Abstract
p27[KIP1] (p27) is a cyclin dependent kinase inhibitor, involved in the negative regulation of G1 progression in response to a number of anti-proliferative signals. In this study we show, in growing mouse hybridoma (7TD1) and human myeloma (U266) cell lines, that p27 is highly expressed but slightly upregulated when cells are arrested, regardless to the phases of the cell cycle. In contrast, the specific blockade of these cells in early G1 phase reveals the induction of a protein of 23 kDa (p23) specifically recognized by polyclonal anti-p27 antibodies raised against the NH2 terminal part of p27 but not by anti-p21[CIP1] antibodies. Experiments using caspase inhibitors strongly suggest that p23 results from the proteolysis of p27 by a 'caspase-3-like' protease. This cleavage leads to the cytosolic sequestration of p23 but does not alter its binding properties to CDK2 and CDK4 kinases. Indeed, p23 associated in vivo with high molecular weight complexes and coprecipitated with CDK2 and CDK4. We demonstrate by transfection experiments in SaOS-2 cells that p23 induces a G1 phase growth arrest by inhibition of cyclin/CDK2 activity. In summary we describe here a caspase-cleaved form of p27, induced in absence of detectable apoptosis and likely involved in cell cycle regulation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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8-Bromo Cyclic Adenosine Monophosphate / pharmacology
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Amino Acid Chloromethyl Ketones / pharmacology
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Animals
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Apoptosis / drug effects
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CDC2-CDC28 Kinases*
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Caspase 3
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Caspase Inhibitors
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Caspases / genetics
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Caspases / immunology
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Caspases / metabolism*
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Cell Cycle Proteins*
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Cell Division / drug effects
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Cell Division / physiology
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Cell Line
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclin-Dependent Kinase Inhibitor p27
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Cyclin-Dependent Kinases / metabolism
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Cyclins / immunology
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Cyclins / metabolism
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Cysteine Proteinase Inhibitors / pharmacology
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Cytosol / metabolism
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Dimethyl Sulfoxide / pharmacology
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G1 Phase / drug effects
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G1 Phase / physiology*
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Humans
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Hybridomas / drug effects
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Hybridomas / metabolism
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Hybridomas / pathology
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Interleukin-6 / metabolism
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Mice
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Microtubule-Associated Proteins / drug effects
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Microtubule-Associated Proteins / immunology
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Microtubule-Associated Proteins / metabolism*
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Molecular Weight
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Multiple Myeloma / drug therapy
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Multiple Myeloma / metabolism
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Multiple Myeloma / pathology
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Oligopeptides / pharmacology
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Peptide Fragments / immunology
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins*
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Time Factors
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Tumor Cells, Cultured
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Tumor Suppressor Proteins*
Substances
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Amino Acid Chloromethyl Ketones
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CDKN1A protein, human
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Caspase Inhibitors
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Cdkn1a protein, mouse
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Cdkn1b protein, mouse
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Cell Cycle Proteins
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins
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Cysteine Proteinase Inhibitors
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Interleukin-6
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Microtubule-Associated Proteins
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Oligopeptides
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Peptide Fragments
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Proto-Oncogene Proteins
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Tumor Suppressor Proteins
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acetyl-aspartyl-glutamyl-valyl-aspartal
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benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
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Cyclin-Dependent Kinase Inhibitor p27
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8-Bromo Cyclic Adenosine Monophosphate
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Protein Serine-Threonine Kinases
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CDC2-CDC28 Kinases
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CDK2 protein, human
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CDK4 protein, human
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Cdk2 protein, mouse
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Cdk4 protein, mouse
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinases
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CASP3 protein, human
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Casp3 protein, mouse
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Caspase 3
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Caspases
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Dimethyl Sulfoxide