Activation and translocation of PKCδ is necessary for VEGF-induced ERK activation through KDR in HEK293T cells

Masamitsu Kuriyama, Taizo Taniguchi, Yasuhito Shirai, Atsuo Sasaki, Akihiko Yoshimura, Naoaki Saito

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-γ-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 PKCδ knock-down using siRNA method showed that PKCδ 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 PKCδ specific inhibitor, or human PKCδ siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCδ-knockdown HEK293T cells, but co-expression of rat PKCδ-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCδ-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCδ-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCδ-knockdown HEK293T cell. These results indicate that PKCδ is involved in VEGF-KDR-induced ERK activation via C1B domain.

Original languageEnglish
Pages (from-to)843-851
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume325
Issue number3
DOIs
Publication statusPublished - 2004 Dec 17
Externally publishedYes

Keywords

  • C1B domain
  • ERK
  • GFP
  • HEK293T cells
  • KDR/Flk-1
  • Myristoylated alanine-rich C kinase substrate
  • Phospholipase C-γ
  • Protein kinase C
  • Vascular endothelial growth factor-A

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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