Activation of protein kinase Cα increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct
Activation of protein kinase Cα increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct
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DOI：
10.1152/ajpcell.00171.2014
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发表时间：
2015-11-01
影响因子：
5.5
通讯作者：
Sands, Jeff M.
中科院分区：
文献类型：
--
作者：
Blount, Mitsi A.;Cipriani, Penelope;Sands, Jeff M.
高张力增加尿素的转运，以及UT-A1的磷酸化和膜积累，UT-A1是负责尿素在内髓集合管(IMCD)通透性的转运体。高张力通过PKC介导的磷酸化作用刺激尿素转运。为了确定PKC是否磷酸化UT-A1，分别用丙氨酸替换了8个潜在的PKC磷酸化位点，然后将其导入LLC-PK1细胞。在单个突变体中，只有S494位点的去除抑制了PKC激活剂佛波二丁酸酯(PDBu)对总UT-A1磷酸化的诱导。使用一种新产生的抗体证实了这一结果，该抗体专门检测了S494处UT-A1的磷酸化。在S494，高张力增加了UT-A1的磷酸化。相反，cAMP通路激活剂(PKA和EPAC)在S494没有增加UT-A1的磷酸化。PKC和PKA通路的激活都增加了UT-A1的质膜积聚，尽管单独激活PKC不能做到这一点。然而，去除PKC位点S494会降低质膜上UT-A1的丰度。这表明cAMP途径促进UT-A1转运到顶膜，在那里PKC途径可以磷酸化转运蛋白，导致UT-A1在顶膜的滞留增加。综上所述，PKC的激活增加了UT-A1在特定残基S494的磷酸化。虽然与cAMP信号通路没有串扰，但通过PKC对S494的磷酸化可能通过将UT-A1保留在质膜上来增强加压素刺激的尿素通透性。
Hypertonicity increases urea transport, as well as the phosphorylation and membrane accumulation of UT-A1, the transporter responsible for urea permeability in the inner medullary collect duct (IMCD). Hypertonicity stimulates urea transport through PKC-mediated phosphorylation. To determine whether PKC phosphorylates UT-A1, eight potential PKC phosphorylation sites were individually replaced with alanine and subsequently transfected into LLC-PK1 cells. Of the single mutants, only ablation of the S494 site dampened induction of total UT-A1 phosphorylation by the PKC activator phorbol dibutyrate (PDBu). This result was confirmed using a newly generated antibody that specifically detected phosphorylation of UT-A1 at S494. Hypertonicity increased UT-A1 phosphorylation at S494. In contrast, activators of cAMP pathways (PKA and Epac) did not increase UT-A1 phosphorylation at S494. Activation of both PKC and PKA pathways increased plasma membrane accumulation of UT-A1, although activation of PKC alone did not do so. However, ablating the PKC site S494 decreased UT-A1 abundance in the plasma membrane. This suggests that the cAMP pathway promotes UT-A1 trafficking to the apical membrane where the PKC pathway can phosphorylate the transporter, resulting in increased UT-A1 retention at the apical membrane. In summary, activation of PKC increases the phosphorylation of UT-A1 at a specific residue, S494. Although there is no cross talk with the cAMP-signaling pathway, phosphorylation of S494 through PKC may enhance vasopressin-stimulated urea permeability by retaining UT-A1 in the plasma membrane.