Human CNK1 Acts as a Scaffold Protein, Linking Rho and Ras Signal Transduction Pathways

Abstract

Rho family GTPases act as molecular switches to control a variety of cellular responses, including cytoskeletal rearrangements, changes in gene expression, and cell transformation. In the active, GTP-bound state, Rho interacts with an ever-growing number of effector molecules, which promote distinct biochemical pathways. Here, we describe the isolation of hCNK1, the human homologue of Drosophila connector enhancer of ksr, as an effector for Rho. hCNK1 contains several protein-protein interaction domains, and Rho interacts with one of these, the PH domain, in a GTP-dependent manner. A mutant hCNK1, which is unable to bind to Rho, or depletion of endogenous hCNK1 by using RNA interference inhibits Rho-induced gene expression via serum response factor but has no apparent effect on Rho-induced stress fiber formation, suggesting that it acts as a specific effector for transcriptional, but not cytoskeletal, activation pathways. Finally, hCNK1 associates with Rhophilin and RalGDS, Rho and Ras effector molecules, respectively, suggesting that it acts as a scaffold protein to mediate cross talk between the two pathways.

We thank G. Bokoch, M. Motri, and S. Narumiya for constructs, J. Moran for HeLa cells, M. Motri for advice on luciferase assays and the use of a luminometer, A. Self for advice on dot blots, A. Couve and J. Kittler for advice on GST pulldowns, C. Nobes and A. Schmidt for guidance on injections, N. Billon for discussing RNAi strategies, and the members of the Hall lab for helpful discussion.

This work was generously supported by a program grant from Cancer Research UK. Part of the work reported in this paper was undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer to A.B.J.