Phosphorylation of phospholipase Cγ1 on tyrosine residue 783 by platelet-derived growth factor regulates reorganization of the cytoskeleton

It is known that platelet-derived growth factor (PDGF) induces the phosphorylation of phospholipase C (PLC) gamma 1 and that phosphorylation on tyrosine (Tyr) 783 of PLC1 gamma is essential for phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vivo while phosphorylation does not affect the catalytic activity in vitro. To study the roles of Tyr-783 phosphorylation in vivo we developed a polyclonal antibody that recognizes PLC gamma 1 containing phosphotyrosine 783 (alpha PLC gamma 1 PY). Tyr-783-phosphorylated PLC gamma 1 was not detected in the absence of PDGF, appeared after stimulation, increased for 30 min, and then decreased to near the prestimulation level. Immunostaining of cells showed that PDGF-produced Tyr-783-phosphorylated PLC gamma 1 localized predominantly at membrane ruffles and stress fibers where it colocalized with actin filaments within 30 min. Ninety minutes after PDGF stimulation, the actin filaments were disassembled to short fragments, and the levels of Tyr-783-phosphorylated PLC gamma 1 were remarkably decreased in membrane ruffles and cytoskeleton. Furthermore, the depolymerization of actin filaments and membrane ruffling caused by PDGF stimulation were blocked by microinjecting alpha-PLC gamma 1 PY, as occurred following the microinjection of the PLC gamma 1-2SH(2) domain, which is expected to associate with phosphorylated PDGF receptors and to block PLC gamma 1 binding. It is worth noting that the microinjection of tyrosine-phosphorylated peptide (consisting of 13 amino acids containing Tyr-783) induced the disassembly of actin filaments and membrane ruffling as observed in PDGF-stimulated cells, while nonphosphorylated peptide did not cause any effect. These data suggest that the phosphorylation of PLC gamma 1 on tyrosine 783 by PDGF plays an important role in cytoskeletal reorganization in addition to mitogenesis. (C) 1998 Academic Press.