Zhang Jinwei, Siew Keith, Macartney Thomas, O'Shaughnessy Kevin M, Alessi Dario R
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland and
Experimental Medicine and Immunotherapeutics, Department of Medicine, University of Cambridge, Cambridge, UK.
Hum Mol Genet. 2015 Aug 15;24(16):4545-58. doi: 10.1093/hmg/ddv185. Epub 2015 May 20.
The STE20/SPS1-related proline/alanine-rich kinase (SPAK) controls blood pressure (BP) by phosphorylating and stimulating the Na-Cl (NCC) and Na-K-2Cl (NKCC2) co-transporters, which regulate salt reabsorption in the kidney. SPAK possesses a conserved carboxy-terminal (CCT) domain, which recognises RFXV/I motifs present in its upstream activator [isoforms of the With-No-lysine (K) kinases (WNKs)] as well as its substrates (NCC and NKCC2). To define the physiological importance of the CCT domain, we generated knock-in mice in which the critical CCT domain Leu502 residue required for high affinity recognition of the RFXI/V motif was mutated to Alanine. The SPAK CCT domain defective knock-in animals are viable, and the Leu502Ala mutation abolished co-immunoprecipitation of SPAK with WNK1, NCC and NKCC2. The CCT domain defective animals displayed markedly reduced SPAK activity and phosphorylation of NCC and NKCC2 co-transporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in mRNA levels. The SPAK CCT domain knock-in mice showed typical features of Gitelman Syndrome with mild hypokalaemia, hypomagnesaemia, hypocalciuria and displayed salt wasting on switching to a low-Na diet. These observations establish that the CCT domain plays a crucial role in controlling SPAK activity and BP. Our results indicate that CCT domain inhibitors would be effective at reducing BP by lowering phosphorylation as well as expression of NCC and NKCC2.
STE20/SPS1相关富含脯氨酸/丙氨酸激酶(SPAK)通过磷酸化并激活钠氯共转运体(NCC)和钠钾氯共转运体(NKCC2)来控制血压(BP),这两种共转运体调节肾脏中的盐重吸收。SPAK具有一个保守的羧基末端(CCT)结构域,该结构域可识别其上游激活剂[无赖氨酸(K)激酶(WNK)的异构体]及其底物(NCC和NKCC2)中存在的RFXV/I基序。为了确定CCT结构域的生理重要性,我们构建了基因敲入小鼠,其中将RFXI/V基序高亲和力识别所需的关键CCT结构域Leu502残基突变为丙氨酸。SPAK CCT结构域缺陷的基因敲入动物能够存活,并且Leu502Ala突变消除了SPAK与WNK1、NCC和NKCC2的共免疫沉淀。CCT结构域缺陷的动物显示SPAK活性以及NCC和NKCC2共转运体在被SPAK磷酸化的残基处的磷酸化明显降低。这还伴随着NCC和NKCC2蛋白表达的降低,而mRNA水平没有变化。SPAK CCT结构域基因敲入小鼠表现出吉特曼综合征的典型特征,伴有轻度低钾血症、低镁血症、低钙尿症,并且在改用低钠饮食时出现盐消耗。这些观察结果表明,CCT结构域在控制SPAK活性和血压方面起着关键作用。我们的结果表明,CCT结构域抑制剂通过降低NCC和NKCC2的磷酸化以及表达来有效降低血压。