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Biomedical subjects

K Polyak

Publications and source records attributed to K Polyak.

29 records · Page 2Linked to original sources
PubMed1995

Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-beta.

G1 progression in mammalian cells requires the activity of the cyclin D-dependent kinases Cdk4 and/or Cdk6 and the cyclin E-dependent kinase Cdk2. Proliferating Mv1Lu mink lung epithelial cells and human keratinocytes contain high levels of the universal Cdk inhibitor p27Kip1 distributed in complexes with Cdk2, Cdk4, and Cdk6. Addition of the antimitogenic cytokine transforming growth factor-beta (TGF-beta) elevates expression of the Cdk4/6-specific inhibitor p15Ink4B and induces the release of p27 from Cdk4 and Cdk6. In Mv1Lu cells, this release of p27 coincides with increased binding of p27 to Cdk2. Recombinant p15 inhibits p27 binding to Cdk4 in vitro, and p15 overexpression induces the transfer of p27 from Cdk4 to Cdk2 in vivo, suggesting that the release of Cdk4-bound p27 in TGF-beta-treated cells is caused by the surge in p15 levels. In keratinocytes, TGF-beta increases not only p15 but also p21Cip1, which binds to Cdk2. These events correlate with Cdk2 inhibition and cell cycle arrest and occur without a loss of G1 Cdk components. The results suggest that TGF-beta induces G1 arrest in these two epithelial cell types by inhibiting various cyclin-Cdk kinases through the cooperative action of an Ink4 Cdk inhibitor and a Cip/Kip Cdk inhibitor. Subsequent to cell cycle arrest, Cdk2 and Cdk4 levels decline as part of a second set of events that may represent a program of cell adaptation to the quiescent state.

Animals↗
PubMed1995

Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex.

The crystal structure of the human cyclinA-cyclin-dependent kinase2 (CDK2)-ATP complex has been determined at 2.3 A resolution. CyclinA binds to one side of CDK2's catalytic cleft, inducing large conformational changes in its PSTAIRE helix and T-loop. These changes activate the kinase by realigning active site residues and relieving the steric blockade at the entrance of the catalytic cleft.

Adenosine Triphosphate↗
PubMed1995

p27Kip1: chromosomal mapping to 12p12-12p13.1 and absence of mutations in human tumors.

The p27Kip1 gene codes for a cyclin-dependent kinase inhibitor implicated in G1 arrest by transforming growth factor beta, cell-cell contact, agents that elevate cyclic AMP, and the growth-inhibitory drug rapamycin. p27 binds to and inhibits complexes formed by cyclin E-cdk2, cyclin A-cdk2, and cyclin D-cdk4. The involvement of p27 in the negative regulation of cell proliferation suggests that it may also function as a tumor suppressor gene. Using a combination of somatic cell hybrid panels and fluorescence in situ hybridization p27Kip1 has been mapped to the short arm of chromosome 12 at the 12p12-12p13.1 boundary, reported to harbor deletions and rearrangements in leukemia and mesotheliomas. In order to assess potential p27Kip1 gene alterations, we have screened a total of 147 human primary solid tumors and found no detectable cancer-specific mutations. These results argue that the often observed loss of antimitogenic transforming growth factor beta responsiveness in human cancer cells is not due to structural defects in p27Kip1.

Base Sequence↗
PubMed1995

p27KIP1, an inhibitor of cyclin-dependent kinases.

The identification of a family of proteins that stoichiometrically regulate the activation of the G1 cyclin-dependent kinases has added to our understanding of the process of commitment to the mitotic cycle. The properties of p27 as a CDK binding protein, the ability of environmental signals to regulate the expression of p27, and the observation that p27 may link the accumulation of G1 CDK complexes with activation of the CDK2 kinase, suggest it may have a critical role in establishing a threshold for G1 cyclin/CDK accumulation prior to activation of CDK2 kinase and entry into the mitotic cycle.

Cell Cycle↗
PubMed1995

Mammalian antiproliferative signals and their targets.

The effect of mitogens on the mammalian cell cycle is opposed by the action of antimitogens, such as TGF-beta, cAMP agonists, and various antiproliferative drugs. The recent identification of TGF-beta receptors that initiate antimitogenic signals and of cell cycle kinase inhibitors that respond to these signals has provided new insights into this process. The evidence argues that mitogenic and antimitogenic signals confront each other by regulating in opposite ways the activity of cyclin-dependent kinases that control cell commitment to DNA replication.

Animals↗
PubMed1994

Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin.

