling. In addition, insulin-like growth factor I receptor (IGF-IR) endocytosis is facilitated by β-arrestin-1, and internalization is necessary for IGF-I-stimulated mitogen-activated protein (MAP) kinase activation. Here, we report that treatment of cells for 12 h with insulin (100 ng/ml) induces an ∼50% decrease in cellular β-arrestin-1 content due to ubiquitination of β-arrestin-1 and proteosome-mediated degradation. This insulin-induced decrease in β-arrestin-1 content was blocked by inhibition of phosphatidylinositol-3 kinase (PI-3 kinase) and MEK with wortmannin and PD98059, respectively. We also found a marked decrease in the association of β-arrestin-1 with the IGF-IR and a 55% inhibition of IGF-I-stimulated MAP kinase phosphorylation. In insulin-treated, β-arrestin-1-downregulated cells, there was complete inhibition of lysophosphatidic acid (LPA) or isoproterenol (ISO)-stimulated MAP kinase phosphorylation. This was associated with a decrease in β-arrestin-1 association with the β2-AR as well as a decrease in β-arrestin-1-Src and Src-β2-AR association. Ectopic expression of wild-type β-arrestin-1 in insulin-treated cells in which endogenous β-arrestin-1 had been downregulated rescued IGF-I- and LPA-stimulated MAP kinase phosphorylation. In conclusion, we found the following. (i) Chronic insulin treatment leads to enhanced β-arrestin-1 degradation. (ii) This downregulation of endogenous β-arrestin-1 is associated with decreased IGF-I-, LPA-, and ISO-mediated MAP kinase signaling, which can be rescued by ectopic expression of wild-type β-arrestin-1. (iii) Finally, these results describe a novel mechanism for heterologous desensitization, whereby insulin treatment can impair GPCR signaling, and highlight the importance of β-arrestin-1 as a target molecule for this desensitization mechanism.

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Cytosolic phospholipase A2 is activated by the hepatocyte growth factor receptor-kinase in Madin Darby canine kidney cells

Journal of Cell Science ◽

10.1242/jcs.110.14.1655 ◽

1997 ◽

Vol 110 (14) ◽

pp. 1655-1663

Author(s):

G.G. Skouteris ◽

C.H. Schroder

Keyword(s):

Arachidonic Acid ◽

Growth Factor ◽

Tyrosine Kinase ◽

Map Kinase ◽

Kinase Activity ◽

Mdck Cells ◽

Tyrosine Kinase Activity ◽

Cytosolic Phospholipase ◽

Acid Release ◽

Hepatocyte Growth

The hepatocyte growth factor/scatter factor (HGF/SF) receptor which is a transmembrane protein encoded by the Met oncogene, possesses intrinsic tyrosine kinase activity which transduces the mitogenic, morphogenic and the scattering effect of HGF/SF. The pluripotent signal of HGF/SF is transduced through association of the Met receptor with various intracellular adaptors. Phosphorylation of cytosolic phospholipase A2 (cPLA2) is associated with activation of this molecule which in turn leads to arachidonic acid production followed by release of prostaglandins and related compounds exerting their roles onto cell proliferation, chemotaxis and vascular motility. Arachidonic acid and its metabolites were shown to be involved in processes like liver regeneration where growth factor receptors possessing tyrosine kinase activity are implicated. In this study we examined whether stimulation of the HGF/SF-receptor's tyrosine kinase activity would involve changes in the phosphorylation state and the activity of cPLA2 in MDCK cells, where HGF/SF is known to induce scattering responses rather than mitogenesis. The activated p145betaMET was shown to associate with and to phosphorylate cPLA2 on tyrosine residues, this leading to subsequent release of arachidonic acid. cPLA2 was also phosphorylated in serine residues and such a role has been so far assigned to the mitogen activated protein (MAP) kinase. Our data have also shown that MAP kinase is associated and phosphorylated on tyrosine by the activated p145betaMET. Immunodepletion of MAP kinase via electroporation of an anti-MAP kinase antibody, did not significantly decrease arachidonic acid release in HGF/SF-stimulated MDCK cells. It is therefore emerging that phosphorylation of cPLA2 on tyrosine by the HGF/SF receptor kinase is capable of triggering arachidonic acid release and that MAP kinase is contributing to full, but does not drive, the activity of cPLA2. The release of arachidonic acid by MDCK cells following HGF/SF stimulation is establishing this fatty acid and its metabolites as major components involved in the transduction of MET-driven signals and at the same time in the amplification of such signals.

