Identification of the Protein Kinases Pyk3 and Phg2 as Regulators of the STATc-Mediated Response to Hyperosmolarity
Identification of the Protein Kinases Pyk3 and Phg2 as Regulators of the STATc-Mediated Response to Hyperosmolarity
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DOI：
10.1371/journal.pone.0090025
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发表时间：
2014-02-25
期刊：
影响因子：
3.7
通讯作者：
Eichinger, Ludwig
中科院分区：
文献类型：
--
作者：
Linh Hai Vu;Araki, Tsuyoshi;Eichinger, Ludwig
细胞对环境渗透压变化的适应对于细胞存活至关重要。在网骨藻中，STATc是高渗胁迫下转录应答的关键调节因子。它的磷酸化和随后的激活由两个信号分支控制，一个是cGMP依赖性的，另一个是Ca 2+依赖性的，其中许多信号成分尚未被鉴定。STATc应激信号通路通过上调STATc和STATc调节基因的表达来反馈自身。基于微阵列研究，我们选择了两个酪氨酸激酶样蛋白，Pyk 3和Phg 2，作为可能的STATc磷酸化的调节剂，并产生单和双敲除突变体。在pyk 3（-）、phg 2（-）和pyk 3（-）/phg 2（-）细胞中，STATc和STATc依赖基因的转录调控受到干扰。Pyk 3和/或Phg 2的缺失导致响应于山梨醇、8-Br-cGMP和Ca 2+释放剂BHQ的STATc依赖性基因的转录减少或完全消除。此外，磷酸化STATc水平在pyk 3（-）和phg 2（-）细胞中显著降低，并且在pyk 3（-）/phg 2（-）细胞中甚至进一步降低。GFP-STATc核转位的显著延迟反映了磷酸化的减少。蛋白酪氨酸磷酸酶3（PTP 3），去磷酸化和抑制STATc，被抑制应激诱导的磷酸化S448和S747。使用磷酸丝氨酸特异性抗体显示Phg 2而不是Pyk 3参与S747上的PTP 3的磷酸化。在下拉试验中，Phg 2和PTP 3直接相互作用，表明Phg 2在体内磷酸化S747上的PTP 3。在phg 2（-）细胞中S448的磷酸化没有改变。我们发现，Phg 2和一个，尚未知道，S448蛋白激酶是负责PTP 3磷酸化，因此它的抑制，Pyk 3参与的STATc的调节，通过直接或间接激活it. Our结果增加了进一步的复杂性STATc的调节，这大概确保其最佳的激活响应不同的环境线索。
Cellular adaptation to changes in environmental osmolarity is crucial for cell survival. In Dictyostelium, STATc is a key regulator of the transcriptional response to hyperosmotic stress. Its phosphorylation and consequent activation is controlled by two signaling branches, one cGMP-and the other Ca2+-dependent, of which many signaling components have yet to be identified. The STATc stress signalling pathway feeds back on itself by upregulating the expression of STATc and STATc-regulated genes. Based on microarray studies we chose two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators of STATc phosphorylation and generated single and double knock-out mutants to them. Transcriptional regulation of STATc and STATc dependent genes was disturbed in pyk3(-), phg2(-), and pyk3(-)/phg2(-) cells. The absence of Pyk3 and/or Phg2 resulted in diminished or completely abolished increased transcription of STATc dependent genes in response to sorbitol, 8-Br-cGMP and the Ca2+ liberator BHQ. Also, phospho-STATc levels were significantly reduced in pyk3(-) and phg2(-) cells and even further decreased in pyk3(-)/phg2(-) cells. The reduced phosphorylation was mirrored by a significant delay in nuclear translocation of GFP-STATc. The protein tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits STATc, is inhibited by stress-induced phosphorylation on S448 and S747. Use of phosphoserine specific antibodies showed that Phg2 but not Pyk3 is involved in the phosphorylation of PTP3 on S747. In pull-down assays Phg2 and PTP3 interact directly, suggesting that Phg2 phosphorylates PTP3 on S747 in vivo. Phosphorylation of S448 was unchanged in phg2(-) cells. We show that Phg2 and an, as yet unknown, S448 protein kinase are responsible for PTP3 phosphorylation and hence its inhibition, and that Pyk3 is involved in the regulation of STATc by either directly or indirectly activating it. Our results add further complexities to the regulation of STATc, which presumably ensure its optimal activation in response to different environmental cues.