Cip/Kip proteins: more than just CDKs inhibitors.

Members of the Cip/Kip family of cyclin-dependent kinases inhibitors (CKIs) are well characterized for their role as negative regulators of G1-phase cell-cycle progression (Sherr and Roberts 1999). In eukaryotic cells, progression through the cell cycle is governed by a suite of cyclins and cyclin-dependent kinase (CDKs) complexes (Murray 2004). Regulation of cyclin–CDKs complexes occurs at multiple levels, including assembly of cyclin and CDK subunits, inhibitory and activating phosphorylation and dephosphorylation events, and association of cyclin–CDK complexes with CKIs. During these regulatory processes, cyclin–CDK complexes positively drive progression of the cell cycle, whereas by binding to and inactivating cyclin–CDKs, CKIs negatively regulate progression through the cell cycle. Based on their sequence homology and specificity of action, CKIs are divided into two distinct families: INK4 and Cip/Kip (Sherr and Roberts 1999). Members of the INK4 family, namely p15, p16, p18, and p19 specifically inhibit the activity of CDK4 and CDK6, whereas Cip/Kip members, that is, p21, p27, and p57 inhibit a broader spectrum of cyclin– CDK complexes (el-Deiry et al. 1993; Gu et al. 1993; Harper et al. 1993; Polyak et al. 1994; Toyoshima and Hunter 1994; Lee et al. 1995). Until recently, Cip/Kip members were almost solely viewed as nuclear proteins with a principal function of inhibiting cyclin–CDK activity and hence, cell-cycle progression. However, emerging studies now suggest that Cip/Kip proteins play additional roles outside of the nucleus (Coqueret 2003). Indeed, previous reports have linked p27 to the regulation of actin dynamics and cell migration (Nagahara et al. 1998; McAllister et al. 2003). Moreover, several recent papers have shown that p21 can act as a Rho-kinase (ROCK) inhibitor and that p57 modulates subcellular localization of LIMK, a serine/threonine kinase involved in the regulation of actin filaments (Tanaka et al. 2002; Yokoo et al. 2003; Lee and Helfman 2004). In this issue of Genes & Development, a report by Besson et al. (2004) shows that cytoplasmic p27 modulates actin dynamics by direct regulation of the small GTPase RhoA pathway. p27 was originally identified as an inhibitor of cyclin–CDK complexes in cells arrested by transforming growth factor-beta (TGF; Polyak et al. 1994; Slingerland et al. 1994). Forced expression of p27 results in a G1 phase cell-cycle arrest in most cell types by associating with complexes of cyclins D1–D3:CDK4 or CDK6 and cyclin E or cyclin A:CDK2 (Polyak et al. 1994; Toyoshima and Hunter 1994). Growth arrest by contact inhibition is also thought to be mediated in part by p27 (Polyak et al. 1994). In normal cells, p27 protein levels are highest during G0 and early G1 phases, then rapidly decline in late G1 and S phases (Nourse et al. 1994; Reynisdottir et al. 1995). p27 abundance is primarily controlled by phosphorylation-regulated ubiquitin-mediated proteolysis (Pagano et al. 1995). Phosphorylation of p27 on Thr-187 by CDK2 allows for SCF ubiquitin-protein ligase recognition and subsequent degradation (Carrano et al. 1999; Sutterluty et al. 1999; Tsvetkov et al. 1999). p27 harbors its cyclin–CDK inhibition and binding in an N-terminal domain that contacts both the cyclin and the CDK subunits (Russo et al. 1996). Importantly, to exert its CDK inhibitory functions, p27 is required to be localized in the nucleus. p27 contains a phosphorylation-regulated nuclear localization signal (NLS) harbored in the C terminus (Zeng et al. 2000). Transport of p27 between the nucleus and the cytoplasm is also regulated by phosphorylation events. Upon mitogen stimulation, the human kinase interacting stathmin (hKIS) phosphorylates p27 on Ser 10 to signal its nuclear export into the cytoplasm (Ishida et al. 2000; Rodier et al. 2001; Boehm et al. 2002). Furthermore, phosphorylation of p27 on the NLS Thr-157 by AKT disables its nuclear localization capacity (Liang et al. 2002; Shin et al. 2002; Viglietto et al. 2002). Thus, nuclear binding of cyclin–CDK complexes appears to account for most or all of the previously associated properties of p27 in negative regulation of cell-cycle progression. However, emerging data have suggested a role for cytoplasmic p27 in the regulation of cell migration independent of cyclin–CDK inhibition. Nagahara et al. (1998) first demonstrated that delivery of a transducible form of p27 (TATp27) into HepG2 hepatocellular carcinoma cells induced cell migration or scattering to a similar extent as HGF. Subsequent work found that 1Corresponding author. E-MAIL sdowdy@ucsd.edu; FAX (858) 534-7797. Article and publication are at http://www.genesdev. org/cgi/doi/10.1101/ gad.1205304.