C-PHOSPHORYLATION AND ACTIVATION OF THE IGF-I RECEPTOR IN SRC-TRANSFORMED CELLS

Using a panel of src mutants partially defective for malignant transfo rmation, our laboratory has previously identified the insulin-like gro wth factor (IGF-I) receptor as a protein whose tyrosine phosphorylatio n correlates with transformation by src in embryonic chick cells (Kozm a ct al., 1990; Kozma and Weber, 1990). It has not been clear, however , whether are-induced phosphorylation altered the enzymatic or signali ng properties of the IGF-I receptor and thus whether the IGF-I recepto r could be a functionally significant target for pp(60v.src). To exami ne the effect of src expression on the activity of the IGF-I receptor, the human IGF-I receptor was expressed in Rat-1 fibroblasts co-expres sing the temperature-sensitive v-src mutant, tsLA29, The IGF-I recepto r exhibited an elevated level of tyrosine phosphorylation in are trans formed cells even in the absence of IGF-I treatment. Increased recepto r phosphorylation occurred rapidly when cells expressing a temperature -conditional are mutant were shifted from the restrictive to the permi ssive temperature. Src-induced phosphorylation of the receptor was cor related with an increase in the in vitro tyrosine kinase activity of t he receptor, both toward itself and exogenous substrates. The are-indu ced increase in receptor activity was shown to be dependent on tyrosin e phosphorylation, as treatment with a tyrosine-specific phosphatase l owered receptor activity. A kinase-defective mutant of the IGF-I recep tor also became constitutively phosphorylated in are-transformed cells , ruling out a possible autocrine mechanism for this phosphorylation. Collectively these data indicate that pp(60v.src) induces ligand-indep endent phosphorylation and activation of the IGF-I receptor by an intr acellular mechanism, consistent with the possibility that receptor pho sphorylation could contribute to the genesis of the transformed phenot ype.