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.

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 of Cdk-inhibitory activity associated with a 28-kilodalton protein (p28lck1) 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 p28lck1 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 cyclin-dependent kinase (Cdk) inhibitor p2lWan/Cipl/sdil, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability ofp21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

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 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.

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.

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p27

p27Kip1 restrains cell proliferation by binding to and inhibiting cyclin-dependent kinases. To investigate the mechanisms of p27 translational regulation, we isolated a complete p27 cDNA and identified an internal ribosomal entry site (IRES) located in its 5UTR. The IRES allows for efficient p27 translation under conditions where cap-dependent translation is reduced. Searching for possible regulators of IRES activity we have identified the neuronal ELAV protein HuD as a specific binding factor of the p27 5UTR. Increased expression of HuD or the ubiquitously expressed HuR protein specifically inhibits p27 translation and p27 IRES activity. Consistent with an inhibitory role of Hu proteins in p27 translation, siRNA mediated knockdown of HuR induced endogenous p27 protein levels as well as IRES-mediated reporter translation and leads to cell cycle arrest in G1.