Protein Kinase C-Dependent Phosphorylation of Human Pleckstrin Reduces its Capacity for Self-Association and Impairs its Ability to Bind Phosphoinositide

Pleckstrin is a major substrate of protein kinase C in lymphocytes, macrophages, monocytes, granulocytes and platelets. In these cells, pleckstrin is expressed at high levels and represents approximately 1 % of total cellular protein. Pleckstrin plays an important role in protein kinase C-dependent secretion, and abberant pleckstrin phosphorylation has been associated with disease. The mechanism, however, by which phosphorylation regulates pleckstrin function is unknown. Here, we show that native pleckstrin self-associates to form dimers that reduce its binding capacity to phosphoinositides. Phosphomimetic amino acid substitutions at residues normally phosphorylated by protein kinase C result in significantly reduced pleckstrin dimerization. Although phosphomimetic forms of pleckstrin exhibit subtle conformational changes they retain most of their phosphoinositide binding properties. These findings suggest that phosphorylation regulates pleckstrin interaction with membranes via a dimerization-dependent mechanism.