PLECKSTRIN INHIBITS PHOSPHOINOSITIDE HYDROLYSIS INITIATED BY G-PROTEIN-COUPLED AND GROWTH-FACTOR RECEPTORS - A ROLE FOR PLECKSTRINS PH DOMAINS

Pleckstrin is a 40-kDa protein present in platelets and leukocytes tha t contains two PH domains separated by a 150-residue intervening seque nce. Pleckstrin is a major substrate for protein kinase C, but its fun ction is unknown. The present studies examine the effects of pleckstri n on second messenger generation. When expressed in cos-1 or HEK-293 c ells, pleckstrin inhibited 1) the G(alpha)-mediated activation of phos pholipase C-beta initiated by thrombin, M1-muscarinic acetylcholine, a nd angiotensin II receptors, 2) the stimulation of phospholipase C-bet a by constitutively active G(q alpha), 3) the G(beta gamma)-mediated a ctivation of phospholipase C-beta caused by alpha(2A)-adrenergic recep tors, and 4) the tyrosine phosphorylation-media ted activation of phos pholipase C-gamma caused by Trk A. However, pleckstrin had no effect o n either the stimulation or inhibition of adenylyl cyclase, The inhibi tion of phosphoinositide hydrolysis caused by pleckstrin was similar i n 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-termi nal PH domain had no effect, deleting the N-terminal PH domain abolish ed activity (but not expression) and mutating a highly conserved trypt ophan residue within the N-terminal PH domain decreased activity by on e-third, Notably, however, a pleckstrin variant in which the N-termina l PH domain was replaced with a second copy of the C-terminal PH domai n was nearly as active as native pleckstrin, These results show that: 1) pleckstrin can inhibit pathways leading to both phospholipase C-bet a- and phospholipase C-gamma-mediated phosphoinositide hydrolysis, 2) this inhibition affects activation of phospholipase C-beta mediated by either G(alpha) or G(beta gamma) but does not affect the regulation o f adenylyl cyclase activity by G(alpha) or G(beta gamma), 3) although pleckstrin is a substrate for protein kinase C, the effects of pleckst rin and PMA are at least partially independent, 4) the inhibition caus ed by pleckstrin appears to be mediated by the PH domain at the N term inus, rather than the C terminus of the molecule, and 5) location of t he two PH domains within the molecule clearly contributes to their ind ividual activity, These results do not appear to be readily attributab le to an interaction between pleckstrin and G(beta gamma), but they ar e consistent with a recent report showing an association between PH do mains and phosphatidylinositol 4,5-bisphosphate in vitro.