Heat Shock Activates c-Src Tyrosine Kinases and Phosphatidylinositol 3-Kinase in NIH3T3 Fibroblasts*
- From the Veterans Affairs Palo Alto Health Care System and Geriatrics Research, Education and Clinical Center, Palo Alto, California 94304 and Department of Medicine, Stanford University, Stanford, California 94305
Abstract
There is increasing evidence that cellular responses to stress are in part regulated by protein kinases, although specific mechanisms are not well defined. The purpose of these experiments was to investigate potential upstream signaling events activated during heat shock in NIH3T3 fibroblasts. Experiments were designed to ask whether heat shock activates p60 c-Src tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Using in vitro protein kinase activity assays, it was demonstrated that heat shock stimulates c-Src and PI 3-kinase activity in a time-dependent manner. Also, there was increased PI 3-kinase activity in anti-phosphotyrosine and anti-c-Src immunoprecipitated immunocomplexes from heated cells. Heat shock activated mitogen-activated protein kinase (MAPK) and p70 S6 kinase (S6K) in these cells. The role of PI 3-kinase in regulating heat shock activation of MAPK and p70 S6K was investigated using wortmannin, a specific pharmacological inhibitor of PI 3-kinase. The results demonstrated that wortmannin inhibited heat shock activation of p70 S6K but only partially inhibited heat activation of MAPK. A dominant negative Raf mutant inhibited activation of MAPK by heat shock but did not inhibit heat shock stimulation of p70 S6K. Genistein, a tyrosine kinase inhibitor, and suramin, a growth factor receptor inhibitor, both inhibited heat shock stimulation of MAPK activity and tyrosine phosphorylation of MAPK. Furthermore, a selective epidermal growth factor receptor (EGFR) inhibitor, tryphostin AG1478, and a dominant negative EGFR mutant also inhibited heat shock activation of MAPK. Heat shock induced EGFR phosphorylation. These results suggest that early upstream signaling events in response to heat stress may involve activation of PI 3-kinase and tyrosine kinases, such as c-Src, and a growth factor receptor, such as EGFR; activation of important downstream pathways, such as MAPK and p70 S6K, occur by divergent signaling mechanisms similar to growth factor stimulation.
Footnotes
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↵* This work was supported in part by National Institute of Health Grant HL41315.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ R. Z. Lin was supported by a Pharmaceutical Research and Manufacturers of America Foundation Fellowship for Careers in Clinical Pharmacology. Present address: Department of Pharmacology, University of Texas Health Sciences Center, San Antonio, TX. 78284.
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↵§ To whom correspondence should be addressed: Veterans Affairs Medical Center, GRECC 182B, 3801 Miranda Ave., Palo Alto, CA 94304. Tel.: 415-858-3933; Fax: 415-855-9437; E-mail: bbh{at}icon.palo-alto.med.va.gov.
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↵1 The abbreviations used are: MAPK, mitogen-activated protein kinase; hsp, heat shock protein; MAPKAPK-2, MAPK activated protein kinase-2; MBP, myelin basic protein; PI 3-kinase, phosphatidylinositol 3-kinase; S6K, S6 kinase; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; EGF, epidermal growth factor; EGFR, EGF receptor; HSF, heat shock transcription factor.
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- Received March 26, 1997.
- Revision received September 12, 1997.
