Phorbol esters modulate the ras exchange factor RasGRP3

RasGRP represents the prototype of a new class of guanine nucleotide exchan ge factors that activate small GTPases, The guanyl nucleotide-releasing pro tein (GRP) family members contain catalytic domains related to CDC25, the R as exchange factor of Saccharomyces cerevisiae, They also contain a motif r esembling a pair of calcium-binding EF-hands and a C1 domain similar to the diacylglycerol interaction domain of protein kinase C, The sequence of KIA A0846, identified in a human brain cDNA library, encodes a member of the GR P family that we refer to as RasGRP3, We show here that RasGRP3 bound phorb ol esters with high affinity. This binding depended on anionic phospholipid s, 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 Ras(GTP) and phos phorylation of the extracellular-regulated kinases, Finally, both phorbol 1 2-myristate Ij-acetate and the diacylglycerol analogue 1,2-dioctanoyl-sn-gl ycerol induced redistribution of RasGRP3 to the plasma membrane and/or peri nuclear area in HEK-293 cells, as demonstrated using a green fluorescent fu sion protein. We conclude that RasGRP3 serves as a PKC-independent pathway to link the tumor-promoting phorbol esters with activation of pas GTPases.