Planificación del proyecto

The CIP/KIP family includes p21^^^ (El-Deiry et a l, 1993; Gu et a l, 1993; Harper et a l, 1993; Noda et a l, 1994), p27^^^ (Hengst et a l, 1994; Polyak et a l,

1994a; Polyak et a l, 1994b; Toyoshima and Hunter, 1994) and p57*^“’^ (Lee et a l,

1995; Matsuoka et a l, 1995). p21^^^^ was cloned via a number of approaches, implicating it in a wide range of cellular activities, such as checkpoint control (Gu

et a l, 1993; Harper et a l, 1993; Xiong et a l, 1993), differentiation (Halevy et a l.

1995; Jiang and Fisher, 1993; Steinman et al., 1994), senescence (Noda et al. 1994; Tahara et a l, 1995; Zhang et a l, 1994) and apoptosis (El-Deiry et a l, 1994; El- Deiry et a l, 1993). p27™*^ appears to be primarily responsible for regulating CDK activity in response to extracellular growth inhibitory signals (Polyak et a l, 1994b; Slingerland et a l, 1994; Toyoshima and Hunter, 1994) and restricting cellular proliferation during the course of development (Fero et a l, 1996; Kiyokawa et a l,

1996; Nakayama et a l, 1996). p21^^^ and p27^* are widely expressed in tissues, unlike p57^^^ which is highly tissue specific (Lee et a l, 1995; Matsuoka et a l,

1995). There is a good correlation between p57™^ expression and the differentiated state of cells, suggesting that p57^^^ plays a role in cell cycle exit associated with terminal differentiation during mouse development (Matsuoka et a l, 1995).

The proteins are grouped together largely because they share a 60 amino acid domain of similarity (39% to 47%) in the amino-terminal half of each protein which is necessary for cyclin binding and CDK inhibitory functions. They also have nuclear localisation signals near the carboxy terminus, but outside these regions, the three proteins have no resemblance except for a short segment of similarity near the carboxy-terminus of p27^^^^ and p57^^^^ (Chen et a l, 1995; Luo et a l, 1995; Toyoshima and Hunter, 1994).

The CIP/KIP proteins exhibit a broad specificity for cyclin-CDK complexes

in vitro, binding efficiently to cyclin A-CDK2, cyclin E-CDK2, cyclin D-CDK4 and less efficiently to cyclin B-cdc2 (El-Deiry et a l, 1993; Harper et a l, 1993; Lee

et a l, 1995; Matsuoka et a l, 1995; Polyak et a l, 1994b; Toyoshima and Hunter, 1994). p21^“’* and p27^^^^ bind to cyclin-CDK complexes, making contacts with both subunits but do not associate with kinase subunits unless a cyclin is present (Chen et a l, 1995; Hall et a l, 1995; Harper et a l, 1995). CKI association with cyclin-CDK complexes in vitro is correlated with a reduction in CDK kinase activity (Harper et a l, 1993; Lee et a l, 1995; Matsuoka et a l, 1995; Polyak et a l,

1994a; Polyak et a l, 1994b; Toyoshima and Hunter, 1994; Xiong et a l, 1993). The overexpression of CIP/KIP proteins in normal fibroblasts results in a G l arrest (Harper et a l, 1993; Lee et a l, 1995; Matsuoka et a l, 1995; Polyak et a l, 1994a; Polyak et a l, 1994b; Toyoshima and Hunter, 1994; Xiong et a l, 1993) suggesting a

role as inhibitors of G l cyclin-CDK complexes. However, cell lines engineered for inducible expression of p21^“’* elicit both a G l and a G2/M arrest (Bates et al.,

1998b; Cayrol et al., 1998; Medema et al., 1998), and recent reports have indicated other functional activities for the CIP/KIP proteins apart from CDK inhibition (see below).

Surprisingly for a protein which blocks cell cycle progression, p21^^^^ expression was also shown to be induced when quiescent cells were stimulated to proliferate (Li et al., 1994a; Noda et al., 1994), and cyclin-CDK complexes in proliferating cells have been reported to include p21^°*^ (Harper et al., 1995; Zhang

et al., 1994). This apparent paradox led to the suggestion that p21^*^ can exist in active and inactive cyclin complexes depending on the concentration of p21^^^^ (Zhang et al., 1994). It was proposed that cyclin-CDK complexes are active when associated with one molecule of p21^^^ and inactive when they contain multiple p21^^^ molecules, at least for cyclin D-CDK4 complexes (Harper et a l, 1995; Zhang et al., 1994). These results run contrary to the crystallographic data on cyclin A-CDK-p27*^^^* ternary complexes (Russo et al., 1996). The structure reveals that p2 7 Kipi separate binding sites on the cyclin and CDK subunits consistent with

cooperative binding, but indicates that complexes containing multiple p27^* molecules would be thermodynamically unstable (Russo et al., 1996). Subsequent

in vivo data confirmed the structural indications, that cyclin A-CDK2 complexes are fully inhibited by one molecule of p21 (Hengst et al., 1998).

However, it may not be appropriate to extrapolate data for cyclin A-CDK2 complexes to cyclin D-CDK4 complexes. The association of cyclin D with CDK4 may require an assembly factor (Kato et a l, 1994; Matsushime et al., 1994) and there is evidence that the CIP/KIP proteins may perform such a function promoting kinase activity at low concentrations and inhibiting kinase activity at higher concentrations (LaBaer et al., 1997). p21^^' and p27™’* appear to target cyclin D l- CDK4 to the nucleus (Cheng et a l, 1999; LaBaer et a l, 1997; Reynisdôttir and Massagué, 1997) and in the absence of p 2 l‘^°’* and p27^^’’\ the majority of the cyclin D l remains in the cytoplasm during the cell cycle (Cheng et a l, 1999). The

current data indicate that although CIP/KIP proteins can exist in both CDK4 and CDK2 complexes the latter are the prime targets for inhibition.

Although there have been reports that p21^^^^ expression during the cell cycle is controlled by the E2F family of transcription factors (Hiyama et al., 1998; and see later in section 1.4.3.) the major regulator of p21^“** is believed to be the p53 tumour suppressor (see section 1.6.). Thus, p53 binding sites are present in the p21^^^ promoter and it was cloned in a screen for p53 responsive genes (El-Deiry et al. 1993; El-Deiry gr a/. 1995).

In contrast, p27’“ ’* transcription is constant throughout the cell cycle but the protein is more stable in quiescent cells. p27™'^ is regulated in part by proteolysis through the ubiquitin/proteasome pathway, and it has been suggested that quiescent cells contain lower amounts of ubiquitinating activity compared to proliferating cells (Pagano etal., 1995).