PHOSPHORYLATION OF PLATELET PLECKSTRIN ACTIVATES INOSITOL POLYPHOSPHATE 5-PHOSPHATASE-I

Pleckstrin is the major substrate phosphorylated on serine and threoni ne in response to stimulation of human platelets by thrombin (Abrams, C. S., Zhao, W., Belmonte, E., and Brass, L. F. (1995) J. Biol. Chem. 270, 23317-23321). We now show that pleckstrin in platelets is in a co mplex with inositol polyphosphate 5-phosphatase I (5-phosphatase I). T his enzyme hydrolyzes the 5-phosphate from inositol 1,4,5-trisphosphat e and inositol 1,3,4,5-tetrakisphosphate and thus serves as a calcium signal-terminating enzyme, since the substrates but not the products m obilize intracellular calcium. Pleckstrin co immunoprecipitates with 5 -phosphatase I in homogenates of platelets. Platelet homogenates fract ionated by anion exchange chromatography show coelution of pleckstrin and 5-phosphatase I. Fractions containing phosphorylated pleckstrin ha ve 7-fold greater 5-phosphatase activity than those containing unphosp horylated pleckstrin. Mixing experiments with recombinant 5-phosphatas e I and pleckstrin in, vitro show that they form a stoichiometric comp lex. A mutant form of pleckstrin, in which the serine and threonine re sidues that are phosphorylated by protein kinase C are substituted wit h glutamic acid (pseudophosphorylated pleckstrin), activates recombina nt 5-phosphatase I 2-3-fold while native unphosphorylated pleckstrin d oes not stimulate the enzyme. Thus pleckstrin functions to terminate c alcium signaling in platelets when it is phosphorylated by binding to and activating 5-phosphatase I.