The tumor suppressor Deleted in Liver Cancer 1 (DLC1) is frequently down-regulated in various human cancers, including breast, prostate, liver, and non-small cell lung cancer (NSCLC). Although the tumor suppressor function of DLC1 depends on its Rho-GAP (GTPase activating protein) activity, which converts Rho from the active GTP-bound form to the inactive GDP-bound form, the mechanisms regulating this activity are poorly understood. Here we determined that CDK5, a predominantly cytoplasmic serine/threonine kinase, negatively regulates Rho-GTP in normal and transformed cells that express DLC1, and that this regulation is attributable to the ability of CDK5 to activate DLC1. We found that CDK5, phosphorylates four serine residues (S120, S205, S422, S509) in the N-terminal region of DLC1, which reduces the binding of the N-terminus to the Rho-GAP domain. The phosphorylation of these serine residues stimulate the tumor suppressor activity of DLC1 - as determined by anchorage-independent growth and tumor xenograft formation - by increasing its Rho-GAP activity and facilitating its binding of several ligands. Serine-to-alanine mutations of the 4 serines increased binding between the DLC1 N-terminus and its Rho-GAP domain, resulting in a mutant protein that lacked these activities. We infer that the N-terminus of DLC1 functions as an auto-inhibitory domain that binds the Rho-GAP domain, and that phosphorylation of the N-terminal 4 serines by CDK5 reduces this interaction, activateing the Rho-GAP and tumor suppressor activities of DLC1. Interestingly, in NSCLC, the expression of CDK5, which has numerous targets, can be associated with a poor prognosis, suggesting that its activity in NSCLC might select for down-regulation of DLC1. Consistent with this hypothesis, we found that siRNA knock-down of endogenous DLC1 in a NSCLC line greatly increased the ability of CDK5 to stimulate neoplastic growth of the line. We also found, in a publicly available tumor database, that poorly differentiated NSCLC is much more likely to express high levels of CDK5 mRNA together with low levels of DLC1 mRNA than is well differentiated NSCLC. Thus, we have identified negative Rho regulation as a new function for CDK5, found DLC1 is the key target for this regulation, elucidated a CDK5-dependent post-translational modification for activating DLC1, and demonstrated the relevance of these observations to NSCLC.

Citation Format: Brajendra K. Tripathi, Xiaolan Qian, Philipp Mertins, Dunrui Wang, Alex Papageorge, Steven Carr, Douglas R. Lowy. CDK5 negatively regulates Rho by phosphorylating and activating the Rho-GAP and tumor suppressor functions of DLC1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1574. doi:10.1158/1538-7445.AM2014-1574