Sequence Characteristics, Subcellular Localization, and Substrate Specificity of DYRK-related Kinases, a Novel Family of Dual Specificity Protein Kinases

Abstract

DYRK1 is a dual specificity protein kinase presumably involved in brain development. Here we show that the kinase belongs to a new family of protein kinases comprising at least seven mammalian isoforms (DYRK1A, DYRK1B, DYRK1C, DYRK2, DYRK3, DYRK4A, and DYRK4B), the yeast homolog Yak1p, and the Drosophila kinase minibrain (MNB). In rat tissues, DYRK1A is expressed ubiquitously, whereas transcripts for DYRK1B, DYRK2, DYRK3, and DYRK4 were detected predominantly in testes of adult but not prepuberal rats. By fluorescence microscopy and subcellular fractionation, a green fluorescent protein (GFP) fusion protein of DYRK1A was found to accumulate in the nucleus of transfected COS-7 and HEK293 cells, whereas GFP-DYRK2 was predominantly detected in the cytoplasm. DYRK1A exhibited a punctate pattern of GFP fluorescence inside the nucleus and was co-purified with the nuclear matrix. Analysis of GFP-DYRK1A deletion constructs showed that the nuclear localization of DYRK1A was mediated by its nuclear targeting signal (amino acids 105-139) but that its characteristic subnuclear distribution depended on additional N-terminal elements (amino acids 1-104). When expressed in Escherichia coli, DYRK1A, DYRK2, DYRK3, MNB, and Yak1p catalyzed their autophosphorylation on tyrosine residues. The kinases differed in their substrate specificity in that DYRK2 and DYRK3, but not DYRK1A and MNB, catalyzed phosphorylation of histone H2B. The heterogeneity of their subcellular localization and substrate specificity suggests that the kinases are involved in different cellular functions.

Mechanism of dual specificity kinase activity of DYRK1A

“…We have previously found that an expression construct of the yeast kinase Yak1p consisting only of the catalytic domain and the adjacent DYRK homology box is capable of tyrosine autophosphorylation in E. coli [46]. Here we show that analogous constructs of DYRK2, HIPK2 and CLK1 also autophosphorylate in E. coli.…”

“…Mammalian expression plasmids for the DYRKs and CLK3 have been described previously [35,46]. A deletion construct (GFP-DYRK1A-DNDC) was generated that contained only the catalytic domain and the DYRK homology box (amino acids 135-481; numbering in this paper refers to the splicing variants defined as 'canonical' in the UniProt database www.uniprot.org).…”

DYRK1A regulates Hap1–Dcaf7/WDR68 binding with implication for delayed growth in Down syndrome

“…We were interested in whether Hap1 and DYRK1A interact with Dcaf7 competitively or synergistically. First, however, we wanted to examine the subcellular localization of DYRK1A, because studies have demonstrated both cytoplasmic and nuclear distribution of endogenous DYRK1A in neuronal cells, whereas ectopically expressed DYRK1A is found mainly in the nucleus (37)(38)(39)(40)(41). We found via subcellular fractionation and immunostaining that endogenous DYRK1A is localized predominantly in the cytoplasm in mouse brain in vivo and in cultured HEK293 and PC12 cells (Fig.…”

Rapid turnover of cell‐cycle regulators found in Mirk/dyrk1B transfectants

“…1 Mirk is a member of a multigene family, called the minibrain/dyrk family. [2][3][4][5] Mutant minibrain flies are characterized by a marked reduction in size of the optic lobes and central brain hemispheres, 2 suggesting that minibrain plays a role in neuronal survival and/or proliferation. In cell culture systems, dyrk1A mediates neuronal differentiation by phosphorylating CREB in vivo, leading to the stimulation of subsequent CRE-mediated transcription during neuronal differentiation in hippocampal progenitor cells.…”

“…All dyrk family members are serine/threonine kinases that also can autophosphorylate on tyrosine, so they are dual-function kinases. [2][3][4][5] Their activation sequence is YQY or YTY, with both tyrosines phosphorylated. This YxY sequence aligns with the TxY activation sequence in MAPKs.…”