J. W. Harper

p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells.

Terminal differentiation is coupled to withdrawal from the cell cycle. The cyclin-dependent kinase inhibitor (CKI) p21Cip1 is transcriptionally regulated by p53 and can induce growth arrest. CKIs are therefore potential mediators of developmental control of cell proliferation. The expression pattern of mouse p21 correlated with terminal differentiation of multiple cell lineages including skeletal muscle, cartilage, skin, and nasal epithelium in a p53-independent manner. Although the muscle-specific transcription factor MyoD is sufficient to activate p21 expression in 10T1/2 cells, p21 was expressed in myogenic cells of mice lacking the genes encoding MyoD and myogenin, demonstrating that p21 expression does not require these transcription factors. The p21 protein may function during development as an inducible growth inhibitor that contributes to cell cycle exit and differentiation.

The role of protein stability in the cell cycle and cancer

In addition to the examples mentioned above, other important regulators of cell proliferation such as cyclin D, cyclin E, p21, p27 are all potentially controlled by ubiquitin-mediated proteolysis. In several of these, phosphorylation has been shown to play a role in targeting the proteins for degradation. It remains to be seen how important the SCF pathway and ubiquitin-mediated proteolysis, in general, will become in cancer research. However, it appears that we have only just scratched the surface on the role of these pathways in the control of important regulatory genes. We suspect there will be much more to come from analysis of these fascinating pathways.

Purification and analysis of CIP/KIP proteins

Publisher Summary This chapter discusses the purification and analysis of CIP/KIP proteins. It describes methods and reagents for preparation and analysis of cyclin-dependent kinase (Cdk) inhibitors of the CIP/KIP family. It describes (1) structural and functional domains of CIP/KIP proteins, (2) expression systems for CIP/KIP proteins, (3) methods for assaying recombinant CIP/KIP proteins, and (4) production and characterization of monoclonal antibodies against p21 and p57. Based on structural and biochemical characterization, the CIP/KIP family members contain minimally three domains. CIP/KIP proteins have an additional C-terminal domain that is implicated in association with other proteins. A 20-amino acid segment at the C terminus of p21 is sufficient to direct association with the trimeric form of proliferating cell nuclear antigen (PCNA) and alanine scanning mutagenesis indicates that the charged residues in this region contribute substantially to binding.