Abstract
Pax6 is a transcriptional activator that contains two DNA binding domains and a potent transcription activation domain in the C terminus, which regulates organogenesis of the eye, nose, pancreas, and central nervous system. Homeodomain-interacting protein kinase 2 (HIPK2) interacts with transcription factors, including homeoproteins, and regulates activities of transcription factors. Here we show that HIPK2 phosphorylates the activation domain of Pax6, which augments Pax6 transactivation by enhancing its interaction with p300. Mass spectrometric analysis identified three Pax6 phosphorylation sites as threonines 281, 304, and 373. The substitutions of these threonines with alanines decreased Pax6 transactivation, whereas substitutions to glutamic acids increased transactivation in mimicry of phosphorylation. Furthermore, the knock-down of either endogenous or exogenous HIPK2 expression with HIPK2 shRNA markedly inhibited Pax6 phosphorylation and its transactivating function on proglucagon promoter in cultured cells. These results strongly indicate that HIPK2 is an upstream protein kinase for Pax6 and suggest that it modulates Pax6-mediated transcriptional regulation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Alanine / chemistry
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Animals
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Binding Sites
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Carrier Proteins / chemistry
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Carrier Proteins / physiology*
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Cell Line
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Cell Line, Tumor
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DNA Mutational Analysis
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Eye Proteins / genetics*
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Eye Proteins / metabolism
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Eye Proteins / physiology*
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Glucagon / chemistry
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Glutamic Acid / chemistry
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Green Fluorescent Proteins / metabolism
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Homeodomain Proteins / genetics*
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Homeodomain Proteins / metabolism
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Homeodomain Proteins / physiology*
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Humans
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Immunohistochemistry
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Luciferases / metabolism
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Mass Spectrometry
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Mice
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Mutagenesis, Site-Directed
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Mutation
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PAX6 Transcription Factor
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Paired Box Transcription Factors / genetics*
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Paired Box Transcription Factors / metabolism
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Paired Box Transcription Factors / physiology*
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Phosphorylation
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Promoter Regions, Genetic
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Protein Binding
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Protein Serine-Threonine Kinases / chemistry
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Protein Serine-Threonine Kinases / physiology*
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Protein Structure, Tertiary
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RNA, Small Interfering / chemistry
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RNA, Small Interfering / metabolism
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Repressor Proteins / genetics*
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Repressor Proteins / metabolism
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Repressor Proteins / physiology*
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Reverse Transcriptase Polymerase Chain Reaction
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Threonine / chemistry
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Transcription, Genetic
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Transcriptional Activation
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Transfection
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p300-CBP Transcription Factors / metabolism
Substances
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Carrier Proteins
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Eye Proteins
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Homeodomain Proteins
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PAX6 Transcription Factor
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PAX6 protein, human
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Paired Box Transcription Factors
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Pax6 protein, mouse
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RNA, Small Interfering
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Repressor Proteins
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Green Fluorescent Proteins
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Threonine
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Glutamic Acid
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Glucagon
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Luciferases
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p300-CBP Transcription Factors
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HIPK2 protein, human
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Protein Serine-Threonine Kinases
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Alanine