Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells.
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 tyrosine-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.
Kuriyama
Masamitsu
M
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.
Taniguchi
Taizo
T
Shirai
Yasuhito
Y
Sasaki
Atsuo
A
Yoshimura
Akihiko
A
Saito
Naoaki
N
D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D048049:Extracellular Signal-Regulated MAP Kinases