The SAM domain of the RhoGAP DLC1 binds EF1A1 to regulate cell migration

Abstract

Deleted in liver cancer 1 (DLC1) is a multi-modular Rho-GTPase-activating protein (RhoGAP) and a tumor suppressor. Besides its RhoGAP domain, functions of other domains in DLC1 remain largely unknown. By protein precipitation and mass spectrometry, we identified eukaryotic elongation factor 1A1 (EF1A1) as a novel partner for the sterile alpha motif (SAM) domain of DLC1 but not the SAM domain of DLC2. The solution structure of DLC1 SAM revealed a new monomeric fold with four parallel helices, similar to that of DLC2 SAM but distinct from other SAM domains. Mutating F38, L39 and F40 within a hydrophobic patch retained its overall structure but abolished its interaction with EF1A1 with F38 and L39 forming an indispensable interacting motif. DLC1 SAM did not localize to and was not required for DLC1 to suppress the turnover of focal adhesions. Instead, DLC1 SAM facilitated EF1A1 distribution to the membrane periphery and ruffles upon growth factor stimulation. Compared with wild-type DLC1, the non-interactive DLC1 mutant is less potent in suppressing cell migration, whereas overexpression of the DLC1 SAM domain alone, but not the non-interactive mutant SAM or DLC2 SAM, greatly enhanced cell migration. This finding reveals a novel contribution of the SAM-EF1A1 interaction as a potentially important GAP-independent modulation of cell migration by DLC1.

Epidermal Growth Factor Activates the Rho GTPase-activating Protein (GAP) Deleted in Liver Cancer 1 via Focal Adhesion Kinase and Protein Phosphatase 2A

“…Plasmid Construction-DLC1 was cloned into FLAG-and GFP-pXJ40 mammalian expression vectors (15). The truncation, deletion, and point mutants of DLC1 were generated using specific primers.…”

“…1A). The DLC1-R677E mutant is not capable of inactivating the active RHOA in the cells because of the mutation in the catalytic arginine finger, which is indispensable for its RhoGAP activity (9,15,29). As expected, WT but not the mutant DLC1 inactivated RHOA.…”

“…In addition to its RHOGAP domain, DLC1 contains the sterile alpha motif (SAM) and steroidogenic acute regulatory protein (StAR)-related lipid transfer protein modules and a unique serinerich region (SRR). The START (12,13), RHOGAP (14), SAM (15,16) and SRR regions have been implicated in the regulation of cell morphology, migration, and tumor suppression.…”

PKA-induced dimerization of the RhoGAP DLC1 promotes its inhibition of tumorigenesis and metastasis

“…In addition, mouse hepatoma cells with silenced DLC1 exhibit increased active RhoA levels and actin stress fibre formation, which provides overwhelming evidence for the activation of Rho as a consequence of deregulated DLC1 and stresses the importance of DLC1 RhoGAP activity in tumorigenesis in vivo. Nevertheless, RhoGAP-independent signalling may also contribute to the biological activities of DLC1 in different cellular contexts 6,7,[15][16][17] . Therefore, it is important to comprehend how the RhoGAP activity of DLC1 is regulated and associated with its functional effects in cancer cells.…”

Loss of DLC1 is an independent prognostic factor in patients with oral squamous cell carcinoma

“…21,30,31 Several binding partners of DLC1 have been identified, including members of the tensin family of focal adhesion proteins (eg, tensin1, tensin2, and C-terminal tensin like (cten)), p120RasGAP, elongation factor 1A1 (EF1A1), and S100A10, which further delineates its mechanism and its role in inhibiting cancer cell migration, proliferation, and metastasis. 30,[32][33][34][35] DLC1 gene silencing promotes pro-angiogenic responses through upregulation of vascular endothelial growth factor, accompanied by the accumulation of hypoxia-inducible factor 1 alpha and its nuclear localization. 36 Since its discovery, compelling evidence has accumulated that demonstrates a role for DLC1 as a bonafide tumor suppressor gene in different types of human cancers.…”