Farnesylation of Cenp-F

Protein farnesylation involves the covalent addition of a 15carbon isoprenoid lipid moiety to the cysteine residue of a Cterminal CAAX motif (reviewed by Sinensky, 2000; Crul et al., 2001). Farnesylated proteins include the Ras GTPase, prelamin A, lamin B, fungal pheromones and rhodopsin kinase. Although most farnesylated proteins are found associated with membranes, farnesylation may also mediate protein-protein interactions. Because farnesylation is required for Ras signalling (Willumsen et al., 1984; Kato et al., 1992) and because activating mutations in Ras have been detected in many human tumours, farnesyl transferase inhibitors (FTIs) have been developed as potential anti-cancer agents. While FTIs can suppress growth of tumour cells with Ha-Ras mutations by inducing G1 growth arrest (Sinensky, 2000; Crul et al., 2001), many other tumour cells exhibit G2/M delays in response to FTIs (Vogt et al., 1997; Ashar et al., 2001; Crespo et al., 2001). The mechanisms underlying FTI-induced G2/M delays are unknown. Chromosome segregation in mitosis depends on kinetochores, complex protein structures that assemble at the centromeres of chromosomes (Rieder and Salmon, 1998). Kinetochores not only tether and move chromosomes along microtubule fibres but they also play a central role in the checkpoint mechanism that delays anaphase onset until all the chromosomes achieve correct, bipolar attachments (Amon, 1999). Although a number of kinetochore components have been identified, their precise roles are only partially defined (Maney et al., 2000; Pidoux and Allshire, 2000). Cenp-F was first identified as a human autoantigen that localises to kinetochores during mitosis (Rattner et al., 1993). During interphase Cenp-F is part of the nuclear matrix but upon entry into mitosis Cenp-F is released into the cytosol and a sub-pool becomes detectable at kinetochores (Casiano et al., 1993; Liao et al., 1995; Zhu et al., 1995a). Cenp-F remains kinetochore associated until anaphase onset when it re-localises to the spindle midzone. Thus, Cenp-F belongs to the family of ‘chromosome passenger’ proteins (Earnshaw and Bernat, 1991). Cenp-F is a cell-cycle-regulated protein, reaching maximum levels in G2/M followed by rapid degradation after mitosis (Liao et al., 1995; Zhu et al., 1995a). The 3210 amino acid protein is mainly coiled coil sequence, containing two internal repeats, several leucine heptad repeats and a bipartite nuclear localisation sequence. Expression of epitope-tagged deletion mutants indicates that the kinetochore localisation domain resides in the C-terminal region of the protein (Zhu et al., 1995b). Yeast two hybrid screens show that the C-terminal domain of Cenp-F is also capable of interacting with itself, the kinetochore-associated kinesin-related motor protein Cenp-E, and the spindle checkpoint component Bub1 (Zhu et al., 1995b; Chan et al., 1998; Jablonski et al., 1998). Although these observations suggest that Cenp-F may play a role in kinetochore assembly and/or the spindle checkpoint, the function of Cenp-F remains obscure. Significantly, Cenp-F and Cenp-E end with CAAX farnesylation sequences and recently both have been shown to be farnesylated (Ashar et al., 2000). These observations raise the possibility that the G2/M effects induced by FTIs are due to inhibition of Cenp-E and Cenp-F. To determine whether Cenp-F plays a role in the spindle checkpoint we generated cell lines expressing a C-terminal Cenp-F mutant. Rather than compromising spindle checkpoint function, expression of this mutant delays progression through G2/M. Significantly, this effect requires an intact CAAX farnesylation motif. We also show that the CAAX motif and farnesyl transferase activity are required for Cenp-F localisation to kinetochores, the nuclear envelope at the G2/M 3403