Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets - Phosphorylation-induced actin polymerization causedby Hsp27 mutants

Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mito gen activated protein kinase-activated protein kinase (MAPKAP) 2 is associa ted with signaling events involved in platelet aggregation and regulation o f microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for t he inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time -dependent phosphorylation of Hsp27. This is supported by the ability of cG K to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-medi ated phosphorylation of its established substrate vasodilator-stimulated ph osphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phos phorylating Hsp27 and interfering with the MAP-KAP kinase phosphorylation o f Hsp27, the known MAP-KAP kinase 2-phosphorylation sites (Ser(15), Ser(78) , and Ser(82)) as well as Thr(143) were replaced by negatively charged amin o acids, which are considered to mimic phosphate groups, and tested in acti n polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylatio n sites led to mutants with a stimulating effect on actin polymerization, M utation of the cGK-specific site Thr(143) alone had no effect on actin poly merization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, th is mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regula tion of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.