9. Protein degradation is important in mitosis

27.8 G0/G1 and G1/S transitions involve cdk inhibitors


An important insight into control of the cell cycle at G1 has been provided by the identification of tumor suppressor genes that code for products that interact with cdk-cyclin complexes or with the downstream circuitry. Tumor suppressors are generally identified as genes in which loss of function causes tumor formation, either as seen by transformation of cells in culture, or by association of loss-of-function mutations with tumors in animals (see 28 Oncogenes and cancer).




Figure 27.23 A block to the cell cycle is released when RB is phosphorylated by cdk-cyclin.

The product of the tumor suppressor RB is a substrate for cdk-cyclin D complexes, and exerts its effects during the part of G1 that precedes the restriction point. Figure 27.23 shows the basic circuit. In quiescent cells, or during the first part of G1, RB is bound to the transcription factor E2F. This has two effects. First, some genes whose products are essential for S phase depend upon the activity of E2F. By sequestering E2F, RB ensures that S phase cannot initiate. Second, the E2F-RB complex represses transcription of other genes. This may be the major effect in RB’s ability to arrest cells in G1 phase (for review see Weinberg, 1995).


The nonphosphorylated form of RB forms a complex with cdk-cyclins. The complex with cdk4,6-cyclin D1,2,3 is the most prominent, but RB is also a substrate for cdk2-cyclin E. At or close to the restriction point, RB is phosphorylated by cdk4,6-cyclin D kinases. The phosphorylation causes RB to release E2F, which then activates transcription of the genes whose functions are required for S phase, and also releases repression of genes by the E2F-RB complex. The importance of E2F is seen by the result that expression of E2F in quiescent cells enables them to synthesize DNA.


There is an especially close relationship between RB and cyclin D1. Over-expression of D1 causes cells to enter S phase early. Inhibition of expression of D1 arrests cells before S phase. The sole role of cyclin D1 could be to inactivate RB and permit entry into S phase.


There are several related transcription factors in the E2F family, sharing the property that all recognize genes with the same consensus element. RB binds three of these factors. Two further proteins, p107 and p130, which are related to RB, behave in a similar way, and bind the other members of the E2F group. So together RB and p107 may control the activity of the E2F group of factors.


RB is a target for several pathways that inhibit growth, and may be the means by which growth inhibitory signals maintain cells in G1 (or G0). Several of these signals, including the growth inhibitory factor TGFβ , act through inhibitors of cdk-cyclin kinases. The inhibitors are called ckis. They are found as proteins bound to cdk-cyclin dimers in inactive complexes, for example, in quiescent cells. By maintaining the cdk-cyclin complexes in inactive form, they prevent the phosphorylation of RB, making it impossible to release cells to enter S phase.


The cki proteins fall into two classes. The INK4 family is specific for cdk4 and cdk6, and has four members: p16INK4A, p15INK4B, p18INK4C, and p19INK4D. The Kip family inhibit all G1 and S phase cdk enzymes, and have three members: p21Cip1/WAF1, p27Kip1, and p57Kip2. (Each protein is identified by its size, with the casual name used as a superscript (for review see Sherr and Roberts, 1995).




Figure 27.24 p16 binds to cdk4 and cdk6 and to cdk4,6-cyclin dimers. By inhibiting cdk-cyclin D activity, p16 prevents phosphorylation of RB and keeps E2F sequestered so that it is unable to initiate S phase.

INK4 protein binds specifically to cdk4 and cdk6. This suggests a connection with the G0/G1 transition. p16 cannot inhibit proliferation of cells that lack RB, which suggests that it functions by preventing cdk-cyclin kinase activity from using RB as a substrate, as illustrated in Figure 27.24. By binding to the cdk subunits, INK4 proteins inhibit both cdk4-cyclin D and cdk6-cyclin D activities. As exemplified by p16 and p19, they bind next to the ATP binding site of cdk6. This both inhibits catalytic activity and triggers a conformational change that prevents cyclin from binding (the conformational change is propagated to the cyclin-binding site).


p21 is a universal cdk inhibitor, binding to all complexes of cdk2, 4, 6. This suggests that it is likely to block progression through all stages of G1/S. In primary cultured cells (taken directly from the animal), cdk-cyclin dimers are usually found in the form of quaternary complexes that contain two further components. One is PCNA, a component of DNA polymerase δ , which may provide a connection with DNA replication. The other is the inhibitor p21. It may seem paradoxical that an inhibitor is consistently associated with the cdk-cyclin dimer, but it turns out that at a stoichiometry of 1:1 the p21 is not inhibitory. An increase in the number of p21 subunits associated with the cdk-cyclin dimer inhibits kinase function. In transformed cells (from lines that have been successfully perpetuated in culture), cdk-cyclin complexes lack p21 and PCNA. This suggests the possibility that p21 is involved in G1/S control, and that relaxation of this control is necessary for cells to be perpetuated in culture.




