No Yi Ran, He Peijian, Yoo Byong Kwon, Yun C Chris
Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia;
Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; Winship Cancer Institute, Emory University, Atlanta, Georgia
Am J Physiol Cell Physiol. 2015 Jul 1;309(1):C14-21. doi: 10.1152/ajpcell.00067.2015. Epub 2015 Apr 8.
Na(+)/H(+) exchange by Na(+)/H(+) exchanger 3 (NHE3) is a major route of sodium absorption in the intestine and kidney. We have shown previously that lysophosphatidic acid (LPA), a small phospholipid produced ubiquitously by all types of cells, stimulates NHE3 via LPA5 receptor. Stimulation of NHE3 activity by LPA involves LPA5 transactivating EGF receptor (EGFR) in the apical membrane. EGFR activates proline-rich tyrosine kinase 2 (Pyk2) and ERK, both of which are necessary for NHE3 regulation. However, Pyk2 and ERK are regulated by EGFR via independent pathways and appear to converge on an unidentified intermediate that ultimately targets NHE3. The p90 ribosomal S6 kinase (RSK) family of Ser/Thr protein kinases is a known effector of EGFR and ERK. Hence, we hypothesized that RSK may be the convergent effector of Pyk2 and ERK although it is not known whether Pyk2 regulates RSK. In this study, we show that Pyk2 is necessary for the maintenance of phosphoinositide-dependent kinase 1 (PDK1) autophosphorylation, and knockdown of Pyk2 or PDK1 mitigated LPA-induced phosphorylation of RSK and stimulation of NHE3 activity. Additionally, we show that RSK2, but not RSK1, is responsible for NHE3 regulation. RSK2 interacts with NHE3 at the apical membrane domain, where it phosphorylates NHE3. Alteration of S663 of NHE3 ablated LPA-induced phosphorylation of NHE3 and stimulation of the transport activity. Our study identifies RSK2 as a new kinase that regulates NHE3 activity by direct phosphorylation.
由钠氢交换体3（NHE3）介导的钠氢交换是肠道和肾脏中钠吸收的主要途径。我们之前已经表明，溶血磷脂酸（LPA）是一种由所有类型细胞普遍产生的小磷脂，它通过LPA5受体刺激NHE3。LPA对NHE3活性的刺激涉及LPA5在顶端膜中转活表皮生长因子受体（EGFR）。EGFR激活富含脯氨酸的酪氨酸激酶2（Pyk2）和细胞外信号调节激酶（ERK），这两者都是NHE3调节所必需的。然而，Pyk2和ERK由EGFR通过独立途径调节，并且似乎汇聚于一个最终靶向NHE3的未知中间体。丝氨酸/苏氨酸蛋白激酶的p90核糖体S6激酶（RSK）家族是EGFR和ERK的已知效应器。因此，我们假设RSK可能是Pyk2和ERK的汇聚效应器，尽管尚不清楚Pyk2是否调节RSK。在本研究中，我们表明Pyk2对于维持磷酸肌醇依赖性激酶1（PDK1）的自磷酸化是必需的，并且敲低Pyk2或PDK1可减轻LPA诱导的RSK磷酸化和NHE3活性的刺激。此外，我们表明是RSK2而非RSK1负责NHE3的调节。RSK2在顶端膜结构域与NHE3相互作用，在那里它使NHE3磷酸化。NHE3的S663位点改变消除了LPA诱导的NHE3磷酸化和转运活性的刺激。我们的研究确定RSK2是一种通过直接磷酸化调节NHE3活性的新激酶。
