Specificity determinants of substrate recognition by the protein kinase DYRK1A
Specificity determinants of substrate recognition by the protein kinase DYRK1A
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DOI：
10.1074/jbc.275.4.2431
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发表时间：
2000-01-28
影响因子：
4.8
通讯作者：
Becker, W
中科院分区：
文献类型：
--
作者：
Himpel, S;Tegge, W;Becker, W
DYRK 1A是一种双特异性蛋白激酶，被认为与大脑发育有关。我们确定了一个单一的磷酸化氨基酸残基DYRK底物组蛋白H3（苏氨酸45）的质谱，磷酸化氨基酸分析和蛋白质测序。将苏氨酸45替换为丙氨酸可消除DYRK 1A和相关激酶DYRK 1B、DYRK 2和DYRK 3对组蛋白H3的磷酸化，但不消除CLK 8对组蛋白H3的磷酸化。对这些肽中磷酸盐掺入的评价鉴定了DYRK 1A是一种脯氨酸导向的激酶，其磷酸化共有序列（RPX（S/T）P）与ERK 2（PX（S/T）P）相似。根据最佳底物序列设计的肽（DYRKtide）被DYRK 1A有效磷酸化（K-m = 35 μ m），但不通过ERK 2，ERK 2和DYRK 1A都磷酸化髓鞘碱性蛋白，而只有ERK 2，而不是DYRK 1A磷酸化促分裂原活化蛋白激酶底物ELK-1，DYRK 1A和ERK 2之间底物特异性的这种显著差异可以通过在P-ERK 2上需要精氨酸来解释。3位点及其与天冬氨酸247的相互作用。
DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK8, In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K-m = 35 mu m) but not by ERK2, Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A phosphorylated the mitogen-activated protein kinase substrate ELK-1, This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P -3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.