Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3.
Members of the RasGRP family of Ras activators have C1 domains that bind diacylglycerol (DAG) and DAG analogs such as the tumor-promoting phorbol esters. RasGRP members could be responsible for some of the DAG signaling processes that have previously been attributed to protein kinase C (PKC). We found that RasGRP3 is selectively expressed in B cells, suggesting that RasGRP3 might function downstream of the B-cell receptor (BCR). Indeed, stimulation of Ramos B cells with the DAG analog phorbol ester myristate (PMA) results in the association of RasGRP3 with the membrane fraction. However, we also made the unexpected observation that RasGRP3 is phosphorylated, coincident with Ras activation after stimulation. When inhibitors of PKC are present, Ras activation is attenuated, and this attenuation correlates with an inhibition of RasGRP3 phosphorylation. RasGRP3 is phosphorylated in vitro by PKC-theta and PKC-beta2. When ectopically coexpressed in HEK-293 cells, a dominant-activated mutant of PKC-theta phosphorylates RasGRP3 and enhances Ras-Erk signaling. These results provide the first indication for a functional interaction between a RasGRP family member and a dissimilar DAG binding protein. A convergent DAG signaling system could be important in fine-tuning Ras signaling during B-cell development or during the humoral immune response.
Teixeira
Christine
C
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Stang
Stacey L
SL
Zheng
Yong
Y
Beswick
Naomi S
NS
Stone
James C
JC
D000818:Animals; D002051:Burkitt Lymphoma / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010703:Phorbol Esters / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins / Q000378:metabolism
D004075:Diglycerides; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D010703:Phorbol Esters; C413212:RASGRP3 protein, human; D011994:Recombinant Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D019319:Okadaic Acid; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins