Abstract
The abundant, cytoplasmic 90-kDa heat-shock protein associates transiently with the Rous sarcoma virus oncogenic protein tyrosine kinase, pp60v-src, directs its cellular trafficking and negatively regulates its kinase activity. Here we report that the serine/threonine phosphatase inhibitor, okadaic acid, destabilized the heat-shock protein 90-pp60v-src chaperone complex in v-src-transfected cells. Concomitant with complex destabilization by okadaic acid, phosphoserine was doubled and phosphothreonine was increased 20-fold in the heat-shock protein 90. Although phosphorylation of the total pool of immunoprecipitable pp60v-src was unchanged, okadaic acid slightly increased phosphoserine and phosphothreonine levels specifically in pp60v-src bound to heat-shock protein 90. The low level of tyrosine phosphorylation in the pp60v-src complexed with heat-shock protein 90 was further decreased by okadaic acid. Interestingly, okadaic acid-stabilized hyperphosphorylation of the heat-shock protein 90-pp60v-src complex lowered the level of pp60v-src in cell membranes, the functional location for pp60v-src. We suggest that serine/threonine phosphorylation of heat-shock protein 90 and/or pp60v-src functions as a regulatory molecular trigger to release pp60v-src from the chaperone complex at the inner surface of cell membranes.
Highlights
It has been established that nascent pp60v-src protein is synthesized on cytosolic free polyribosomes [5], the mature kinase localizes predominantly to the cytoplasmic surface of the plasma membrane where it is anchored via a myristic
Association of hsp90 with the pp60v-src Oncoprotein—TSvsrc3T3 cells express a mutant temperature-sensitive RSV pp60v-src protein that, like certain other temperature-sensitive mutants, associates with hsp90 to a greater degree than does wild-type RSV pp60v-src [9, 24]. Immunoprecipitation of this mutant pp60v-src protein from these cells with an anti-src mAb coprecipitated hsp90, as determined by Western immunoblotting with a monoclonal antibody specific for the hsp90 family of stress proteins (AC88, 830 mAb, StressGen) (Fig. 1, lanes 1–3)
okadaic acid (OA) Disrupts the v-Src Protein-hsp90 Heterocomplex—In preliminary experiments, we found that preformed pp60v-src-hsp90 complex did not dissociate when treated with alkaline phosphatase or the serine/threonine phosphatases PP1 and PP2A
Summary
It has been established that nascent pp60v-src protein is synthesized on cytosolic free polyribosomes [5], the mature kinase localizes predominantly to the cytoplasmic surface of the plasma membrane where it is anchored via a myristic. Complexation with hsp sharply diminishes the intrinsic kinase activity of pp60v-src, and both autophosphorylation of the v-Src protein at tyrosine 416 and phosphorylation of cellular substrate proteins on tyrosine are minimalized until after pp60v-src is attached to the plasma membrane [12]. We were interested in the role of phosphorylation/dephosphorylation of either hsp or pp60v-src in the molecular interactions of these two proteins. Toward this end, we attempted to manipulate the phosphate status of both proteins by treating v-src-transfected cells with okadaic acid (OA), a potent inhibitor of the serine/threonine protein phosphatases PP1 and PP2A (see Ref. 14 for review). OA has been shown to stabilize the hyperphosphorylation of many cellular proteins, notably retinoblastoma protein and p53 [15], and to reverse the neoplastic phenotypic characteristics of v-src-transformed 3T3 cells [16]
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