NHE3 function and phosphorylation are regulated by a calyculin A-sensitive phosphatase
NHE3 function and phosphorylation are regulated by a calyculin A-sensitive phosphatase
复制标题
DOI：
10.1152/ajprenal.00182.2009
复制
发表时间：
2010-03-01
影响因子：
4.2
通讯作者：
Kocinsky, Hetal S.
中科院分区：
文献类型：
--
作者：
Dynia, Diane W.;Steinmetz, Amy G.;Kocinsky, Hetal S.
Dynia DW, Steinmetz AG, Kocinsky HS。NHE3的功能和磷酸化受一种钙调蛋白a敏感磷酸酶的调控。[J] .中国生物医学工程学报，2016,31(5):559 - 563。首次发表于2009年12月16日；doi: 10.1152 / ajprenal.00182.2009。-Na(+)/H（+）交换器3 （NHE3）被磷酸化并受多种激酶调控，包括PKA、SGK1和CK2；然而，磷酸酶在NHE3的去磷酸化和调控中的作用仍然未知。本研究的目的是确定丝氨酸/苏氨酸磷酸酶是否改变NHE3活性和磷酸化，如果是，在哪些位点。为此，我们首先研究了calyculin A[一种组合蛋白磷酸酶1 （PP1）和PP2A抑制剂]和冈田酸（PP2A抑制剂）对一般和位点特异性NHE3磷酸化的影响。Calyculin A诱导磷酸化依赖的NHE3凝胶迁移，并增加了552和605丝氨酸的NHE3磷酸化。冈田酸处理后NHE3磷酸化未见变化。在转染野生型或突变型（S552A, S605G, S661A, S716A）大鼠NHE3的calyculin a处理的COS-7细胞中，NHE3凝胶迁移也明显改变。由于NHE3凝胶迁移发生在已知磷酸化位点突变的情况下，因此必须存在新的磷酸化位点。接下来，我们检测了花青素A和冈田酸对NHE3活性的影响，发现花青素A对NHE3活性的抑制作用为24%，而冈田酸对NHE3活性没有影响。当所有已知的NHE3磷酸化位点发生突变时，calyculin A诱导NHE3活性的刺激，证明了对新磷酸化位点的功能意义。最后，我们在体外建立了PP1催化亚基可以直接使免疫纯化的NHE3去磷酸化。总之，我们的数据表明，一种calyculin a敏感的磷酸酶，最有可能是PP1，参与了NHE3在已知和新位点的调控和去磷酸化。
Dynia DW, Steinmetz AG, Kocinsky HS. NHE3 function and phosphorylation are regulated by a calyculin A-sensitive phosphatase. Am J Physiol Renal Physiol 298: F745-F753, 2010. First published December 16, 2009; doi:10.1152/ajprenal.00182.2009.-Na(+)/H(+) exchanger 3 (NHE3) is phosphorylated and regulated by multiple kinases, including PKA, SGK1, and CK2; however, the role of phosphatases in the dephosphorylation and regulation of NHE3 remains unknown. The purpose of this study was to determine whether serine/threonine phosphatases alter NHE3 activity and phosphorylation and, if so, at which sites. To this end, we first examined the effects of calyculin A [a combined protein phosphatase 1 (PP1) and PP2A inhibitor] and okadaic acid (a PP2A inhibitor) on general and site-specific NHE3 phosphorylation. Calyculin A induced a phosphorylation-dependent NHE3 gel mobility shift and increased NHE3 phosphorylation at serines 552 and 605. No change in NHE3 phosphorylation was detected after okadaic acid treatment. An NHE3 gel mobility shift was also evident in calyculin A-treated COS-7 cells transfected with either wild-type or mutant (S552A, S605G, S661A, S716A) rat NHE3. Since the NHE3 gel mobility shift occurred despite mutation of known phosphorylation sites, novel sites of phosphorylation must also exist. Next, we assayed NHE3 activity in response to calyculin A and okadaic acid and found that calyculin A induced a 24% inhibition of NHE3 activity, whereas okadaic acid had no effect. When all known NHE3 phosphorylation sites were mutated, calyculin A induced a stimulation of NHE3 activity, demonstrating a functional significance for the novel phosphorylation sites. Finally, we established that the PP1 catalytic subunit can directly dephosphorylate immunopurified NHE3 in vitro. In conclusion, our data demonstrate that a calyculin A-sensitive phosphatase, most likely PP1, is involved in the regulation and dephosphorylation of NHE3 at known and novel sites.