PROTEIN-KINASE-C REGULATES PLECKSTRIN BY PHOSPHORYLATION OF SITES ADJACENT TO THE N-TERMINAL PLECKSTRIN HOMOLOGY DOMAIN

Pleckstrin is a substrate for protein kinase C in activated platelets that contains at its N and C termini two of the pleckstrin homology (P H) domains that have been proposed to mediate protein-protein and prot ein-lipid interactions. We have recently shown that pleckstrin can inh ibit agonist-induced phosphoinositide hydrolysis and that this inhibit ion requires an intact N-terminal PH domain (residues 6 to 99). In the present studies, we have identified the sites of phosphorylation in p leckstrin and examined their contribution to pleckstrin function. In h uman platelets activated with thrombin or phorbol esters, and in COS-1 cells expressing pleckstrin, a combination of phosphopeptide analysis and site directed mutagenesis shows that three residues in the interv ening sequence between the two pleckstrin PH domains become phosphoryl ated: Ser(113), Thr(114), and Ser(117). Replacing all three of these s ites with glycine decreased phosphorylation by >90% and reduced plecks trin's ability to inhibit phosphoinositide hydrolysis by as much as 80 %. Replacing the phosphorylation sites with alanine residues had a sim ilar effect, while substitution with aspartate, glutamate, or lysine r esidues produced pleckstrin variants that were fully active even in th e absence of phosphorylation, These results suggest that phosphorylati on enhances pleckstrin's activity by introducing a cluster of charges into a region adjacent to, but not within, the N-terminal PH domain. T his may have an allosteric effect on the N-terminal PH domain, regulat ing its interaction with other molecules necessary for the inhibition of phosphoinositide hydrolysis.