Serine/Threonine Dephosphorylation May Be Involved in Tyrosine Phosphorylation: A New Mode of Signal Transduction in Platelets

Abstract

Platelet signal transduction involves not only reversible phosphorylation of proteins on both tyrosine and serine/threonine residues, but also mechanisms of cross-talk to coordinate different pathways. We have, therefore, investigated the effect of okadaic acid, a potent inhibitor of serine/threonine protein phosphatases type 1 and type 2A (PP1 and PP2A), to better understand the interplay that must exist between serine/threonine and tyrosine phosphorylations during platelet activation.

Okadaic acid drastically inhibits thrombin-induced platelet aggregation, secretion, and thromboxane synthesis. The inhibition is accompanied by a marked increase in the phosphorylation of at least 5 proteins (230, 210, 74, 57, and 50 to 52 kDa). However, protein kinase C activity is not modified because thrombinand phorbol-12-myristate-13-acetate-induced phosphorylation of pleckstrin is still occurring, although slightly decreased.

Inhibition of platelet function and extent of the phosphorylation of the 5 substrates in the presence of okadaic acid are concentration and time dependent, suggesting a relation between the accumulation of one or more phosphoproteins and the inhibitory effect of okadaic acid.

Okadaic acid inhibits thrombin-induced tyrosine phosphorylation in a concentration-dependent manner. According to Brautigan and Pinault, the inhibition of protein phosphatases in kidney cells resulted in the activation of a 55-kDa-tyrosine phosphatase and the tyrosine phosphatase activity was synergistically increased when okadaic acid acted in concert with prostaglandin I₂ (PGI₂). Interestingly, in agreement with these results, the okadaic acid-induced phosphosphorylation of the 50-kDa substrate, which occurs without a cyclic adenosine monophosphate increase in platelets, has the same molecular weight as the platelet membrane tyrosine phosphatase isolated by Dawicki and Steiner.

Furthermore, we also found that thrombin-induced tyrosine phosphorylation was markedly inhibited in the presence of low concentrations of both okadaic acid and PGI₂, therefore explaining the synergistic inhibition of platelet aggregation and secretion. The results greatly support the notion of a cross-talk between stimulation of serine/threonine kinases (in response to inhibition of serine/threonine PP) and inhibition of tyrosine phosphorylations and emphasize the role of the 50-kDa substrate in regulating platelet activation.

Keywords:

Serine/threonine phosphatase - okadaic acid - tyrosine phosphorylation - platelet aggregation - thrombin