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.

[1]  A. F. Castro,et al.  Identification of Guanine Nucleotide Exchange Factors (GEFs) for the Rap1 GTPase , 2000, The Journal of Biological Chemistry.

[2]  J. Stone,et al.  RasGRP is essential for mouse thymocyte differentiation and TCR signaling , 2000, Nature Immunology.

[3]  N. Hayward,et al.  Characterization of RasGRP2, a Plasma Membrane-targeted, Dual Specificity Ras/Rap Exchange Factor* , 2000, The Journal of Biological Chemistry.

[4]  M. Matsuda,et al.  CalDAG-GEFIII Activation of Ras, R-Ras, and Rap1* , 2000, The Journal of Biological Chemistry.

[5]  Christian Rosenmund,et al.  Regulation of transmitter release by Unc-13 and its homologues , 2000, Current Opinion in Neurobiology.

[6]  P. Blumberg,et al.  RasGRP links T-cell receptor signaling to Ras. , 2000, Blood.

[7]  P. Blumberg,et al.  The guanine nucleotide exchange factor RasGRP is a high -affinity target for diacylglycerol and phorbol esters. , 2000, Molecular pharmacology.

[8]  D. Wilton,et al.  Anionic phospholipids, interfacial binding and the regulation of cell functions. , 2000, Biochimica et biophysica acta.

[9]  V. Najfeld,et al.  Identification of novel chromosomal rearrangements in acute myelogenous leukemia involving loci on chromosome 2p23, 15q22 and 17q21 , 1999, Leukemia.

[10]  T. Morimoto,et al.  Endomembrane Trafficking of Ras The CAAX Motif Targets Proteins to the ER and Golgi , 1999, Cell.

[11]  C. Marshall,et al.  New Insights into the Interaction of Ras with the Plasma Membrane , 1999, Cell.

[12]  C. Der,et al.  Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain , 1998, Molecular and Cellular Biology.

[13]  A. Graybiel,et al.  A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J. Stone,et al.  RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs. , 1998, Science.

[15]  M. Suyama,et al.  Prediction of the coding sequences of unidentified human genes. XII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. , 1998, DNA research : an international journal for rapid publication of reports on genes and genomes.

[16]  E. Blennow,et al.  The germinal center kinase gene and a novel CDC25-like gene are located in the vicinity of the PYGM gene on 11q13 , 1997, Human Genetics.

[17]  H. Saito,et al.  Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells , 1997, Leukemia.

[18]  D. Bar-Sagi,et al.  The role of the PH domain in the signal‐dependent membrane targeting of Sos , 1997, The EMBO journal.

[19]  M. Moran,et al.  Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras , 1997, Molecular and cellular biology.

[20]  J. Bos,et al.  Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras , 1997, Oncogene.

[21]  Y. Nishizuka,et al.  Taxonomy and function of C1 protein kinase C homology domains , 1997, Protein science : a publication of the Protein Society.

[22]  C. Der,et al.  Guanine nucleotide exchange factors: Activators of Ras superfamily proteins , 1995, BioEssays : news and reviews in molecular, cellular and developmental biology.

[23]  M. Marshall,et al.  Ras target proteins in eukaryotic cells , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[24]  Anirvan Ghosh,et al.  Calcium activation of Ras mediated by neuronal exchange factor Ras-GRF , 1995, Nature.

[25]  M. Valentine,et al.  Sequence Correction , 1995, Science.

[26]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[27]  M. Zerial,et al.  Association of Rap1a and Rap1b proteins with late endocytic/phagocytic compartments and Rap2a with the Golgi complex. , 1994, Journal of cell science.

[28]  D N Shapiro,et al.  Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. , 1994, Science.

[29]  M. Wigler,et al.  Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. , 1993, Science.

[30]  C. Monfries,et al.  A novel functional target for tumor-promoting phorbol esters and lysophosphatidic acid. The p21rac-GTPase activating protein n-chimaerin. , 1993, The Journal of biological chemistry.

[31]  A. Newton,et al.  Interaction of protein kinase C with phosphatidylserine. 2. Specificity and regulation. , 1992, Biochemistry.

[32]  J. de Gunzburg,et al.  Association of the Ras-antagonistic Rap1/Krev-1 proteins with the Golgi complex. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[33]  R. Bell,et al.  Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding, and binding to mixed micelles. , 1989, The Journal of biological chemistry.

[34]  Y. Nishizuka,et al.  Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[35]  P. Blumberg,et al.  Highly lipophilic phorbol esters as inhibitors of specific [3H]phorbol 12,13-dibutyrate binding. , 1985, Cancer research.

[36]  T. Kenakin.  The classification of drugs and drug receptors in isolated tissues. , 1984, Pharmacological reviews.