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EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway

Journal of Cell Science ◽

10.1242/jcs.111.5.615 ◽

1998 ◽

Vol 111 (5) ◽

pp. 615-624 ◽

Cited By ~ 8

Author(s):

H. Xie ◽

M.A. Pallero ◽

K. Gupta ◽

P. Chang ◽

M.F. Ware ◽

...

Keyword(s):

Growth Factor ◽

Cell Motility ◽

Map Kinase ◽

Signaling Pathway ◽

Focal Adhesion ◽

Kinase Activity ◽

Egf Receptor ◽

Focal Adhesions ◽

Short Term ◽

Egfr Kinase

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

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Involvement of Ras/MAP Kinase in the Regulation of Ca2+ Channels in Adult Bullfrog Sympathetic Neurons by Nerve Growth Factor

Journal of Neurophysiology ◽

10.1152/jn.1998.80.3.1352 ◽

1998 ◽

Vol 80 (3) ◽

pp. 1352-1361 ◽

Cited By ~ 21

Author(s):

Saobo Lei ◽

William F. Dryden ◽

Peter A. Smith

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Protein Kinase ◽

Map Kinase ◽

Kinase Inhibitors ◽

Sympathetic Neurons ◽

Mitogen Activated Protein Kinase ◽

Nerve Growth ◽

Channel Current ◽

Mitogen Activated Protein

Lei, Saobo, William F. Dryden, and Peter A. Smith. Involvement of Ras/MAP kinase in the regulation of Ca2+ channels in adult bullfrog sympathetic neurons by nerve growth factor. J. Neurophysiol. 80: 1352–1361, 1998. The cellular mechanisms that underlie nerve growth factor (NGF) induced increase in Ca2+-channel current in adult bullfrog sympathetic B-neurons were examined by whole cell recording techniques. Cells were maintained at low density in neuron-enriched, defined-medium, serum-free tissue culture for 6 days in the presence or absence of NGF (200 ng/ml). The increase in Ba2+ current ( I Ba) density induced by NGF was attenuated by the RNA synthesis inhibitor cordycepin (20 μM), by the DNA transcription inhibitor actinomycin D (0.01 μg/ml), by inhibitors of Ras isoprenylation (perillic acid 0.1–1.0 mM or α-hydroxyfarnesylphosphonic acid 10–100 μM), by tyrosine kinase inhibitors genistein (20 μM) or lavendustin A (1 μM), and by PD98059 (10–100 μM), an inhibitor of mitogen-activated protein kinase kinase. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) pathway (wortmannin, 100 nM, or LY29400, 100 μM) were ineffective as were inhibitors of phospholipase Cγ (U73122 or neomycin, both 100 μM). The effect of NGF persisted in Ca2+-free medium that contained 1.8 mM Mg2+ and 2 mM ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid. It was mimicked by a Trk antibody that was capable of inducing neurite outgrowth in explant cultures of bullfrog sympathetic ganglion. Antibodies raised against the low-affinity p75 neurotrophin receptor were ineffective in blocking the effect of NGF on I Ba. These results suggest that NGF-induced increase in Ca2+ channel current in adult sympathetic neurons results, at least in part, from new channel synthesis after Trk activation of Ras and mitogen activated protein kinase by a mechanism that is independent of extracellular Ca2+.

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