Yeo E J, Provost J J, Exton J H
Department of Molecular Physiology, Vanderbilt University School of Medicine, Nashville, TN 37232-0295, USA.
Biochim Biophys Acta. 1997 May 27;1356(3):308-20. doi: 10.1016/s0167-4889(97)00006-2.
Platelet-derived growth factor (PDGF) stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP2) via phospholipase C-gamma1 (PLC-gamma1) in Swiss 3T3 cells. Treatment of cells with the protein kinase C (PKC) inhibitor Ro-31-8220 greatly decreased PDGF-induced tyrosine phosphorylation of PLC-gamma1, but paradoxically enhanced the production of inositol phosphates (InsPs). The inhibitor also caused an increase of PDGF receptor tyrosine phosphorylation at later times. The changes in phosphorylation of the receptor were correlated with alterations in PLC-gamma1 translocation to the particulate fraction. Thus, although activation of PLC-gamma1 was associated with phosphorylation of the receptor and translocation of the enzyme to the particulate fraction, it was dissociated from its tyrosine phosphorylation. A non-receptor-associated, cytosolic tyrosine kinase also was found to phosphorylate PLC-gamma1 in a PDGF-dependent manner, but was not inhibited by Ro-31-8220 in vitro. PKC depletion by phorbol ester treatment decreased the tyrosine phosphorylation of PLC-gamma1 induced by PDGF and slowed the translocation of PLC-gamma1, but Ro-31-8220 produced further effects. The effect of Ro-31-8220 to enhance the production of InsPs could not be attributed to inhibition of PKC since InsPs production with PDGF was decreased in PKC-depleted cells and a stimulatory effect of the inhibitor was still evident. Interestingly, Ro-31-8220 decreased the radioactivity in phosphatidylinositol and increased that in phosphatidylinositol 4-phosphate and PtdInsP2 in cells labeled with myo[3H]inositol. The increased synthesis of PtdInsP2 could contribute to the increased production of InsPs induced by Ro-31-8220. In summary, these results support the conclusion that the activation of PLC-gamma1 in response to PDGF requires autophosphorylation of the receptor and membrane association of PLC-gamma1, but not phosphorylation of the enzyme. Furthermore, the effects of Ro-31-8220 on the tyrosine phosphorylation and activity of PLC-gamma1, and on PtdInsP2 synthesis cannot be attributed to inhibition of PKC.
血小板衍生生长因子（PDGF）通过磷脂酶C-γ1（PLC-γ1）刺激瑞士3T3细胞中磷脂酰肌醇4,5-二磷酸（Ptd InsP2）的水解。用蛋白激酶C（PKC）抑制剂Ro-31-8220处理细胞，可大大降低PDGF诱导的PLC-γ1酪氨酸磷酸化，但矛盾的是却增强了肌醇磷酸（InsPs）的产生。该抑制剂在后期还导致PDGF受体酪氨酸磷酸化增加。受体磷酸化的变化与PLC-γ1向颗粒部分的转位改变相关。因此，尽管PLC-γ1的激活与受体的磷酸化以及该酶向颗粒部分的转位有关，但它与其酪氨酸磷酸化是分离的。还发现一种非受体相关的胞质酪氨酸激酶以PDGF依赖的方式使PLC-γ1磷酸化，但在体外不受Ro-31-8220抑制。佛波酯处理使PKC耗竭，降低了PDGF诱导的PLC-γ1酪氨酸磷酸化并减缓了PLC-γ1的转位，但Ro-31-8220产生了进一步的影响。Ro-31-8220增强InsPs产生的作用不能归因于对PKC的抑制，因为在PKC耗竭细胞中PDGF诱导的InsPs产生减少，而该抑制剂的刺激作用仍然明显。有趣的是，Ro-31-8220降低了用肌醇[3H]标记的细胞中磷脂酰肌醇的放射性，并增加了磷脂酰肌醇4-磷酸和PtdInsP2中的放射性。PtdInsP2合成的增加可能导致Ro-31-8220诱导的InsPs产生增加。总之，这些结果支持以下结论：响应PDGF时PLC-γ1的激活需要受体的自磷酸化和PLC-γ1的膜结合，但不需要该酶的磷酸化。此外，Ro-31-8220对PLC-γ1酪氨酸磷酸化和活性以及对PtdInsP2合成的影响不能归因于对PKC的抑制。
