Phorbol esters modulate the Ras exchange factor RasGRP3.
RasGRP represents the prototype of a new class of guanine nucleotide exchange factors that activate small GTPases. The guanyl nucleotide-releasing protein (GRP) family members contain catalytic domains related to CDC25, the Ras exchange factor of Saccharomyces cerevisiae. They also contain a motif resembling a pair of calcium-binding EF-hands and a C1 domain similar to the diacylglycerol interaction domain of protein kinase C. The sequence of KIAA0846, identified in a human brain cDNA library, encodes a member of the GRP family that we refer to as RasGRP3. We show here that RasGRP3 bound phorbol esters with high affinity. This binding depended on anionic phospholipids, which is characteristic of phorbol ester binding to C1 domain proteins. In addition, phorbol esters also caused activation of the RasGRP3 exchange activity in intact cells, as determined by an increase in RasGTP and phosphorylation of the extracellular-regulated kinases. Finally, both phorbol 12-myristate 13-acetate and the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol induced redistribution of RasGRP3 to the plasma membrane and/or perinuclear area in HEK-293 cells, as demonstrated using a green fluorescent fusion protein. We conclude that RasGRP3 serves as a PKC-independent pathway to link the tumor-promoting phorbol esters with activation of Ras GTPases.
Lorenzo
P S
PS
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.
Kung
J W
JW
Bottorff
D A
DA
Garfield
S H
SH
Stone
J C
JC
Blumberg
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002273:Carcinogens / Q000378:metabolism / Q000494:pharmacology*; D002352:Carrier Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002874:Chromosome Mapping; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D010703:Phorbol Esters / Q000378:metabolism / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011955:Receptors, Drug / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism / Q000494:pharmacology; D020663:ras Guanine Nucleotide Exchange Factors
D029742:Caenorhabditis elegans Proteins; D002273:Carcinogens; D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D010703:Phorbol Esters; C413212:RASGRP3 protein, human; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D020663:ras Guanine Nucleotide Exchange Factors; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020559:Monomeric GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate