N-terminal Dbl domain of the RhoGEF, Kalirin

Abstract

Guanine nucleotide exchange factors (GEF) promote the release of GDP from GTPases, thus allowing the free GTPase molecule to bind the more abundant GTP molecule. In the GTPbound state, the GTPase elicits signal transduction by acting on its effector proteins. Spontaneous release of GDP is a slow process and the catalysis of the GDP release by a GEF is generally a prerequisite for efficient signaling (Vetter and Wittinghofer 2001). The structurally related GEFs form subfamilies that regulate a specific family of GTPase proteins. GEFs that activate Rho GTPases have been implicated in cancer and mental retardation. RhoGEFs are a relatively large family, and many of the ~69 human RhoGEFs were discovered based on their oncogenic activation in cancer and cancer models. The catalytic components of RhoGEFs are referred to as Dbl homology domains, after the screen that identified the protein Dbl encoded by the diffuse B-cell lymphoma (dbl) oncogene (Eva and Aaronson 1985). Thus the RhoGEF family is a potential target for treating tumors and cancer.RhoGEFs contain either the Dbl-homology (DH) domain followed by a pleckstrinhomology (PH) domain or the structurally unrelated domain in the DOCK (dedicator of cytokinesis) family. A preliminary mechanism of GEF action on GTPase was proposed based mainly on available X-ray structures of the complexes GEF/GTPase or GDP/GTPase, but the biophysical basis for GEF activity remains elusive (Vetter and Wittinghofer 2001). Crystallographic studies have shown that the binding site of RhoGEFs for GTPases is located on the DH domain. Upon binding, the DH domain displaces the switch 1 and 2 loops, and the P loop of a RhoGTPase from the orientation required to hold GDP in the catalytic pocket. As a result of these conformational changes in the RhoGTPase, the affinity for a nucleotide decreases and GDP dissociates (Vetter and Wittinghofer 2001). However, in some cases the presence of an adjacent PH domain facilitates, or is even essential for full catalytic activity of the DH domain (Rossman et al. 2005).In this work we describe studies on a DH domain from rat Kalirin, a large multifunctional protein found primarily in the nervous system (Ma et al. 2001;Rabiner et al. 2005). Kalirin is involved in axon and dendritic spine formation, regulation of neuropeptide formation and induction of nitric-oxide synthase (iNOS) activity. Several isoforms of Kalirin have been found that differ in the C-terminus by the presence of additional domains: the second DH/ * Corresponding author. hoch@uchc.edu. Database accession numbersThe chemical shift assignments, NOEs, RDCs and the coordinates of the Kalirin DH1 domain have been deposited to BMRB and RCSB, entries ID 16632 and 2KR9, respectively. et al. 2006), and propose a mechanism of action for CPEPD based on our observations. NIH Public Access Methods and results Protein expression and purificationIsotopically 15 N-and 15 N, 13 C-labeled rat Kalirin DH1 domains and unlabeled Rac1 were overexpressed in E. coli. as GST fusion proteins. Purification was perf...