Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors: A role for Pleckstrin's PH domains

Abstract

Pleckstrin is a 40-kDa protein present in platelets and leukocytes that contains two PH domains separated by a 150-residue intervening sequence. Pleckstrin is a major substrate for protein kinase C, but its function is unknown. The present studies examine the effects of pleckstrin on second messenger generation. When expressed in cos-1 or HEK-293 cells, pleckstrin inhibited 1) the Gα-mediated activation of phospholipase Cβ initiated by thrombin, M1-muscarinic acetylcholine, and angiotensin II receptors, 2) the stimulation of phospholipase Cβ by constitutively active Gqα, 3) the Gβγ-mediated activation of phospholipase Cβ caused by α2A-adrenergic receptors, and 4) the tyrosine phosphorylation-mediated activation of phospholipase Cγ caused by Trk A. However, pleckstrin had no effect on either the stimulation or inhibition of adenylyl cyclase. The inhibition of phosphoinositide hydrolysis caused by pleckstrin was similar in magnitude to that caused by activating protein kinase C with phorbol 12-myristate 13-acetate (PMA). When combined, pleckstrin and PMA had an additive effect, inhibiting phosphoinositide hydrolysis by as much as 90%. Structure-function analysis highlighted the role of pleckstrin's N-terminal PH domain in these events. Although deleting the C-terminal PH domain had no effect, deleting the N-terminal PH domain abolished activity (but not expression) and mutating a highly conserved tryptophan residue within the N-terminal PH domain decreased activity by one-third. Notably, however, a pleckstrin variant in which the N-terminal PH domain was replaced with a second copy of the C-terminal PH domain was nearly as active as native pleckstrin. These results show that: 1) pleckstrin can inhibit pathways leading to both phospholipase Cβ- and phospholipase Cγ-mediated phosphoinositide hydrolysis, 2) this inhibition affects activation of phospholipase Cβ mediated by either Gα or Gβγ, but does not affect the regulation of adenylyl cyclase activity by Gα or Gβγ, 3) although pleckstrin is a substrate for protein kinase C, the effects of pleckstrin and PMA are at least partially independent, 4) the inhibition caused by pleckstrin appears to be mediated by the PH domain at the N terminus, rather than the C terminus of the molecule, and 5) location of the two PH domains within the molecule clearly contributes to their individual activity. These results do not appear to be readily attributable to an interaction between pleckstrin and Gβγ, but they are consistent with a recent report showing an association between PH domains and phosphatidylinositol 4,5-bisphosphate in vitro.