Phosphorylation of RasGRP3 on threonine 133 provides a mechanistic link between PKC and Ras signaling systems in B cells.
B-cell receptor (BCR) signaling activates a number of intracellular signaling molecules including phospholipase C-gamma2 (PLC-gamma2), which generates membrane diacylglycerol (DAG). DAG recruits both protein kinase C (PKC) and RasGRP family members to the membrane and contributes to their activation. We have hypothesized that membrane colocalization facilitates activation of RasGRP3 by PKC. Here we demonstrate that PKC phosphorylates RasGRP3 on Thr133 in vitro, as determined by mass spectrometry. RasGRP3 with a Thr133Ala substitution is a poor PKC substrate in vitro and a poor Ras activator in vivo. Antiphosphopeptide antibodies recognize Thr133-phosphorylated RasGRP3 in B cells after BCR stimulation or DAG analog treatment, but much less so in resting cells. PKC inhibitors block RasGRP3 Thr133 phosphorylation and Ras-extracellular signal-related kinase (Erk) signaling with a similar pattern. After stimulation of T-cell receptor (TCR) or DAG analog treatment of T cells, PKC-catalyzed phosphorylation of RasGRP1 occurs on the homologous residue, Thr184. These studies shed light on the proposed "PKC-Ras pathway" and support the hypothesis that RasGRP phosphorylation by PKC is a mechanism that integrates DAG signaling systems in T and B cells. PKC-mediated regulation of RasGRPs in lymphocytes may generate cooperative signaling in response to increases in DAG. The mast- and myeloid-selective family member RasGRP4 is regulated by different means.
Zheng
Yong
Y
Department of Biochemistry, University of Alberta, Edmonton AB, Canada.
Liu
Huaizhi
H
Coughlin
Jason
J
Zheng
Jing
J
Li
Liang
L
Stone
James C
JC
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D001665:Binding Sites; D045744:Cell Line, Tumor; D004075:Diglycerides / Q000378:metabolism / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D019169:Jurkat Cells; D013058:Mass Spectrometry; D051379:Mice; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism; D020663:ras Guanine Nucleotide Exchange Factors; D018631:ras Proteins / Q000378:metabolism*
D004075:Diglycerides; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; C413212:RASGRP3 protein, human; D020663:ras Guanine Nucleotide Exchange Factors; D011493:Protein Kinase C; D018631:ras Proteins