The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site
The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site
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DOI：
10.1042/bj3550597
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发表时间：
2001-05-01
影响因子：
4.1
通讯作者：
Cohen, P
中科院分区：
文献类型：
--
作者：
Woods, YL;Rena, G;Cohen, P
横纹肌肉瘤中的叉头蛋白（FKHR）是一种转录因子，参与胰岛素对基因表达的控制，以及生存因子对细胞凋亡的调节。这些信号分子通过磷酸肌醇3-激酶依赖性途径触发蛋白激酶B（PK B）催化的FKHR三个残基（Thr（24）、Ser（256）和Ser（319））磷酸化，导致该转录因子的核退出和失活。在这里，我们已经确定了一个保守的残基（Ser（329））作为一个新的体内磷酸化位点的FKHR。Ser（329）磷酸化也降低了FKHR刺激基因反式激活的能力，并降低了细胞核中FKHR的比例。然而，与PKB靶向的残基不同，Ser（329）在未刺激的HEK-293细胞中被磷酸化，并且磷酸化不会通过用胰岛素样生长因子-1刺激或通过用3-磷酸肌醇依赖性蛋白激酶-1转染而增加。我们还从兔骨骼肌中纯化了一种蛋白激酶，该蛋白激酶在Ser（329）特异性磷酸化FKHR，并将其鉴定为DYRK 1A（双特异性酪氨酸磷酸化和调节激酶1A）。我们发现，FKHR和DYRK 1A共定位在细胞核的离散区域，可以从细胞提取物中共免疫沉淀。这些实验表明DYRK 1A可以在体内使FKHR的Ser（329）磷酸化。
Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signers trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr(24), Ser(256) and Ser(319)) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser(329)) as a novel in vivo phosphorylation site on FKHR. Ser(329) phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser(329) is phosphorylated in unstimulated HEK-293 cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser(329) specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser(329) in vivo.