We previously found that IP6K1 is enriched in the plasma membrane ( 8) and wondered whether its deletion alters protein expression in the cell membrane. Furthermore, evidence for the existence of endogenous negative regulators of Na +/K +-ATPase, such as endogenous digitalis-like factors, has existed for decades ( 17), but the identification of such molecules has been elusive. However, the regulatory mechanisms controlling Na +/K +-ATPase homeostatic turnover are unclear. Subsequently, adaptor protein 2 (AP2) is recruited to drive clathrin-mediated endocytosis ( 15) and downstream proteasomal and lysosomal degradation of Na +/K +-ATPase ( 12, 16). Endocytosis of Na +/K +-ATPase under pathological conditions involves phosphorylation of the α subunit ( 13) and interaction with phosphatidylinositol 3-kinase (PI3K) p85α ( 14). Steady-state turnover of Na +/K +-ATPase requires coordinated endocytosis from, and insertion into, the plasma membrane ( 12). It generates electrochemical gradients across the cell membrane that are crucial for cell volume maintenance, signal transduction, and secondary transport of various nutrients ( 11).
Na +/K +-transporting adenosine triphosphatase (Na +/K +-ATPase) is universally expressed in the plasma membrane of animal cells, constituting a substantial proportion of total membrane proteins and consuming a large percentage of cytoplasmic ATP ( 10). To further understand the physiological function of IP6K1, we performed a gel-based proteomic screen of membrane proteins and found that deletion of IP6K1 elicits a twofold enrichment of Na +/K +-ATPase-α1 in the plasma membrane. Both the distinct phenotypic regulation and the differential localization suggest a model whereby individual IP6Ks generate pools of 5-InsP 7 that function in discrete, localized areas where the kinase is enriched ( 5, 9). Moreover, IP6Ks exhibit an isoform-specific cellular localization: IP6K1/K3 localizes to the cytoplasm and plasma membrane, whereas IP6K2 localizes to the nucleus ( 7, 8). IP6K1 knockout (KO) mice are lean and resistant to high-fat–induced obesity, IP6K2 KO mice are more susceptible to carcinogen-induced aerodigestive tract carcinoma, and IP6K3 KO mice display impaired motor learning and coordination ( 5, 6). Deleting different IP6K isoforms elicits distinct phenotypes. 5-InsP 7 regulates disparate cell biology processes, including nuclear dynamics, vacuole biogenesis, vesicular trafficking, cellular morphology, cell death, and growth factor and cytokine signaling ( 4, 5). 5-Diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP 7), which is generated by a family of three inositol hexakisphosphate kinases (IP6Ks), is the most abundant inositol pyrophosphate in mammalian cells ( 3). Higher inositol phosphates with energetic pyrophosphate bonds participate in essential cell signaling pathways by binding or pyrophosphorylating target proteins ( 2). d- myo-inositol 1,4,5-trisphosphate, the most studied inositol phosphate, releases calcium from intracellular stores ( 1).
Inositol phosphates are indispensable signaling molecules in animal and plant cells. Our study identifies 5-InsP 7 as an endogenous negative regulator of Na +/K +-ATPase-α1. This recruits adaptor protein 2 (AP2) and triggers the clathrin-mediated endocytosis of Na +/K +-ATPase-α1. Using a suite of synthetic chemical biology tools, we found that 5-InsP 7 binds the RhoGAP domain of phosphatidylinositol 3-kinase (PI3K) p85α to disinhibit its interaction with Na +/K +-ATPase-α1. Deletion of IP6K1 elicits a twofold enrichment of Na +/K +-ATPase-α1 in plasma membranes of multiple tissues and cell types. Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP 7), generated by inositol hexakisphosphate kinase 1 (IP6K1), promotes physiological endocytosis and downstream degradation of Na +/K +-ATPase-α1. Mechanisms of regulating Na +/K +-ATPase homeostatic turnover are unknown. Endogenous negative regulators have long been postulated to play an important role in regulating the activity and stability of Na +/K +-ATPase, but characterization of these regulators has been elusive. Sodium/potassium-transporting adenosine triphosphatase (Na +/K +-ATPase) is one of the most abundant cell membrane proteins and is essential for eukaryotes.
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