DIFFERENTIAL ASSOCIATION OF PROTEIN SER THR PHOSPHATASE TYPE-1 AND TYPE-2A WITH THE CYTOSKELETON UPON PLATELET ACTIVATION/

The association of protein Ser/Thr phosphatase type 1(PP1) and type 2A (PP2A) with the cytoskeleton (Triton X-100 insoluble residue) during human platelet activation was investigated. In unstimulated platelets, 40% of total PP1-like activity was present in the Triton-insoluble cy toskeleton, while only 10% of the total PP2A-like activity was present in this fraction. Stimulation with 1 U/ml thrombin produced a 1.8-fol d increase in PP1-like activity and a 7-fold increase in PP2A-like act ivity, respectively, in the cytoskeletal fraction, under aggregating c onditions. Immunoblot analysis revealed that thrombin treatment increa sed association of PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamm a, PP1 delta) and PP2A catalytic subunit with the cytoskeleton, with c oncomitant decrease of these enzymes in Triton-soluble fractions. The amounts of cytoskeleton-associated PP1 and PP2A depended on the dose o f thrombin which could activate platelets. Agonist-induced redistribut ion of PP1 and PP2A into the cytoskeleton was inhibited by OP-41483 (a prostaglandin I-2 analog). Interaction of PP2A with cytoskeletal prot eins strongly correlates with aggregation, whereas the association of PP1 with cytoskeleton can be detected upon platelet activation, even i n the absence of aggregation. Co-extraction of protein kinase C and my osin light chain kinase with the cytoskeleton eventually translocated to the cytoskeleton, but only during aggregation. These results sugges t that differential translocation of PP1 and PP2A to the cytoskeleton is involved in platelet activation, and their association with cytoske letal proteins may regulate phosphorylation levels together with prote in kinases in platelets.