Interactions of p27 with Other Proteins

There are 2 questions being addressed in the laboratory on this issue. First, cell cycle exit is accompanied by a reduction in G1 cyclin-cdk activity due to a rapid accumulation of a member or members of the stoichiometric cdk-inhibitor families, the Ink4 family, or the Cip/Kip family. The patterns of cdk-inhibitor expression demonstrate that these proteins overlap in many tissues. The biochemical activities and the expression patterns, both in mouse development and in in vitro differentiation systems, implicated these proteins as effectors of the signaling pathways that control cell cycle exit. However, if these proteins only act within the differentiation process to arrest cells, it is not clear why more than 1 accumulates during differentiation.

We have been focusing on the role of cdk-inhibitor proteins of the Cip/Kip family in controlling the cell cycle, and previously showed that 2 cdk inhibitors from the Cip/Kip family (p21 and p27) accumulated during the differentiation of oligodendrocytes; and 1 from the Ink4 family (p16) was present in both precursors and differentiated progeny. Oligodendrocytes are the myelinating cells of the central nervous system. Progenitor cells can be isolated from various regions of the neonatal mouse, such as the optic nerve and cortical regions, and differentiated in culture following withdrawal of serum. Consequently, cortical OPCs were a suitable model to study the role of cki in differentiation and to assess if any have a function independent of growth arrest.

In this study, using mice deficient in each cdk inhibitor expressed during OPC differentiation, we showed that p27 and p21 carry out separate and distinct functions in the differentiation program, which together mediate cell cycle withdrawal and morphologic differentiation, respectively. Current efforts are directed towards understanding what p21 is doing.

In a parallel program, we are investigating the question of how p27 and p21 interact with cyclin-dependent kinases. This is a question that has been asked and answered many times, however, with quite conflicting results. Our approach is to generate a series of chimeric molecules and use these to determine the interactions with cyclin-dependent kinases and the biologic activity in the oligodendrocyte system above.

Second, we can formally predict that there are at least 4 biologic roles where cdk inhibition or negative regulation of S-phase are required (see Figure):

• in the exit of cells from the cell cycle
• in maintaining cells in a growth-arrested state
• in regulating the rate of G1 transit in the cell cycle
• in preventing continuation of cells through the cycle once committed (i.e., checkpoints)

Intriguingly, p130 has been implicated in maintaining quiescence and p107 in the regulation of G1. As we placed p27 as a function in the exit of cells from the cell cycle, we have begun to probe its genetic relationship to p130 and p107. We have generated mice deficient in both p130 and p27, and p107and p27; and we are characterizing their phenotypes and the growth and transformation properties of cells obtained from these mice.