Summary
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 PPl-like activity was present
in the Triton-insoluble cytoskeleton, while only 10% of the total PP2A-like activity
was present in this fraction. Stimulation with 1 U/ml thrombin produced a 1.8-fold
increase in PPl-like activity and a 7-fold increase in PP2A-like activity, respectively,
in the cytoskeletal fraction, under aggregating conditions. Immunoblot analysis revealed
that thrombin treatment increased association of PP1 catalytic subunit isozymes (PPlα,
PPlβ, PP1γ) and PP2A catalytic subunit with the cytoskeleton, with concomitant decrease
of these enzymes in Triton-soluble fractions. The amounts of cytoskeleton-associated
PP1 and PP2A depended on the dose of thrombin which could activate platelets. Agonist-induced
redistribution of PP1 and PP2A into the cytoskeleton was inhibited by OP-41483 (a
prostaglandin I2 analog). Interaction of PP2A with cytoskeletal proteins strongly correlates with
aggregation, whereas the association of PP1 with cytoskeleton can be detected upon
platelet activation, even in the absence of aggregation. Co-extraction of protein
kinase C and myosin light chain kinase with the cytoskeleton eventually translocated
to the cytoskeleton, but only during aggregation. These results suggest that differential
translocation of PP1 and PP2A to the cytoskeleton is involved in platelet activation,
and their association with cytoskeletal proteins may regulate phosphorylation levels
together with protein kinases in platelets.