HEAT-SHOCK ACTIVATES C-SRC TYROSINE KINASES AND PHOSPHATIDYLINOSITOL 3-KINASE IN NIH3T3 FIBROBLASTS

There is increasing evidence that cellular responses to stress are in part regulated by protein kinases, although specific mechanisms are no t well defined, The purpose of these experiments was to investigate po tential upstream signaling events activated during heat shock in NIH3T 3 fibroblasts. Experiments were designed to ask whether heat shock act ivates p60 c-Src tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Using in vitro protein kinase activity assays, it was demon strated 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 immunocompl exes 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 i nvestigated using wortmannin, a specific pharmacological inhibitor of PI 3-kinase. The results demonstrated that wortmannin inhibited heat s hock activation of p70 S6K but only partially inhibited heat activatio n 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 r eceptor inhibitor, both inhibited heat shock stimulation of MAPK activ ity and tyrosine phosphorylation of MAPK. Furthermore, a selective epi dermal 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 sugges t that early up stream 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 sign aling mechanisms similar to growth factor stimulation.