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 |
|---|---|
| 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 |
Fingerprint
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
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Activation and translocation of PKCδ is necessary for VEGF-induced ERK activation through KDR in HEK293T cells. / Kuriyama, Masamitsu; Taniguchi, Taizo; Shirai, Yasuhito; Sasaki, Atsuo; Yoshimura, Akihiko; Saito, Naoaki.
In: Biochemical and Biophysical Research Communications, Vol. 325, No. 3, 17.12.2004, p. 843-851.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Activation and translocation of PKCδ is necessary for VEGF-induced ERK activation through KDR in HEK293T cells
AU - Kuriyama, Masamitsu
AU - Taniguchi, Taizo
AU - Shirai, Yasuhito
AU - Sasaki, Atsuo
AU - Yoshimura, Akihiko
AU - Saito, Naoaki
PY - 2004/12/17
Y1 - 2004/12/17
N2 - 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.
AB - 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.
KW - C1B domain
KW - ERK
KW - GFP
KW - HEK293T cells
KW - KDR/Flk-1
KW - Myristoylated alanine-rich C kinase substrate
KW - Phospholipase C-γ
KW - Protein kinase C
KW - Vascular endothelial growth factor-A
UR - http://www.scopus.com/inward/record.url?scp=8444241833&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=8444241833&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2004.10.102
DO - 10.1016/j.bbrc.2004.10.102
M3 - Article
C2 - 15541367
AN - SCOPUS:8444241833
VL - 325
SP - 843
EP - 851
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 3
ER -