Phorbol Esters Modulate the Ras Exchange Factor Rasgrp3
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[CANCER RESEARCH 61, 943–949, February 1, 2001] Phorbol Esters Modulate the Ras Exchange Factor RasGRP3 Patricia S. Lorenzo, Justin W. Kung, Drell A. Bottorff, Susan H. Garfield, James C. Stone, and Peter M. Blumberg1 Laboratory of Cellular Carcinogenesis and Tumor Promotion [P. S. L., J. W. K., P. M. B.], and Laboratory of Experimental Carcinogenesis [S. H. G.], National Cancer Institute, Bethesda, Maryland 20892-4255, and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada [D. A. B., J. C. S.] ABSTRACT for Rap1 and has been referred to as CalDAGI (11) or GRP2 (12). In a recent study, Clyde-Smith et al. (13) described an alternative spliced RasGRP represents the prototype of a new class of guanine nucleotide variant of this Rap GEF, named RasGRP2, which possesses GEF exchange factors that activate small GTPases. The guanyl nucleotide- activity for N-Ras, K-Ras, and Rap1. RasGRP2 is located in chromo- releasing protein (GRP) family members contain catalytic domains re- lated to CDC25, the Ras exchange factor of Saccharomyces cerevisiae. some 11p13, a region frequently amplified in human tumors (10, 13). They also contain a motif resembling a pair of calcium-binding EF-hands Finally, the KIAA0846 sequence (14) was reported to encode a GEF and a C1 domain similar to the diacylglycerol interaction domain of (CalDAGIII or GRP3), which can activate both Ras and Rap1 (12, protein kinase C. The sequence of KIAA0846, identified in a human brain 15). We refer to this third member of the family as RasGRP3. Despite cDNA library, encodes a member of the GRP family that we refer to as the differences in substrate activity, all of the GRP members share RasGRP3. We show here that RasGRP3 bound phorbol esters with high similar overall domain structure. They have the Ras GEF signature affinity. This binding depended on anionic phospholipids, which is char- motif CDC25 (Ras GEF of Saccharomyces cerevisiae), a pair of acteristic of phorbol ester binding to C1 domain proteins. In addition, atypical EF-hands (a calcium-binding motif), and the C1 domain. phorbol esters also caused activation of the RasGRP3 exchange activity in Recent studies have revealed that RasGRP functions as a critical intact cells, as determined by an increase in RasGTP and phosphorylation molecule in thymocyte differentiation and T-cell activation, linking of the extracellular-regulated kinases. Finally, both phorbol 12-myristate 13-acetate and the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol in- the T-cell receptor and diacylglycerol messengers to Ras signaling duced redistribution of RasGRP3 to the plasma membrane and/or perinu- (16, 17). Moreover, phorbol esters can directly interact with RasGRP clear area in HEK-293 cells, as demonstrated using a green fluorescent with nanomolar affinity, promote the EF activity, and thereby activate fusion protein. We conclude that RasGRP3 serves as a PKC-independent Ras signaling in T cells. In this study, we present evidence that pathway to link the tumor-promoting phorbol esters with activation of RasGRP3 is also a high-affinity receptor for phorbol esters. Not only Ras GTPases. did RasGRP3 bind phorbol esters with nanomolar affinity but also its GEF activity for Ras was increased by phorbol ester treatment in the INTRODUCTION intact cell. In addition, RasGRP3 redistributed to particulate compart- ments in response to phorbol ester treatment. Taken together, these Phorbol esters are natural products first recognized as potent tumor- findings suggest that RasGRP3 serves as a high-affinity target for the promoting agents in skin. The first described cellular target for phor- tumor-promoting phorbol esters, inducing PKC-independent activa- 2 bol esters was PKC, , and still today, most of the actions of these tion of Ras. The widespread tissue distribution of RasGRP3 and its compounds are considered to be mediated by PKC pathways. How- ability to modulate the small GTPase Ras may be relevant in the ever, emerging evidence has revealed new cellular receptors, which context of the phorbol ester tumor-promoting activity. respond both in vivo and in vitro with sensitivity similar to PKC. The chimerins (1) and Munc-13 proteins (2) are non-PKC members of the phorbol ester receptor family. They all have a C1 domain, the signa- MATERIALS AND METHODS ture motif that is involved in the recognition of the phorbol ester molecule and its physiological counterpart, the second messenger Plasmids. The coding sequence of human RasGRP3 (KIAA0846) was subcloned into the vector pMAL-c2 (New England Biolabs, Beverly, MA) diacylglycerol (3, 4). RasGRP (5, 6), which also possesses a C1 motif, downstream from the MBP gene. The construct, which coded for the fusion represents a novel class of phorbol ester targets that functions as protein MBP-RasGRP3, was used for bacterial expression and purification. To GEFs. produce a construct suitable for mammalian expression and confocal studies, The GEFs are proteins that catalyze the dissociation of GDP from the cDNA of RasGRP3 was amplified by PCR and subcloned into the pQBI25 Ras GTPases, to allow the formation of the active GTP-bound con- vector (Quantum Biotechnologies Inc., Montreal, Canada) using the NheI site. formation (7). Examples of GEFs for Ras include the well-character- This generated a fusion protein (RasGRP3-GFP) between the COOH terminus ized GEF SOS (8) and the calcium-dependent RasGRF (9). RasGRP of RasGRP3 and the NH2 terminus of the red-shifted GFP tag. RasGRP3 was represents the prototype of a new class of GEFs that is composed of also subcloned in the retroviral vector pBabePuro (5) for generation of a at least three members. RasGRP was the first member characterized as RasGRP3-Puro virus. a GEF for Ras (5). The related coding sequence HDC25L, described Cell Culture. HEK-293 cells were propagated in Eagle’s MEM adjusted to contain 0.1 mM nonessential amino acids (American Type Culture Collection, in 1997 as a potential Ras activator (10), was later shown to be a GEF Manassas, VA) and supplemented with 10% heat-inactivated horse serum (Life Technologies, Gaithersburg, MD). Cell transfections with pQBI25 or Ras- Received 3/17/00; accepted 11/20/00. GRP3-pQBI25 constructs were performed using LipofectAMINE Plus (Life The costs of publication of this article were defrayed in part by the payment of page Technologies). For generation of stable transformants expressing either Ras- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. GRP3-GFP or the GFP protein alone, cells were selected in the presence of 500 1 To whom requests for reprints should be addressed, at 37 Convent Drive MSC 4255, g/ml of the antibiotic G-418 (Life Technologies) for 2 weeks. For generation Building 37/Room 3A01, Bethesda, MD 20892-4255. Phone: (301) 496-3189; Fax: (301) of stable cell lines of RasGRP3 in rat2 fibroblasts, cells were infected with the 496-8709; E-mail: [email protected]. 2 RasGRP3-Puro virus and selected in the presence of puromycin. The abbreviations used are: PKC, protein kinase C; GEF, guanine nucleotide ex- 3 3 3 change factor; MBP, maltose-binding protein; GFP, green fluorescent protein; PDBu, Binding of [ H]PDBu. Binding of [ H] [ H]PDBu (777 Gbq/mmol; New phorbol 12, 13-dibutyrate; POPS, 1-palmitoyl-2-oleoyl-phosphatidylserine; POPA, England Nuclear, Boston, MA) was measured as described elsewhere (18). The 1-palmitoyl-2-oleoyl-phosphatidic acid; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcho- assay mixture contained 50 mM Tris-HCl (pH 7.4), 1 mg/ml IgG, 0.1 mM line; PMA, phorbol 12-myristate 13-acetate; RBD, Ras-binding domain; GST, glutathione 14 14 CaCl , RasGRP3 protein, and the corresponding lipid mixture. Incubations S-transferase; DOG, 1,2-dioctanoyl-sn-glycerol; [ C]DPPC, L-[1- C]dipalmitoylphos- 2 phatidylcholine; ERK, extracellular signal-regulated kinase; FISH, fluorescence in situ were carried out at 18°C. Nonspecific binding was measured using an excess hybridization; DAG, 1,2-diacyl-sn-glycerol; GRP, guanyl nucleotide-releasing protein. of PDBu (30 M), and specific binding was done in triplicate at each ligand 943 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2001 American Association for Cancer Research. RasGRP3 AND PHORBOL ESTERS concentration (0.31–20 nM). Recombinant RasGRP3 produced in Escherichia peroxidase-conjugated antimouse antibody (Bio-Rad). Aliquots of total lysates coli was used for the assays. Briefly, BL-21 bacteria, transformed with the (20 g) were also analyzed for quantitation of total Ras. For the rat2 cells RasGRP3-pMAL-p2 construct, were induced using 0.3 mM isopropyl--D- transfected with the RasGRP3-Puro virus, cells were plated in a 10-cm dish thiogalactopyranoside for2hat37°C. The bacterial culture was then centri- overnight and then treated with PMA or solvent. Lysates were incubated for 30 fuged for 20 min at 4,000 ϫ g at 4°C, and the pellet was resuspended in min with GST-RasRBD coupled to gluthathione beads. Samples were run on column buffer [20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM SDS-PAGE, blotted onto nitrocellulose membranes and probed with a mixture -mercaptoethanol, and protease inhibitors] and frozen overnight at Ϫ20°C. of a panRas R02120 antibody 1:500 dilution (BD Biosciences, Los Angeles, After thawing, cell lysis was completed by six 15-s pulses of sonication over CA) and K-Ras antibody 1:100 dilution (Santa Cruz Biotechnologies, Santa 2 min. The lysate was centrifuged at 4°C for 20 min at 14,000 ϫ g, and the Cruz, CA) followed by horseradish peroxidase antimouse antibody. supernatant was used as the crude extract for the subsequent purification. The Confocal Microscopy Studies. HEK-293 cells were seeded onto 40-mm MBP-RasGRP3 fusion protein was purified using an amylose resin according to circular glass coverslips at a density of 5–10 ϫ 104 cells/coverslip.