The Rac1 C-terminal Polybasic Region Regulates the Nuclear Localization and Protein Degradation of Rac1

Abstract

We observed evolutionary conservation of canonical nuclear localization signal sequences (K(K/R)X(K/R)) in the C-terminal polybasic regions (PBRs) of some Rac and Rho isoforms. Canonical D-box sequences (RXXL), which target proteins for proteasome-mediated degradation, are also evolutionarily conserved near the PBRs of these small GTPases. We show that the Rac1 PBR (PVKKRKRK) promotes Rac1 nuclear accumulation, whereas the RhoA PBR (RRGKKKSG) keeps RhoA in the cytoplasm. A mutant Rac1 protein named Rac1 (pbrRhoA), in which the RhoA PBR replaces the Rac1 PBR, has greater cytoplasmic localization, enhanced resistance to proteasome-mediated degradation, and higher protein levels than Rac1. Mutating the D-box by substituting alanines at amino acids 174 and 177 significantly increases the protein levels of Rac1 but not Rac1(pbrRhoA). These results suggest that Rac1 (pbrRhoA) is more resistant than Rac1 to proteasomemediated degradative pathways involving the D-box. The cytoplasmic localization of Rac1(pbrRhoA) provides the most obvious reason for its resistance to proteasome-mediated degradation, because we show that Rac1(pbrRhoA) does not greatly differ from Rac1 in its ability to stimulate membrane ruffling or to interact with SmgGDS and IQGAP1-calmodulin complexes. These findings support the model that nuclear localization signal sequences in the PBR direct Rac1 to the nucleus, where Rac1 participates in signaling pathways that ultimately target it for degradation.

Rac1/osmosensing scaffold for MEKK3 contributes via phospholipase C-γ1 to activation of the osmoprotective transcription factor NFAT5

“…Rac1 is located in the nucleus as well as in the cytoplasm (22). siRNA against Rac1 knocks it down more in the nucleus than in the cytoplasm (Fig.…”

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

“…Localization of Rac1 to the membrane and cytoplasm is important for Rac1 activity and function in promoting lamellipodia formation [75][76][77]. Nuclear accumulation of Rac1 has been shown to affect stability of Rac1 and eventually lead to Rac1 protein degradation [77,82,83]. Both the disruption of lamellipodia and localization of Rac1 to the nucleus in P19 cells expressing miR-124 suggest that miR-124 may indirectly downregulate the activity of Rac1 in the cytoplasm.…”

The Rac1 hypervariable region in targeting and signaling

“…The Rac1 PBR comprises, in contrast to that of RhoA, a nuclear localization signal and Rac1 mutants lacking the PBR, do not localize to the nucleus. 13 These authors further proposed that smgGDS-promoted Rac1 activation stimulated nuclear translocation of the complex, whereas smgGDS binding to and activating RhoA would promote its cytoplasmic accumulation, due to the absence of a NLS in RhoA.…”

“…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.…”

“…The Rac1 C-terminus harbors a nuclear localization signal (NLS) represented by its PBR. 13,23 A fusion protein in which the Rac1 C-terminus is coupled to GFP shows a nuclear localization, indicating that the Rac1 C-terminus is sufficient to drive proteins into the nucleus of MDCK, COS-1, porcine aortic endothelial (PAE) cells as well as ECV304 human bladder carcinoma, HeLa and NIH 3T3 cells. 24 The NLS is inactive when Rac1 is bound to RhoGDI, which explains its cytosolic localization.…”