The amount of p27 is a critical determinant for the decision of cells in G1
to either withdraw from or commit to the cell cycle and enter S phase. p27 inhibits cyclin E-cdk2 (56
). This kinase is both necessary and rate limiting for S-phase entry (42
) and increases threefold as G1
cells commit to DNA replication (11
). Once activated in mid-G1
, it triggers a positive feedback loop, both inactivating Rb (22
) and promoting p27 degradation (41
), ultimately culminating in the transition to S phase.
Small changes in the amount of p27 protein can have dramatic phenotypic consequences: mice heterozygous for p27 have half the wild-type amount of protein and display intermediate growth phenotypes (27
). Furthermore, carcinogen-induced tumor development is similar in p27 heterozygous mice and in animals completely lacking p27 (16
). These consequences can be attributed to the role of p27 as a mediator of antimitogenic signals (7
). In the absence of p27, cells exposed to signals that induce growth arrest fail to withdraw from the cell cycle in a timely fashion, undergoing more mitotic divisions until other pathways mediate their withdrawal from the cell cycle (7
). The nature of these collaborating or redundant pathways is not always clear; however, other cdk inhibitors and the Rb-like protein p130 have been implicated in fibroblasts, at least with regard to inactivation of cyclin E-cdk2 (9
Regardless of the potential for redundancy, the failure of p27−/−
cells to respond appropriately to growth arrest signals leads to disease. In luteal cells, the lack of p27 leads to a perturbation of estradiol signaling following conception and prevents embryo implantation (59
). The organization of the ear, specifically the ability to hear, also becomes compromised (8
), and p27-deficient animals develop tumors (10
). Thus, an understanding of how the availability of p27 is controlled would impact our understanding of how tissue organization occurs and how cells communicate with each other.
p27 protein is most abundant in G1
cells and decreases precipitously as cells enter S phase, remaining low throughout the remainder of the cell cycle (35
). The expression of p27 can be controlled at the levels of gene transcription (29
), translation (1
), sequestration (57
), nuclear localization (58
), and proteolysis (41
). Proteolysis of p27 is dependent on cdk2 (41
) and possibly skp2 (6
), which conspire to regulate ubiquitin-dependent proteolytic degradation of p27, a phenomenon that might insure irreversibility of the commitment decision, as these proteins are activated or produced just prior to or contemporaneously with the G1
/S transition. A number of groups have suggested that signals promoting growth arrest may act by directly interfering with p27 proteolysis; however, the cause-and-effect relationship is not entirely clear because p27 proteolysis is dependent on proteins and activities that occur once cells are committed to S phase.
On the other hand, growth arrest is accompanied by an increase in the translation of p27 mRNA above a basal state observed in asynchronous cells. In quiescent tetradecanoyl phorbolacetate (TPA)-treated HL-60 cells, the synthesis of p27 protein is increased, correlating with an increase in the amount of p27 mRNA associated with polysomes (35
). Likewise, the rate of p27 synthesis is increased in cells arrested in mid-G1
by lovastatin (23
). Additionally, translation of p27 mRNA continues into S phase (and presumably G2
phase), but proteolysis of the protein prevents its accumulation (35
). Thus, the translation rate of p27 mRNA can vary in a signal-dependent manner: a basal rate in growing cells and an elevated rate (induced) in growth-arrested cells.
The following observations prompted us to look at the translational regulation of p27 mRNA as a mechanism contributing to growth arrest in G1 cells. First, the steady-state amount of p27 is critical to the commitment process, and this is the sum of the synthesis and degradation rates. Second, since proteolysis is dependent on cdk2 activity and skp2, both of which appear following commitment to the cell cycle, it would seem that they could not effectively control p27 accumulation in the early G1 cell, which is deciding between proliferation and growth arrest. However, if translation could be induced in a cell cycle phase-dependent manner, one would expect that the change in synthesis rate might overcome the proteolytic barrier and p27 would accumulate. In this report, we demonstrate that p27 mRNA translation in both basal (proliferating) and induced (nonproliferating) states requires a U-rich sequence in the 5′ untranslated region (5′UTR) of p27 mRNA. This sequence promotes polysome association of the mRNA. Two proteins, designated p33 and p40/41, in cytosolic extracts from asynchronous cells could be cross-linked to the 5′UTR. These factors were enriched in nocadazole-treated cells (G2/M arrest) and lovastatin-treated cells (G1 arrest) compared to hydroxyurea-treated cells (G1/S-phase arrest). We identify p33 as HuR, which binds to the U-rich element independently of other proteins, and p40/41 as hnRNP C1/C2. We discuss the cell cycle-regulated formation of these RNPs in light of the role that translational regulation of p27 may have in the response to antimitogenic signals.