Published Online: 28 April, 2008 | Supp Info: http://doi.org/10.1083/jcb.200801047 JCB: ARTICLE Downloaded from jcb.rupress.org on May 9, 2018 Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division David Michaelson , 2,5 Wasif Abidi , 2,5 Daniele Guardavaccaro , 4,5 Mo Zhou, 3,5 Ian Ahearn , 3,5 Michele Pagano , 4,5 and Mark R. Philips 1,2,3,5 1 Department of Medicine, 2 Department of Cell Biology, 3 Department of Pharmacology, 4 Department of Pathology, and the 5 New York University Cancer Institute, New York University School of Medicine, New York, NY 10016 ac1 regulates a wide variety of cellular processes. zation of cells stably expressing low levels of GFP-Rac1, The polybasic region of the Rac1 C terminus func- and time-lapse microscopy of asynchronous cells revealed Rtions both as a plasma membrane – targeting motif Rac1 accumulation in the nucleus in late G2 and exclu- and a nuclear localization sequence (NLS). We show that sion in early G1. Although constitutively active Rac1 re- a triproline N-terminal to the polybasic region contributes stricted to the cytoplasm inhibited cell division, activated to the NLS, which is cryptic in the sense that it is strongly Rac1 expressed constitutively in the nucleus increased the inhibited by geranylgeranylation of the adjacent cysteine. mitotic rate. These results show that Rac1 cycles in and out Subcellular fractionation demonstrated endogenous Rac1 of the nucleus during the cell cycle and thereby plays a in the nucleus and Triton X-114 partition revealed that this role in promoting cell division. pool is prenylated. Cell cycle – blocking agents, synchroni- Introduction Rac1 is among the most extensively characterized members of a major role in regulating the signaling output of promiscuous the Rho family of small GTPases. Like all GTPases, Rac1 func- regulatory proteins such as Rac1 ( Mor and Philips, 2006 ). tions as a molecular switch regulated by GTP/GDP exchange. Like all Rho proteins, Rac1 is targeted within cells by Rac1 regulates a wide variety of cellular functions including ac- posttranslational modifi cation of a C-terminal CAAX motif by tin remodeling for cell ruffl ing, adherens junction formation, prenylation, proteolysis, and carboxyl methylation and by as- cell motility, and polarity. Other functions of Rac1 include tran- sociation with a cytosolic chaperone, Rho guanosine nucleo- scriptional activation and regulation of the NADPH oxidase tide dissociation inhibitor (RhoGDI; Michaelson et al., 2001 ). ( Jaffe and Hall, 2005 ). Rac1 has also been implicated in cellular In resting cells, Rac1 is found in the cytosol as a soluble 1:1 transformation and may promote cell cycle progression through complex with RhoGDI. Upon activation, Rac1 is discharged THE JOURNAL OF CELL BIOLOGY induction of cyclin D1 ( Westwick et al., 1997 ). from RhoGDI and displays affi nity for the plasma membrane Rac1 is regulated by numerous guanine nucleotide ex- ( Michaelson et al., 2001 ). This affi nity can be explained by the change factors (GEFs) and several GTPase-activating proteins geranylgeranyl modifi cation of the Rac1 C terminus that func- (GAPs) and signals by interacting with a large set of effectors tions in conjunction with a strong polybasic region immedi- ( Jaffe and Hall, 2005 ). The specifi cities of the several GEFs and ately adjacent to the prenylcysteine ( Michaelson et al., 2001 ). GAPs and numerous effectors that interact with Rac1 may ex- In its plasma membrane – binding capacity, Rac1 behaves like plain its myriad functions. However, differential regulation of K-Ras4B, which also has a strong polybasic region. The poly- signaling by Rac1 in different contexts is poorly understood. basic region binds via electrostatic interactions with the nega- Increasing evidence suggests that subcellular localization plays tively charged inner leafl et of the plasma membrane ( Yeung et al., 2006 ). Recently, we have shown that the plasma mem- D. Michaelson and W. Abidi contributed equally to this paper. brane localization of Rac1 is modulated during phagocytosis Correspondence to M.R. Philips: [email protected] by loss of the negative charge on the inner leafl et of the mem- Abbreviations used in this paper: GAP, GTPase-activating protein; GEF, guanine brane ( Yeung et al., 2006 ). nucleotide exchange factor; IF, immunofl uorescent; NLS, nuclear localization In addition to the cytosol and plasma membrane, GFP-Rac1 sequence; PAE, porcine aortic endothelial; RhoGDI, Rho guanosine nucleotide dissociation inhibitor. has been localized to the nuclear envelope ( Kraynov et al., 2000 ; The online version of this paper contains supplemental material. Michaelson et al., 2001 ) and nucleoplasm ( Michaelson et al., 2001 ; © 2008 Michaelson et al. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 181 No. 3 485–496 www.jcb.org/cgi/doi/10.1083/jcb.200801047 JCB 485 Lanning et al., 2003 ). Lanning et al., (2003, 2004) identifi ed the type, we studied the distribution of these proteins in multiple polybasic sequence of the Rac1 hypervariable region as a nu- cell lines including MDCK, COS-1, and porcine aortic endo- clear localization sequence (NLS), raising the question of how thelial (PAE) cells ( Fig. 1, B and C ) and ECV304 human blad- a single motif can target a protein to two distinct compartments, der carcinoma, HeLa, and NIH 3T3 (not depicted). GFP-Rac1 the plasma membrane and the nucleus. These investigators ranged from 5 ± 2% nuclear in MDCK cells to 30 ± 5% nu- also found that the NLS of Rac1 was partially responsible clear in COS-1 cells ( Fig. 1 C ). Constitutively GTP-bound for the accumulation in the nucleus of the armadillo repeat GFP-Rac1L61 was distributed in a pattern similar to that of proteins smgGDS and p120 catenin ( Lanning et al., 2003 ) and wild-type GFP-Rac1 in each of the cell types. Moreover, GFP was required for efficient proteosomal degradation of Rac1 extended with the 11 C-terminal amino acids of Rac1 showed ( Lanning et al., 2004 ). In these studies, a constitutively GTP- a higher degree of nuclear localization relative to the full- bound form of Rac1 was slightly more effi cient in nuclear entry length protein in all cell lines studied. These observations sug- ( Lanning et al., 2003, 2004 ). Rac1, in association with MgcRac- gest that the polybasic 11 – amino acid C-terminal sequence GAP, has also been implicated in the nuclear import of STAT5 contains an NLS and that the GTP-binding state of the mole- ( Kawashima et al., 2006 ). Spatiotemporal studies of Rac1 acti- cule does not affect the NLS. vation in live cells using fl uorescence resonance energy transfer Because the canonical NLS of the polyomavirus large (FRET)-based biosensors have revealed confl icting results with T antigen begins with a diproline, we sought to determine if the regard to the activation state of nuclear Rac1. Kraynov et al., unusual triproline motif immediately upstream of the polybasic (2000 ) found a large pool of GFP-Rac1 in the nucleoplasm that region in the Rac1 C terminus contributes to the NLS. GFP ex- remained inactive. In contrast, Wong and Isberg ( 2005 ) detected tended with the C-terminal 21 amino acids of Rac1 that in- active GFP-Rac1 in the nucleus but only in cells infected with cluded the triproline sequence was signifi cantly more nuclear Yersinia strains that secrete YopT, a prenylcysteine endoprote- that either GFP extended with the 11 – amino acid tail or full- ase that relocated Rac1 to the nucleus. Yoshizaki et al., (2003 ) length Rac1 in all cell types ( Fig. 1 C ). Indeed, in PAE cells, used an intramolecular FRET probe that assesses the balance this construct was nuclear in 80 ± 5% of cells. In contrast, of GEFs and GAPs for Rac1 in cells undergoing mitosis and when a 21 – amino acid tail was used in which the triproline found that the balance favored inactive Rac1 in the region of the motif was mutated to trialanine, the sequence was no more po- mitotic spindle. tent in directing GFP into the nucleus than was the 11 – amino We have studied the structure and regulation of the Rac1 acid tail. Thus, the triproline motif immediately N-terminal to NLS and the basis for the seemingly stochastic nature of its en- the polybasic sequence contributes signifi cantly to the NLS of gagement. We show for the fi rst time that a pool of endogenous Rac1. Neither GFP-Rac2 nor GFP-Rac3 was observed in the Rac1 is nuclear. We fi nd that a triproline motif adjacent to the nucleus ( Michaelson et al., 2001 ; Chan et al., 2005 ). The for- polybasic sequence contributes to the NLS and that the NLS is mer has a PQP motif upstream of the polybasic region and the cryptic in the sense that it is inhibited by the adjacent geranyl- later retains the same triproline sequence as Rac1. However, geranyl modifi cation. Despite the inhibitory effect of prenyl- both have weak polybasic sequences relative to that of Rac1. ation on the NLS, we found that endogenous nuclear Rac1 is Substitution of the K-Ras4B polybasic region (KKKKKKSKTK) lipidated. We found that the distinct populations of cells with for that of Rac1 (VKKRKRK) did not reconstitute the NLS, and without nuclear expression of Rac1 could be explained by a despite retention of the triproline sequence ( Fig. 1 D ). Thus, a cell cycle dependence on nuclear import: Rac1 accumulated in polyproline sequence does not confer NLS activity upon all poly- the nucleus in late G2 and was excluded from this compartment basic sequences and there appears to be specificity in the in early G1. Although nuclear-targeted GTP-bound Rac1 accel- polybasic sequence of Rac1 that is preceded by a hydrophobic erated cell division, GTP-bound Rac1 restricted to the cyto- valine and has a net charge of +6.