PKCdelta associates with and is involved in the phosphorylation of RasGRP3 in response to phorbol esters.
RasGRP is a family of guanine nucleotide exchange factors that activate small GTPases and contain a C1 domain similar to the one present in protein kinase C (PKC). In this study, we examined the interaction of RasGRP3 and PKC in response to the phorbol ester PMA. In Chinese hamster ovary or LN-229 cells heterologously expressing RasGRP3, phorbol 12-myristate 13-acetate (PMA) induced translocation of RasGRP3 to the perinuclear region and a decrease in the electrophoretic mobility of RasGRP3. The mobility shift was associated with phosphorylation of RasGRP3 on serine residues and seemed to be PKCdelta-dependent because it was blocked by the PKCdelta inhibitor rottlerin as well as by a PKCdelta kinase-dead mutant. Using coimmunoprecipitation, we found that PMA induced the physical association of RasGRP3 with PKCdelta and, using in situ methods, we showed colocalization of PKCdelta and RasGRP3 in the perinuclear region. PKCdelta phosphorylated RasGRP3 in vitro. Previous studies suggest that ectopic expression of RasGRP3 increases activation of Erk1/2. We found that overexpression of either PKCdelta or RasGRP3 increased the activation of Erk1/2 by PMA. In contrast, coexpression of PKCdelta and RasGRP3 yielded a level of phosphorylation of Erk1/2 similar to that of control vector cells. Our results suggest that PKCdelta may act as an upstream kinase associating with and phosphorylating RasGRP3 in response to PMA. The interaction between RasGRP3 and PKCdelta points to the existence of complex cross-talk between various members of the phorbol ester receptors which can have important impact on major signal transduction pathways and cellular processes induced by phorbol esters or DAG
Brodie
Chaya
C
Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel. chaya@mail.biu.ac.il
Steinhart
Rivka
R
Kazimirsky
Gila
G
Rubinfeld
Hadara
H
Hyman
Tehila
T
Ayres
Jolene N
JN
Hur
Gang Min
GM
Toth
Attila
A
Yang
Dazhi
D
Garfield
Susan H
SH
Stone
James C
JC
Blumberg
Peter M
PM
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D004586:Electrophoresis; D005260:Female; D020662:Guanine Nucleotide Exchange Factors / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D020663:ras Guanine Nucleotide Exchange Factors
D020662:Guanine Nucleotide Exchange Factors; C413212:RASGRP3 protein, human; D020663:ras Guanine Nucleotide Exchange Factors; D012694:Serine; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate