Abstract 3928: Structural basis for the failure of the C1 domain of RasGRP2 to bind phorbol ester
Authors: Agnes Czikora, Daniel J. Lundberg, Nancy E. Lewin, Noemi Kedei, Peter M. Blumberg
Journal: Cancer Research (2015)

Abstract C1 domains represent the recognition motif for the second messenger diacylglycerol (DAG) and for the phorbol esters in protein kinase C, RasGRP, and several other families of signaling proteins. In addition to such typical C1 domains, atypical C1 domains have been identified which possess varying levels of sequence and structural homology with the typical C1 domains but fail to bind DAG or phorbol ester. Among RasGRP family members, RasGRP1 and RasGRP3 possess typical C1 domains, whereas the C1 domain of RasGRP2 stands out as being atypical. In order to better understand the structural constraints for ligand binding, we have analyzed the basis for the failure of the C1 domain of RasGRP2 to bind ligands. Using site-directed mutagenesis, we have identified four critical amino acid residues responsible for the lack of phorbol ester sensitivity. We found that the C1 domain of RasGRP2 had weak binding affinity (Kd = 2890 ± 240 nM) in vitro for [3H]phorbol 12,13-dibutyrate ([3H]PDBu). Replacing all four of critical amino acid residues (Asn7, Ser8, Ala19 and Ile21) with the corresponding residues (Thr7, Tyr8, Gly19 and Leu21, respectively) of RasGRP1 resulted in potent [3H]PDBu binding affinity (Kd = 1.47 ± 0.03 nM) and translocation in response to PMA in the LNCaP cell line. The mutant C1 domains incorporating one to three of these critical residues showed intermediate binding and translocation properties. Of the four substitutions, S8Y made the greatest contribution. Phospholipid requirements for [3H]PDBu binding to the mutant C1 domains were determined; at constant total phospholipid, the requirement for the proportion of phosphatidylserine in phosphatidylserine : phosphatidylcholine mixtures decreased in going from the single S8Y mutant to the quadruple mutant. Binding activity for DAG was also restored in the mutant C1 domains, in parallel with that for phorbol ester. The full length RasGRP2 protein containing the mutated C1 domains likewise showed strong phorbol ester binding, albeit modestly weaker than that of the purified C1 domain (Kd = 8.2 ± 1.1 nM for the full length protein containing all four mutations) and displayed translocation in the LNCaP (human prostate adenocarcinoma) cells in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. A Rap1 GTPase pull-down assay was used to compare the guanine nucleotide exchange activity of wild type and mutated full length RasGRP2 in human embryonic kidney 293 (HEK 293) cells in the presence or absence of phorbol ester. These cells were transfected with either the wild type or the mutant RasGRP2. Consistent with the ability of phorbol ester to induce translocation of the full length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Citation Format: Agnes Czikora, Daniel J. Lundberg, Nancy E. Lewin, Noemi Kedei, Peter M. Blumberg. Structural basis for the failure of the C1 domain of RasGRP2 to bind phorbol ester. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2015-3928