The Rac1 Polybasic Region Is Required for Interaction with Its Effector PRK1

Abstract

Protein kinase C-related kinase 1 (PRK1 or PKN) is involved in regulation of the intermediate filaments of the actin cytoskeleton, as well as having effects on processes as diverse as mitotic timing and apoptosis. It is activated by interacting with the Rho family small G proteins and arachidonic acid or by caspase cleavage. We have previously shown that the HR1b of PRK1 binds exclusively to Rac1, whereas the HR1a domain binds to both Rac1 and RhoA. Here, we have determined the solution structure of the HR1b-Rac complex. We show that HR1b binds to the C-terminal end of the effector loop and switch 2 of Rac1. Comparison with the HR1a-RhoA structure shows that this part of the Rac1-HR1b interaction is homologous to one of the contact sites that HR1a makes with RhoA. The Rac1 used in this study included the C-terminal polybasic region, which is frequently omitted from structural studies, as well as the core G domain. The Rac1 C-terminal region reverses in direction to interact with residues in switch 2, and the polybasic region itself interacts with residues in HR1b. The interactions with HR1b do not prevent the polybasic region being available to contact the negatively charged membrane phospholipids, which is considered to be its primary role. This is the first structural demonstration that the C terminus of a G protein forms a novel recognition element for effector binding.

Nuclear Factor of Activated T Cells c1 Mediates p21-activated Kinase 1 Activation in the Modulation of Chemokine-induced Human Aortic Smooth Muscle Cell F-actin Stress Fiber Formation, Migration, and Proliferation and Injury-induced Vascular Wall Remodeling

“…In addition, we have demonstrated that MCP1-induced VSMC migration and proliferation require PKN1 (22), a Rho GTPase effector (30). However, many studies have reported that Pak1, a Cdc42/Rac1 effector, plays an essential role in the regulation of cell migration and proliferation (31,32).…”

The Rac1 hypervariable region in targeting and signaling

“…This small region shows striking sequence diversity among related GTPases, which has been linked to their differential localization to intracellular membranes, nuclear translocation as well as binding to regulatory-and effector-proteins. [12][13][14][15] In the early '90s, a series of studies established the functional importance of the HVR of Rac GTPases by focusing on its role in the activation of the NADPH oxidase complex. This complex was, at the time, already identified as an established Rac effector, responsible for production of reactive oxygen species in granulocytes.…”

“…Intriguingly, the analysis by Modha et al suggested that the Rac1 C-terminus has a rather fixed structure comprising a proline-based α-helix, followed by an extended stretch of large basic residues. 15 Importantly, the C-terminal portion of this region (Lys 183-Arg185) was proposed to loop back to interact with the core G-domain, in particular with portions of the switch II region and the region C-terminal to helix 3. In addition, several of the basic residues in the PBR contact the cognate binding region in PRK1.…”

“…In addition, several of the basic residues in the PBR contact the cognate binding region in PRK1. 15 Since the Rac1 C-terminus is predicted to interact with its switch II region (i.e., Pro73, Gln74), it is likely that GTP binding does affect the relative position of the C-terminus, and thereby the affinity for effectors such as PRK1. Reminiscent of this is our own finding that Rac1 binding to the ubiquitin ligase Nedd4, for which the Rac1 C-terminus is sufficient, is more efficient for activated Rac1 (Q61 mutant) as compared with inactive Rac1 (N17 mutant).…”

“…48 The analysis by scintillation proximity assays and NMR of the interaction between Rac1 and PRK1 supported a model in which the Rac1 PBR mediates specific interactions with effector proteins. 15,49 In these analyses, the relevance of any of the basic residues for the interaction were underscored and a model depicting the specific contribution of the Rac1 C-terminus in PRK1 binding was presented. Intriguingly, the analysis by Modha et al suggested that the Rac1 C-terminus has a rather fixed structure comprising a proline-based α-helix, followed by an extended stretch of large basic residues.…”