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 language | English |
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Pages (from-to) | 843-851 |
Number of pages | 9 |
Journal | Biochemical and Biophysical Research Communications |
Volume | 325 |
Issue number | 3 |
DOIs | |
Publication status | Published - 2004 Dec 17 |
Externally published | Yes |
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