Activation and translocation of PKCδ is necessary for VEGF-induced ERK activation through KDR in FIEK293T cells
Activation and translocation of PKCδ is necessary for VEGF-induced ERK activation through KDR in FIEK293T cells
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DOI：
10.1016/j.bbrc.2004.10.102
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发表时间：
2004-12-17
影响因子：
3.1
通讯作者：
Saito, N
中科院分区：
文献类型：
--
作者：
Kuriyama, M;Taniguchi, T;Saito, N
VEGF-KDR/Flk-1信号利用磷脂酶C-γ-蛋白激酶C(PKC)-Raf-MEK-ERK通路作为主要信号通路，诱导基因表达和cPLA2磷酸化。然而，VEGF-KDR信号诱导的特定PKC亚型的时空激活机制尚不清楚。我们利用瞬时表达KDR的人胚胎肾HEK293T细胞来研究PKC的激活机制。PKC特异性抑制物和人PKC Delta siRNA敲除结果表明，PKC Delta在VEGF-KDR诱导的ERK激活中起重要作用。肉豆蔻酰化的富含丙氨酸的C-激酶底物(MARCKS)依赖于PKC的磷酸化，从质膜转运到细胞质。KDR刺激诱导的MARCKS-GFP转位可被PKCDelta特异性抑制剂rotlerin或人PKCDelta siRNA阻断。人PKC Delta基因敲除的HEK293T细胞中，VEGF-KDR的刺激不能诱导ERK的磷酸化，但大鼠PKC Delta-GFP的共表达可恢复ERK的磷酸化。不能被酪氨酸磷酸化激活但被DAG激活的大鼠PKCDelta-GFP突变体Y311/332F能恢复ERK的磷酸化，而能被酪氨酸磷酸化而不被DAG激活的C1B缺失突变体不能恢复人PKCDelta基因敲除的HEK293T细胞的ERK磷酸化。这些结果表明，PKCDelta通过C1B结构域参与了VEGF-KDR诱导的ERK激活。(C)2004年，爱思唯尔公司出版。
VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyro sine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain. (C) 2004 Published by Elsevier Inc.