MARCKS-Like Protein-1(MLP-1) regulates ENaC Activity in Renal Distal Convoluted Tubule Cells

Chang Song, Qiang Yue, Auriel Moseley, Otor Al-Khalili, Brandi M Wynne, Heping Ma, Lihua Wang, Douglas C Eaton
American Journal of Physiology. Cell Physiology 2020 July 8
ENaC gating is regulated by phosphatidylinositol 4,5-bisphosphate (PIP2 ). The PIP2 -dependent regulation of ENaC is mediated by the myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1). MLP-1 binds to PIP2 at the plasma membrane. We examined MLP-1 regulation of ENaC in DCT-15 cells. MLP-1 consists of a positively charged effector domain that sequesters PIP2 and contains serines that are a PKC targets and controls MLP-1 membrane association; a myristoylation domain promoting membrane association, and a multiple homology 2 domain of unknown function. We constructed several MLP-1 mutants: WT: a full length wild-type protein; S3A: 3 substitutions in the effector domain to prevent phosphorylation; S3D: replaced three serines with aspartates to mimic constitutive phosphorylation; GA: replaced the myristoylation site glycine with alanine so GA could not be myristoylated. Mutants were tagged with N-terminal 3XFLAG or C-terminal m-Cherry or V5. Transfection with MLP mutants modified ENaC activity: S3A activity was highest and S3D lowest; the activity of both was significantly different from WT. In Western blots, when transfected with 3XFLAG tagged MLP-1 mutants, expression of full length MLP-1 at 52 KDa increased in S3A-MLP-1 transfected cells and decreased in S3D-MLP-1 transfected cells. Lower molecular weight bands were detected that correspond to potential protease cleavage products. Confocal imaging shows that mutants localize in different sub-cellular compartments consistent with their preferred location in the membrane or cytosol. Protein kinase C increases phosphorylation of endogenous MLP-1 and reduces ENaC activity. Our results suggest a complicated role for proteolytic processing in MLP-1 regulation of ENaC.

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