ANTISENSE SRC EXPRESSION INHIBITS TYROSINE PHOSPHORYLATION OF SHC ANDITS ASSOCIATION WITH GRB2 AND SOS WHICH LEADS TO MAP KINASE ACTIVATION IN U937 HUMAN LEUKEMIA-CELLS

We constructed a recombinant plasmid which expresses antisense src RNA after dexamethasone (Dexa) treatment, and transfected it into U937 hu man monoblastic leukemia cells (U937-ASRC). Induction of antisense src RNA expression diminished the amounts of c-Src and its protein tyrosi ne kinase (PTK) activity in U937-ASRC cells. The declines in c-Src and its PTK activity subsequently reduced the proliferation of U937-ASRC cells. To elucidate the growth signal transduction pathway downstream of c-Src, tyrosine phosphorylation of She was examined in U937-ASRC ce lls treated with Dexa. The decline in c-Src by induction of antisense src RNA expression decreased the level of tyrosine phosphorylation of She. Immunoprecipitated c-Src directly phosphorylated immunoprecipitat ed She on tyrosine residues in vitro. The amounts of Grb2 and Sos coim munoprecipitated with She were decreased after Dexa treatment. However , the amount of Sos co-immunoprecipitated with Grb2 was apparently not affected by Dexa treatment. These results indicate that Grb2 and Sos constitutively associate with each other in U937 cells. Furthermore, t he level of phosphorylation on tyrosine (204) essential for MAP kinase activation was decreased after Dexa treatment. Taken together with al l these findings, it is suggested that c-Src directly phosphorylates S he on tyrosine residues, which in turn binds to Grb2 constitutively as sociated with Sos to form a Shc-Grb2-Sos complex, and that the complex formation is coupled with MAP kinase activation mediated by Ras activ ation in U937 cells.