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Myristoylated alanine-rich C kinase substrate-like protein-1 regulates epithelial sodium channel activity in renal distal convoluted tubule cells.

ABSTRACT: The epithelial sodium channel (ENaC) regulates blood pressure by fine-tuning distal nephron sodium reabsorption. Our previous work has shown that ENaC gating is regulated by anionic phospholipid phosphates, including 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 and is a reversible source of PIP₂ at the plasma membrane. We examined MLP-1 regulation of ENaC in distal convoluted tubule clonal cell line DCT-15 cells. Wild-type MLP-1 runs at an apparent molecular mass of 52 kDa despite having a predicted molecular mass of 21 kDa. Native MLP-1 consists of several distinct structural elements: an effector domain that is highly positively charged, sequesters PIP₂, contains serines that are the target of PKC, and controls MLP-1 association with the membrane; a myristoylation domain that promotes association with the membrane; and a multiple homology 2 domain of previously unknown function. To further examine MLP-1 in DCT-15 cells, we constructed several MLP-1 mutants: WT, a full-length wild-type protein; S3A, three substitutions in the effector domain to prevent phosphorylation; S3D mimicked constitutive phosphorylation by replacing three serines with aspartates; and GA replaced the myristoylation site glycine with alanine, so GA could not be myristoylated. Each mutant was tagged with either NH₂-terminal 3XFLAG or COOH-terminal mCherry or V5. Transfection with MLP mutants modified ENaC activity in DCT-15 cells: activity was highest in S3A and lowest in S3D, and the activity after transfection with either construct was significantly different from WT. In Western blots, when transfected with 3XFLAG-tagged MLP-1 mutants, the expression of the full length of MLP-1 at 52 kDa increased in mutant S3A-MLP-1-transfected DCT-15 cells and decreased in S3D-MLP-1-transfected DCT-15 cells. Several lower molecular mass bands were also detected that correspond to potential presumptive calpain cleavage products. Confocal imaging shows that the different mutants localize in different subcellular compartments consistent with their preferred location in the membrane or in the cytosol. Activation of 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.

SUBMITTER: Song C

PROVIDER: S-EPMC7509269 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe potassium channel Kir4.1 forms the Kir4.1/Kir5.1 heterotetramer in the basolateral membrane of the distal convoluted tubule (DCT) and plays an important role in the regulation of the thiazide-sensitive NaCl cotransporter (NCC). Kidney-specific deletion of the ubiquitin ligase Nedd4-2 increases expression of NCC, and coexpression of Nedd4-2 inhibits Kir4.1/Kir5.1 in vitro. Whether Nedd4-2 regulates NCC expression in part by regulating Kir4.1/Kir5.1 channel activity in the DCT is unknown.MethodsWe used electrophysiology studies, immunoblotting, immunostaining, and renal clearance to examine Kir4.1/Kir5.1 activity in the DCT and NCC expression/activity in wild-type mice and mice with kidney-specific knockout of Nedd4-2, Kir4.1, or both.ResultsDeletion of Nedd4-2 increased the activity/expression of Kir4.1 in the DCT and also, hyperpolarized the DCT membrane. Expression of phosphorylated NCC/total NCC and thiazide-induced natriuresis were significantly increased in the Nedd4-2 knockout mice, but these mice were normokalemic. Double-knockout mice lacking both Kir4.1/Kir5.1 and Nedd4-2 in the kidney exhibited increased expression of the epithelial sodium channel α-subunit, largely abolished basolateral potassium ion conductance (to a degree similar to that of kidney-specific Kir4.1 knockout mice), and depolarization of the DCT membrane. Compared with wild-type mice, the double-knockout mice displayed inhibited expression of phosphorylated NCC and total NCC and had significantly blunted thiazide-induced natriuresis as well as renal potassium wasting and hypokalemia. However, NCC expression/activity was higher in the double-knockout mice than in Kir4.1 knockout mice.ConclusionsNedd4-2 regulates Kir4.1/Kir5.1 expression/activity in the DCT and modulates NCC expression by Kir4.1-dependent and Kir4.1-independent mechanisms. Basolateral Kir4.1/Kir5.1 activity in the DCT partially accounts for the stimulation of NCC activity/expression induced by deletion of Nedd4-2.

Project description:The myristoylated alanine-rich C-kinase substrate (MARCKS) protein has been at the crossroads of multiple signaling pathways that govern several critical operations in normal and malignant cellular physiology. Functioning as a target of protein kinase C, MARCKS shuttles between the phosphorylated cytosolic form and the unphosphorylated plasma membrane-bound states whilst regulating several molecular partners including, but not limited to calmodulin, actin, phosphatidylinositol-4,5-bisphosphate, and phosphoinositide-3-kinase. As a result of these interactions, MARCKS directly or indirectly modulates a host of cellular functions, primarily including cytoskeletal reorganization, membrane trafficking, cell secretion, inflammatory response, cell migration, and mitosis. Recent evidence indicates that dysregulated expression of MARCKS is associated with the development and progression of hematological cancers. While it is understood that MARCKS impacts the overall carcinogenesis as well as plays a part in determining the disease outcome in blood cancers, we are still at an early stage of interpreting the pathophysiological roles of MARCKS in neoplastic disease. The situation is further complicated by contradictory reports regarding the role of phosphorylated versus an unphosphorylated form of MARCKS as an oncogene versus tumor suppressor in blood cancers. In this review, we will investigate the current body of knowledge and evolving concepts of the physical properties, molecular network, functional attributes, and the likely pathogenic roles of MARCKS in hematological malignancies. Key emphasis will also be laid upon understanding the novel mechanisms by which MARCKS determines the overall disease prognosis by playing a vital role in the induction of therapeutic resistance. Additionally, we will highlight the importance of MARCKS as a valuable therapeutic target in blood cancers and will discuss the potential of existing strategies available to tackle MARCKS-driven blood cancers.

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Myristoylated alanine-rich C kinase substrate-like protein-1 regulates epithelial sodium channel activity in renal distal convoluted tubule cells.

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