Abstract
Calcium/calmodulin-dependent serine kinase (CASK), a causative gene in X-linked mental retardation, acts as a multi-domain scaffold protein and interacts with more than 20 cellular proteins in different subcellular regions of neurons. It is of interest, therefore, to explore whether post-translational modification regulates CASK's protein-protein interactions. Here, we provide evidence that CASK is phosphorylated by protein kinase A (PKA), identifying residue S562 in the PSD-95-Dlg-ZO-1 domain and residue T724 in the guanylate kinase domain as PKA sites by an in vitro PKA kinase reaction and site-directed mutagenesis. Although the role of S562 phosphorylation is not clear, T724 phosphorylation up-regulates the interaction between CASK and T-box transcription factor T-brain-1 (Tbr-1). NMDAR2b, a downstream target of the CASK-Tbr-1 complex, was then used to explore the significance of CASK phosphorylation by PKA. In cultured cortical neurons, the PKA pathway stimulates both the protein expression and the promoter activity of NMDAR2b. Deletion of the Tbr-1-binding sites greatly reduces the 3'-5'-cyclic AMP responsiveness of the NMDAR2b promoter, and the CASK T724A mutation does not promote the 3'-5'-cyclic AMP responsiveness of NMDAR2b. In conclusion, our data provide evidence that PKA phosphorylates CASK, regulates the nuclear function of CASK, and consequently modulates NMDAR2b expression.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Brain / cytology
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Brain / metabolism
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Cell Line
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Cricetinae
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Cricetulus
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Cyclic AMP / metabolism
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Cyclic AMP / pharmacology
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Cyclic AMP-Dependent Protein Kinases / metabolism
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Cyclic AMP-Dependent Protein Kinases / pharmacology*
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Disks Large Homolog 4 Protein
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Enzyme Inhibitors / pharmacology
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Gene Expression Regulation / drug effects*
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Guanylate Kinases / genetics
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Guanylate Kinases / metabolism*
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Immunoprecipitation
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Intracellular Signaling Peptides and Proteins / metabolism
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Isoquinolines / pharmacology
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Membrane Proteins / metabolism
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Mutagenesis, Site-Directed / methods
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Mutation / genetics
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Nerve Tissue Proteins / metabolism
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Neuroblastoma
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Neurons / drug effects
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Neurons / metabolism
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Phosphoproteins / metabolism
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Phosphorylation / drug effects
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Rats
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Receptors, N-Methyl-D-Aspartate / genetics*
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Receptors, N-Methyl-D-Aspartate / metabolism*
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Serine / metabolism
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Signal Transduction / drug effects
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Signal Transduction / physiology
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Sulfonamides / pharmacology
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Syndecan-2 / metabolism
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T-Box Domain Proteins / metabolism
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Tetrodotoxin / pharmacology
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Threonine / genetics
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Threonine / metabolism
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Transfection / methods
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Up-Regulation / drug effects
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Zonula Occludens-1 Protein
Substances
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Disks Large Homolog 4 Protein
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Dlg4 protein, rat
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Enzyme Inhibitors
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Intracellular Signaling Peptides and Proteins
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Isoquinolines
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Membrane Proteins
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NR2B NMDA receptor
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Nerve Tissue Proteins
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Phosphoproteins
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Receptors, N-Methyl-D-Aspartate
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Sulfonamides
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T-Box Domain Proteins
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Tbr1 protein, rat
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Tjp1 protein, rat
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Zonula Occludens-1 Protein
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Syndecan-2
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Threonine
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Tetrodotoxin
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Serine
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Cyclic AMP
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CASK kinases
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Cyclic AMP-Dependent Protein Kinases
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Guanylate Kinases
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N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide