Ludger Hengst

Translational Control of p27Kip1 Accumulation During the Cell Cycle

Cell cycle phase transitions in eukaryotic cells are driven by regulation of the activity of protein kinases known as cyclin-dependent kinases (Cdks). A broad spectrum Cdk-inhibitory activity associated with a 28-kilodalton protein (p28Ick1) was induced in cells treated with the drug lovastatin or upon density-mediated growth arrest and was periodic in the cell cycle, with peak activity in G1. The p28Ick1 protein was shown to be identical to p27Kip1, and the periodic or induced inhibitory activity resulted from a periodic accumulation of the protein. Variations in the amount of p27 protein occurred, whereas the abundance of the p27 messenger RNA remained unchanged. In every instance investigated, the posttranscriptional alteration of p27 protein levels was achieved in part by a mechanism of translational control, although in density-arrested fibroblasts and thymidine-arrested HeLa cells the half-life of the protein was also changed.

The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy

The cyclin-dependent kinase (Cdk) inhibitor p27 (also known as KIP1) regulates cell proliferation, cell motility and apoptosis. Interestingly, the protein can exert both positive and negative functions on these processes. Diverse post-translational modifications determine the physiological role of p27. Phosphorylation regulates p27 binding to and inhibition of cyclin–Cdk complexes, its localization and its ubiquitin-mediated proteolysis. In cancers, p27 is inactivated through impaired synthesis, accelerated degradation and by mislocalization. Moreover, studies in several tumour types indicate that p27 expression levels have both prognostic and therapeutic implications.

Effects of p21Cip1/Waf1 at Both the G1/S and the G2/M Cell Cycle Transitions: pRb Is a Critical Determinant in Blocking DNA Replication and in Preventing Endoreduplication

ABSTRACT It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition. Both G1- and G2-arrested cells were observed in all cell types, with different preponderances. Preponderant G1 arrest in response to p21 expression correlated with the presence of functional pRb. G2 arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest Bs. In addition, DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G2-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.

A novel inhibitor of cyclin-Cdk activity detected in transforming growth factor beta-arrested epithelial cells.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Cyclins E and A in association with Cdk2 have been shown to play a role in the G1-to-S phase transition in mammalian cells. We have studied the effects of TGF-beta-mediated growth arrest on G1/S cyclins E and A. Inhibition of cyclin A-associated kinase by TGF-beta is primarily due to a decrease in cyclin A mRNA and protein. By contrast, while TGF-beta inhibits accumulation of cyclin E mRNA, the reduction in cyclin E protein is minimal. Instead, we find that the activation of cyclin E-associated kinase that normally accompanies the G1-to-S phase transition is inhibited. A novel inhibitor of cyclin-Cdk complexes was detected in TGF-beta-treated cell lysates. Inhibition is mediated by a heat-stable protein that targets both Cdk2 and Cdc2 kinases. In G0-arrested cells, a similar inhibitor of Cdk2 kinase was detected. These data suggest the existence of an inhibitor of cyclin-dependent kinases induced under different conditions to mediate antiproliferative responses.

p27 Phosphorylation by Src Regulates Inhibition of Cyclin E-Cdk2

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.

Cdk-Inhibitory Activity and Stability of p27Kip1 Are Directly Regulated by Oncogenic Tyrosine Kinases

p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.

p27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding

p27 controls cell proliferation by binding and regulating nuclear cyclin-dependent kinases (CDKs). In addition, p27 interacts with other nuclear and cytoplasmic targets and has diverse biological functions. We seek to understand how the structural and dynamic properties of p27 mediate its several functions. We show that, despite showing disorder before binding its targets, p27 has nascent secondary structure that may have a function in molecular recognition. Binding to Cdk2–cyclin A is accompanied by p27 folding, and kinetic data suggest a sequential mechanism that is initiated by binding to cyclin A. p27 regulates CDK–cyclin complexes involved directly in cell cycle control and does not interact with other closely related CDKs. We show that p27-cyclin interactions are an important determinant of this specificity and propose that the homologous cell cycle regulators p21 and p57 function by a similar sequential, folding-on-binding mechanism.

Cyclin A-associated kinase activity is rate limiting for entrance into S phase and is negatively regulated in G1 by p27Kip1.

We have created fibroblast cell lines that express cyclin A under the control of a tetracycline-repressible promoter. When stimulated to reenter the cell cycle after serum withdrawal, these cells were advanced prematurely into S phase by induction of cyclin A. In an asynchronous population, induction of cyclin A caused a decrease in the percentage of cells in G1. These results demonstrate that expression of cyclin A is rate limiting for the G1-to-S transition and suggest that cyclin A can function as a G1 cyclin. Although the level of exogenous cyclin A was constant throughout the cell cycle, its associated kinase activity increased as cells approached S phase. Low kinase activity in early G1 was found to correlate with the presence of p27Kip1 in cyclin A-associated complexes, while high kinase activity in late G1 was correlated with its absence. These results suggest that a function of p27Kip1 in G1 is to prevent premature activation of cyclin A-associated kinase. Cyclin A expression in early G1 led to phosphorylation of the product of the retinoblastoma susceptibility gene (pRb). Thus, cyclin A expression can be rate limiting for pRb phosphorylation, implicating pRb as a physiological substrate of the cyclin A-dependent kinase. Taken together, these results demonstrate that deregulated expression of cyclin A can perturb the normal regulation of the G1-to-S transition.

Endocytosis in yeast: Evidence for the involvement of a small GTP-binding protein (Ypt7p)

From the budding yeast S. cerevisiae, we have cloned a gene, YPT7, that encodes a GTP-binding protein belonging to the Ypt family of ras-related proteins. The 208 amino acid protein shares identical effector domain and C-terminal sequences with the mammalian Rab7 protein. YPT7 gene disruption did not impair cellular growth at temperatures ranging from 17 degrees C to 37 degrees C. ypt7 null mutants are characterized by highly fragmented vacuoles and differential defects of vacuolar protein transport and maturation. The uptake of alpha factor pheromone by wild-type and Ypt7p-deficient cells was found to be indistinguishable, but in mutant cells lacking Ypt7p, degradation of the endocytosed pheromone was severely inhibited. Our findings suggest a role of Ypt7p in protein transport between endosome-like compartments.

G1 control in mammalian cells.

SUMMARY Cyclin-dependent kinases (Cdks) control the major cell cycle transitions in eukaryotic cells. On the basis of a variety of experiments where cyclin function either is impaired or enhanced, D-type cyclins as well as cyclins E and A have been linked to G1 and G1/S phase roles in mammalian cells. We therefore sought to determine if agents that block the G1/S phase transition do so at the level of regulating the Cdk activities associated with these cyclins. A variety of conditions that lead to G1 arrest were found to correlate with accumulation of G1-specific Cdk inhibitors, including treatment of fibroblasts with ionizing radiation, treatment of epithelial cells with TGF-β, treatment of HeLa cells with the drug lovastatin, and removal of essential growth factors from a variety of different cell types. Mechanistically, inhibition of Cdks was found to involve the stoichiometric binding of Cdk inhibitor proteins. p21Waf1/Cip1 was associated with DNA damage induced arrest while p27Kip1/p28Ick1 accumulated under a variety of antiproliferative conditions.