Abstract
Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21(cip1). Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Blotting, Western
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CDC2-CDC28 Kinases*
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Cell Cycle
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Cell Cycle Proteins*
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Cell Line
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Cell Nucleus / metabolism
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Cell Separation
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Cyclin A / metabolism*
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Cyclin E / metabolism*
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclin-Dependent Kinases / metabolism*
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Cyclins / metabolism
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Flow Cytometry
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Glutathione Transferase / metabolism
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Histone Chaperones
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Humans
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Mass Spectrometry
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Microscopy, Fluorescence
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Molecular Sequence Data
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Nuclear Proteins / chemistry*
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Nuclear Proteins / metabolism
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Nuclear Proteins / physiology*
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Peptides / chemistry
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Phosphorylation
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Plasmids / metabolism
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Precipitin Tests
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Protein Binding
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Protein Serine-Threonine Kinases / metabolism*
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Recombinant Fusion Proteins / metabolism
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Repressor Proteins / chemistry
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S Phase
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins*
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Sequence Homology, Amino Acid
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Threonine / chemistry
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Transcription Factors / chemistry*
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Transcription Factors / metabolism
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Transcription Factors / physiology*
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Transfection
Substances
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CDKN1A protein, human
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Cell Cycle Proteins
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Cyclin A
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Cyclin E
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins
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HIR1 protein, S cerevisiae
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HIR2 protein, S cerevisiae
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HIRA protein, human
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Histone Chaperones
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Nuclear Proteins
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Peptides
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Recombinant Fusion Proteins
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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Transcription Factors
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Threonine
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Glutathione Transferase
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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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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases