Splice Variants of the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 4 (DYRK4) Differ in Their Subcellular Localization and Catalytic Activity

Genes and Disease Program, Centre for Genomic Regulation, University Pompeu Fabra, Dr Aiguader 88, 08003 Barcelona, Spain.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2011; 286(7):5494-505. DOI: 10.1074/jbc.M110.157909
Source: PubMed


Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.

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    • "Protein was isolated from the hippocampus and cerebellum of 6 week old mice in RIPA buffer and quantified using a Bradford's assay (Bradford, 1976). A Dyrk1a kinase activity assay was performed as previously published (Papadopoulos et al., 2011; Pons-Espinal et al., 2013) with modifications. Briefly, the protein sample was cleared of any antibodies by pre-incubation with EZ-view Red Protein affinity gel (Sigma-Aldrich, St Louis, MO). "
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    • "Kinase activity of DYRK1A protein was determined from hippocampus (6 mice per group) according to previously published protocol [26] "
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    • "The beads were washed twice with immunoprecipitation buffer, once with immunoprecipitation buffer without protease inhibitors and detergent and once with kinase buffer (25 mM Hepes pH 7.5, 0.5 mM MgCl2, 0.5 mM dithiothreitol). The kinase assays were performed with the biotinylated peptide SAPtide (Bio-RRARKLTATPTPLGG) as described previously [73]. In brief, immunocomplexes were incubated for 30 min at 30°C in 20 µl of kinase buffer, at a final concentration of 10 µm ATP and (γ-32P)ATP (100–150 mCi/pmol) and with 100 µm of the peptide. "
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