Phosphorylation of phospholipase Cgamma1 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) gamma1 and that phosphorylation on tyrosine (Tyr) 783 of PLCgamma1 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 PLCgamma1 containing phosphotyrosine 783 (alpha-PLCgamma1 PY). Tyr-783-phosphorylated PLCgamma1 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 PLCgamma1 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 PLCgamma1 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-PLCgamma1 PY, as occurred following the microinjection of the PLCgamma1-2SH2 domain, which is expected to associate with phosphorylated PDGF receptors and to block PLCgamma1 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 PLCgamma1 on tyrosine 783 by PDGF plays an important role in cytoskeletal reorganization in addition to mitogenesis.
Yu
H
H
Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, 108, Japan.
Fukami
K
K
Itoh
T
T
Takenawa
T
T
D016475:3T3 Cells; D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D051966:Phospholipase C gamma; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D013997:Time Factors; D010738:Type C Phospholipases / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D000199:Actins; D000906:Antibodies; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D014443:Tyrosine; D010738:Type C Phospholipases; D051966:Phospholipase C gamma