Regulation of Urea Transport by PKC-alpha

说明(申请人提供)：加压素通过激活两条依赖环腺苷酸(CAMP)的信号通路来调节尿素转运：蛋白激酶A和由cAMP激活的交换蛋白。这导致UT-A1尿素转运蛋白的磷酸化和顶端质膜积累增加。内髓的高张力可以独立于血管加压素起作用，作为尿素运输的强大刺激。高张力通过增加UT-A1的磷酸化和质膜积累来刺激尿素的运输。然而，这种情况发生的信号通路尚不清楚。我们有新的初步数据表明，高张通过蛋白激酶C(PKC)刺激尿素的通透性。我们也有初步的数据表明，PKC1是参与其中的特定的PKC同工酶，因为：1)高张激活大鼠内髓集合管(IMCDs)中的PKC1；以及2)PKC1基因敲除的小鼠存在尿液浓缩缺陷和UT-A1蛋白丰度降低。因此，最大限度地刺激IMCD末端的尿素转运和UT-A1活性需要cAMP和PKC1的共同刺激。我们的总体目标是研究PKC对UT-A1的调节。我们将检验PKC1通过改变UT-A1磷酸化来刺激UT-A1功能的假设。目的1将确定PKC调节UT-A1磷酸化的机制。目的2将确定UT-A1的S位点和蛋白激酶C的磷酸化的细胞位置。目的3确定PKC和PKA在调节UT-A1膜蓄积、活性、磷酸化和磷酸酶介导的去磷酸化过程中的相互依赖关系。目的4将确定PKC基因敲除小鼠尿浓缩缺陷的机制。我们的研究具有非常重要的意义，因为它们可能为水平衡失调的潜在机制提供新的信息。阐明增加尿素转运的非cAMP机制，从而提高尿浓缩能力，可能为未来先天性肾源性尿崩症的新治疗方法的翻译研究奠定基础。

DESCRIPTION (provided by applicant): Vasopressin regulates urea transport by activating two cyclic AMP (cAMP) dependent signaling pathways: protein kinase A and exchange protein activated by cAMP. This results in increases of both the phosphorylation and apical plasma membrane accumulation of the UT-A1 urea transporter. The hypertonicity present in the inner medulla can act independently of vasopressin as a powerful stimulus of urea transport. Hypertonicity stimulates urea transport by increasing both UT-A1 phosphorylation and plasma membrane accumulation. However, the signaling pathway by which this occurs is unknown. We have new preliminary data showing that hypertonicity stimulates urea permeability via protein kinase C (PKC). We also have preliminary data suggesting that PKC1 is the specific PKC isozyme involved since: 1) hypertonicity activates PKC1 in rat inner medullary collecting ducts (IMCDs); and 2) PKC1 knock-out mice have a urine concentrating defect and a reduction in UT-A1 protein abundance. Thus, maximal stimulation of urea transport and UT-A1 activity in the terminal IMCD requires stimulation by both cAMP and PKC1. Our overall goal is to investigate the regulation of UT-A1 by PKC. We will test the hypothesis that PKC1 stimulates UT-A1 function through changes in UT-A1 phosphorylation. Aim 1 will determine the mechanism by which PKC regulates UT-A1 phosphorylation. Aim 2 will determine the site(s) in UT-A1 and the cellular location of phosphorylation by PKC. Aim 3 will determine the interdependence of PKC and PKA in the regulation of UT-A1 membrane accumulation, activity, phosphorylation, and phosphatase-mediated dephosphorylation. Aim 4 will determine the mechanism for the urine concentrating defect in PKC" knock-out mice. Our proposed studies are highly significant as they are likely to yield new information on mechanisms underlying dysregulation of water homeostasis. Elucidation of non-cAMP mechanisms for increasing urea transport, which in turn would increase urine concentrating ability, could form the basis for future translational studies of novel therapeutic approaches to congenital nephrogenic diabetes insipidus.