Figure 27.25 p21 and p27 inhibit assembly and activity of cdk4,6-cyclin D and cdk2-cyclin E by CAK. They also inhibit cycle progression independent of RB activity. p16 inhibits both assembly and activity of cdk4,6-cyclin D.

p27 has a sequence that is partly related to p21, and also binds promiscuously to cdk-cyclin complexes. Over-expression of p27 blocks progression through S phase, and levels of p27 are increased when cells are sent into a quiescent state by treatment with TGFβ . p21 and p27 block the catalytic subunit of cdk-cyclin dimers from being a substrate for activation by phosphorylation by CAK. They also prevent catalytic activity of the cdk-cyclin complex. The stages at which they function are illustrated in the summary of inhibitory pathways in Figure 27.25.


p21 and p27 are probably partially redundant in their functions. The pathway by which they inhibit the cell cycle is not entirely clear, but we know that it does not depend on controlling RB, because they can inhibit proliferation of cells that lack RB. This may mean that their inhibition of cdk2-cyclin E dimers is critical. Since both are present in proliferating cells, the normal progress of the cell cycle may require the levels of the cdk-cyclin dimers to increase to overcome an inhibitory threshold. p27 appears to be the major connection between extracellular mitogens and the cell cycle, with an inverse correlation between p27 activity and ability to proliferate.


The importance of the pathway from cki proteins to RB is emphasized by the fact that tumor suppressors are found at every stage, including cki proteins, cyclins D1,2, and RB. The implication is that the cki proteins are needed to suppress unrestrained growth of cells. In terms of controlling the cell cycle, this pathway is clearly central. It may be the key pathway by which cells are enabled to undertake a division cycle (by passing the restriction point in G1 if they are already cycling, or by reentering G1 if they are quiescent in G0; for review see Hunter and Pines, 1994).




Figure 27.26 The SCF is an E3 ligase that targets the inhibitor Sic1.


Figure 8.49 The ubiquitin cycle involves three activities. E1 is linked to ubiquitin. E3 binds to the substrate protein. E2 transfers ubiquitin from E1 to the substrate. Further cycles generate polyubiquitin.

The cki proteins provide another level of control. In S. cerevisiae, the cki Sic1 is bound to CDC28-CLB during G1, and this maintains the kinase in an inactive state. Entry into S phase requires degradation of Sic1 to release the kinase. Figure 27.26 shows how Sic1 is targeted for degradation by a ubiquitinating system (see Figure 8.49 for a general description). The Sic1 target is recognized by a complex called the SCF, which functions as an E3 ligase. The SCF complex includes Cdc53, Skp1, and Cdc4. Cdc4 is the targeting component, which, together with Skp1, binds to Sic1. For this reason, the complex is described as SCFCdc4. Skp1 is the connection to Cdc53, which interacts with the E2 ligase (Cdc34). The E2 ligase adds ubiquitin to Sic1, causing it to be degraded.


Cdc4 is a member of a class of proteins called F-box proteins. It uses the F-box motif to bind to Skp1. This is a general paradigm for the construction of SCF complexes. Other SCF complexes exist in which the targeting subunit is a different F-box protein, but the Cdc53 and Skp1 components remain the same. An example relevant to the cell cycle is SCFGrr1 in which the F-box protein Grr1 provides the targeting subunit, and causes the degradation of G1 cyclins (Skowyra et al., 1997).


There are further layers of control in this system. The substrates for the SCF must be phosphorylated to be recognized. The kinases that perform the phosphorylation are the cdk-cyclin complexes that are active at the appropriate stage of the cell cycle. The abundance of the SCF complexes is itself controlled by degrading the F-box subunits. SCFCdc4 targets Cdc4, thus creating an autoregulatory limitation on its activity. The consequence of such feedbacks is to maintain a supply of the Cdc53-Skp1 cores that can be recruited as appropriate by the F-box subunits.




Reviews
Hunter, T. and Pines, J. (1994). Cyclins and cancer II: cyclin D and CDK inhibitors come of age. Cell 79, 573-582.
Sherr, C. J. and Roberts, J. M. (1995). Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 9, 1149-1163.
Weinberg, R. A. (1995). The retinoblastoma protein and cell cycle control. Cell 81, 323-330.

Research
Skowyra, D., et al. (1997). F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91, 209-219.



Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

flylib.com © 2008-2017.
If you may any questions please contact us: flylib@qtcs.net