Sphase

CDK2/Cyclin E Complexes

Upregulation of cyclin E expression begins late in G1 and is maintained into S-phase (reviewed in [24]). The initial CDK2/cyclin E complexes that form are maintained in an inactive state by CIP/KIP inhibitor binding. Complete activation of CDK2/cyclin E complexes requires titration of CIP/KIP proteins from CDK2/cyclin E complexes in mid-G1 inactivating CDK4/cyclin D associations (reviewed in [72]) (Fig. 9.5).

figure 9.5 The balance of CIP/KIP inhibitor binding to CDK/cyclin complexes in G1. (A) During early G1, the majority of CIP/KIP inhibitors are bound to CDK4/cyclin D complexes. Binding of CIP/KIP inhibitors to these complexes does not affect their activity, however, CDK2/cyclin E/CIP/KIP complexes are inactive. (B) Mid-G1 signals a shift of CIP/KIP molecules from CDK4/cyclin D complexes to CDK2/cyclin E complexes. Phosphorylation of CDK2 at this stage partially activates the kinase complex which retains the ability to bind to CIP/KIP. (C) Late in G1 phosphorylation of CIP/KIP molecules targets the proteins for degradation leading to complete activation of CDK2/cyclin E kinases and, hence, progression through S-phase. See Plate 9.5 in Color Plate Section.

figure 9.5 The balance of CIP/KIP inhibitor binding to CDK/cyclin complexes in G1. (A) During early G1, the majority of CIP/KIP inhibitors are bound to CDK4/cyclin D complexes. Binding of CIP/KIP inhibitors to these complexes does not affect their activity, however, CDK2/cyclin E/CIP/KIP complexes are inactive. (B) Mid-G1 signals a shift of CIP/KIP molecules from CDK4/cyclin D complexes to CDK2/cyclin E complexes. Phosphorylation of CDK2 at this stage partially activates the kinase complex which retains the ability to bind to CIP/KIP. (C) Late in G1 phosphorylation of CIP/KIP molecules targets the proteins for degradation leading to complete activation of CDK2/cyclin E kinases and, hence, progression through S-phase. See Plate 9.5 in Color Plate Section.

This event renders the CDK2/cyclin E kinase complex ''active'' to phosphorylate additional residues on pRB (hyperphosphorylated pRB) and permit progression through the G1/S transition [73-75]. These additional phosphorylation events (hyperphosphorylated pRB) result in complete inactivation of pRB's repressive function towards E2F transcription factors in late G1 facilitating the E2F-mediated transcription of genes required for DNA synthesis (refer to Fig. 9.3). In the absence of a functional interaction between cyclin E and CDK2, a cell is not able to pass beyond the restriction point [76].

The importance of CDK2 to cell cycle progression is underscored in cell culture studies that examined the function of dominant negative CDK2 (CDK2DN) protein variants (isoforms). CDK2dn remains consti-tutively inactive despite the presence of negative regulatory factors such as CDK inhibitors and association with cyclins A and E, however, cells are unable to progress beyond G1 [77]. CDK4DN isoforms that assemble into kinase-dead complexes with D-type cyclins are not capable of eliciting similar growth arrest. This suggests that while CDK4 functions to sequester CIP/KIP proteins enabling CDK2/cyclin complex activation, CDK2 activation is critical to cell cycle progression [78]. The decline of CDK2 activity observed at the G1/S transition is controlled mainly by ubiquitin-mediated degradation of cyclin E.

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