The cyclin-dependent kinase (Cdk) enzymes, when associated with the G1 cyclins D and E, are rate-limiting for entry into the S phase of the cell cycle. During T-cell mitogenesis, antigen-receptor signalling promotes synthesis of cyclin E and its catalytic partner, Cdk2, and interleukin-2 (IL-2) signalling activates cyclin E/Cdk2 complexes. Rapamycin is a potent immunosuppressant which specifically inhibits G1-to-S-phase progression, leading to cell-cycle arrest in yeast and mammals. Here we report that IL-2 allows Cdk activation by causing the elimination of the Cdk inhibitor protein p27Kip1, and that this is prevented by rapamycin. By contrast, the Cdk inhibitor p21 is induced by IL-2 and this induction is blocked by rapamycin. Our results show that p27Kip1 governs Cdk activity during the transition from quiescence to S phase in T lymphocytes and that p21 function may be restricted to cycling cells.

Animals↗
PubMed1994

Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation.

Cyclic AMP (cAMP) blocks the mitogenic effects of colony-stimulating factor 1 (CSF-1) in macrophages, inducing cell cycle arrest in mid-G1 phase. Complexes between cyclin D1 and cyclin-dependent kinase 4 (cdk4) assemble in growth arrested cells, but cdk4 is not phosphorylated in vivo by the cdk-activating kinase (CAK) and remains inactive. Although undetectable in lysates of cAMP-treated cells, active CAK is recovered after antibody precipitation, indicating that it is not the direct target of inhibition. Levels of the cdk inhibitor p27Klp1 increase in cAMP-treated cells, and its immunodepletion from inhibitory lysates restores CAK-mediated cdk4 activation. Kip1 does not bind to CAK, but its association with cyclin D-cdk4 prevents CAK from phosphorylating and activating the holoenzyme.

Animals↗
PubMed1994

Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals.

We cloned p27Kip1, a cyclin-dependent kinase inhibitor implicated in G1 phase arrest by TGF beta and cell-cell contact. p27Kip1 associates with cyclin E-Cdk2 complexes in vivo and in vitro, prevents their activation, and inhibits previously activated complexes, and p27Kip1 overexpression obstructs cell entry into S phase. p27Kip1 potently inhibits Rb phosphorylation by cyclin E-Cdk2, cyclin A-Cdk2, and cyclin D2-Cdk4. p27Kip1 is highly conserved and broadly expressed in human tissues, and its mRNA levels are similar in proliferating and quiescent cells. p27Kip1 has a region of sequence similarity to p21Cip1/WAF1, the Cdk inhibitor whose transcription is stimulated by p53. A p27Kip1 peptide corresponding to this region retains Cdk inhibitory activity. We suggest that cell contact, TGF beta, and p53 all restrain cell proliferation through related Cdk inhibitors.

Amino Acid Sequence↗
PubMed1994

p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest.

Cell-cell contact and TGF-beta can arrest the cell cycle in G1. Mv1Lu mink epithelial cells arrested by either mechanism are incapable of assembling active complexes containing the G1 cyclin, cyclin E, and its catalytic subunit, Cdk2. These growth inhibitory signals block Cdk2 activation by raising the threshold level of cyclin E necessary to activate Cdk2. In arrested cells the threshold is set higher than physiological cyclin E levels and is determined by an inhibitor that binds to cyclin E-Cdk2 complexes. A 27-kD protein that binds to and prevents the activation of cyclin E-Cdk2 complexes can be purified from arrested cells but not from proliferating cells, using cyclin E-Cdk2 affinity chromatography. p27 is present in proliferating cells, but it is sequestered and unavailable to interact with cyclin E-Cdk2 complexes. Cyclin D2-Cdk4 complexes bind competitively to and down-regulate the activity of p27 and may thereby act in a pathway that reverses Cdk2 inhibition and enables G1 progression.

Animals↗
PubMed1993

Negative regulation of G1 in mammalian cells: inhibition of cyclin E-dependent kinase by TGF-beta.

Transforming growth factor-beta (TGF-beta) is a naturally occurring growth inhibitory polypeptide that arrests the cell cycle in middle to late G1 phase. Cells treated with TGF-beta contained normal amounts of cyclin E and cyclin-dependent protein kinase 2 (Cdk2) but failed to stably assemble cyclin E-Cdk2 complexes or accumulate cyclin E-associated kinase activity. Moreover, G1 phase extracts from TGF-beta-treated cells did not support activation of endogenous cyclin-dependent protein kinases by exogenous cyclins. These effects of TGF-beta, which correlated with the inhibition of retinoblastoma protein phosphorylation, suggest that mammalian G1 cyclin-dependent kinases, like their counterparts in yeast, are targets for negative regulators of the cell cycle.

Animals↗