The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the tempor...

The influence of reversible protein phosphorylation on nucleosome assembly during DNA replication was analyzed in extracts from human cells. Inhibitor studies and add-back experiments indicated requirements of cyclin A/Cdk2, cyclin E/Cdk2, and protein phosphatase type 1 (PP1) activities for nucleosome assembly during DNA synthesis by chromatin assembly factor 1 (CAF-1). The p60 subunit of CAF-1...

The vaccinia-related kinase (VRK) proteins are a new family with three members in the human kinome. The VRK1 protein phosphorylates several transcription factors and has been postulated to be involved in regulation of cell proliferation. In normal squamous epithelium, VRK1 is expressed in the proliferation area. Because VRK1 can stabilize p53, the expression of the VRK1 protein was analyzed in ...

Silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) is a transcriptional corepressor that participates in diverse signaling pathways and human diseases. However, regulation of SMRT stability remains largely unexplored. We show that the peptidyl-prolyl isomerase Pin1 interacts with SMRT both in vitro and in mammalian cells. This interaction requires the WW domain of Pin1 and...

The human papillomavirus (HPV) E7 protein promotes S-phase reentry in a fraction of postmitotic, differentiated keratinocytes. Here we report that these cells contain an inherent mechanism that opposes E7-induced DNA replication. In organotypic raft cultures of primary human keratinocytes, neither cyclin E nor p21cip1 is detectable in situ. However, E7-transduced differentiated cells not in S p...

Cyclin E/Cdk2 kinase activity is frequently deregulated in human cancers, resulting in impaired apoptosis. Here, we show that cyclin E/Cdk2 phosphorylates and stabilizes the pro-survival Bcl-2 family protein Mcl-1, a key cell death resistance determinant to the small molecule Bcl-2 family inhibitors ABT-199 and ABT-737, mimetics of the Bcl-2 homology domain 3 (BH3). Cyclin E levels were elevate...

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2-cyclin E or A concentration in egg cytosol is ...

Schwann cell proliferation is regulated by multiple growth factors and axonal signals. However, the molecules that control growth arrest of Schwann cells are not well defined. Here we describe regulation of the cyclin-dependent kinase-2 (CDK2) protein, an enzyme that is necessary for the transition from G1 to S phase. Levels of CDK2 protein were elevated in proliferating Schwann cells cultured ...

BACKGROUND The ability of cyclin-dependent kinases (CDKs) to promote cell proliferation is opposed by cyclin-dependent kinase inhibitors (CKIs), proteins that bind tightly to cyclin-CDK complexes and block the phosphorylation of exogenous substrates. Mice with targeted CKI gene deletions have only subtle proliferative abnormalities, however, and cells prepared from these mice seem remarkably no...