Competing for ROCK1

Abstract

The ROCK family of kinases is involved in regulation of the cytoskeleton and cell motility. Cells moving in a 3D environment frequently exhibit an amoeboid type of motility, involving membrane blebbing and movement of rounded cells. Pinner and Sahai identified phosphoinositide-dependent kinase 1 (PDK1) in an RNAi screen for proteins involved in maintenance of a rounded-cell morphology of A375 melanoma cells in collagen gels. In 3D cultures, but not in 2D cultures, reduced abundance of PDK1 in several cancer cell lines resulted in elongated cells and inhibited cell movement, which were similar characteristics to those of cells in which ROCK activity was inhibited. When cells in which PDK1 was knocked down were injected into tail veins of nude mice, the cells tended to be elongated and extravasation of the injected cells into the lung was decreased. Reduction of PDK1 abundance disrupted the organization of myosin light chain at the rear of mobile cells and reduced the accumulation of ROCK1, but not ROCK2, at the plasma membrane. The interaction of PDK1 with ROCK1 did not appear to require phosphorylation of ROCK1 by PDK1 as a catalytically inactive mutant of PDK1 rescued cells from the effects of PDK1 knockdown on morphology, motility, and accumulation of ROCK1 at the plasma membrane. In vitro purified PDK1 interacted with a fragment of ROCK1 that contained the putative PDK1 binding site, and this interaction did not require phosphorylation of ROCK1. ROCK1 was not a substrate for PDK1 in vitro. In vitro experiments suggested that PDK1 and RhoE, a negative regulator of ROCK1, competed for binding to ROCK1, and lysates from cells in which PDK1 was knocked down showed enhanced binding of ROCK1 to RhoE in a pull-down assay. Confirming that RhoE and PDK1 serve as opposing regulators of ROCK1 activity, ROCK1 plasma membrane localization, cellular morphology, and cell motility were restored when both RhoE and PDK1 were depleted using RNAi techniques. Thus, PDK1 appears to have a kinase-independent function in regulation of movement in 3D environments through its ability to recruit ROCK1 to the plasma membrane and prevent the interaction of RhoE with ROCK1, thereby enhancing the activity of ROCK1. S. Pinner, E. Sahai, PDK1 regulates cancer cell motility by antagonizing inhibition of ROCK 1 by RhoE. Nat. Cell Biol. 10 , 127-137 (2008). [PubMed]