Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The topological organization of the Na,K-ATPase alpha subunit is controversial. Detection of extracellular proteolytic cleavage sites would help define the topology, and so attempts were made to find conditions and proteases that would permit digestion of Na,K-ATPase in sealed right-side-out vesicles from renal medulla. The beta subunit is predominantly extracellular and could mask the surface of the alpha subunit. Most of the tested proteases cleaved beta, and some digested it extensively. However, without further disruption of structure, there was still no digestion of the alpha subunit. Reduction (at 50 degrees C) of disulfide bonds that might stabilize the beta subunit fragments, or heating alone at 55 degrees C, permitted tryptic digestion of alpha at a site close to the C terminus, while simultaneously increasing digestion of beta. A 90-kDa N-terminal fragment of alpha was recovered, but the C-terminal fragment was further digested. Heating and reduction resulted in the extracellular exposure of a protein kinase A phosphorylation site, Ser-938, and the C terminus, both of which have been proposed to be located on the intracellular surface. At the same time, access to a distant protein kinase C phosphorylation site was not increased. The data suggest that the harsh treatment simultaneously resulted in alteration of the beta subunit and the extrusion of a segment of alpha that normally spans the membrane, without causing complete denaturation or opening the sealed vesicles. Preincubation with Rb+ was protective, consistent with prior evidence that it stabilizes the protein segments in the C-terminal third of alpha. We conclude that this portion of the alpha subunit contains a transmembrane structure with unique lability to heating.
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PMID:Topology of the Na,K-ATPase. Evidence for externalization of a labile transmembrane structure during heating. 772 85

There is considerable evidence that protein kinases play a role in regulation of the activity of the Na,K-ATPase, but the characteristics of direct kinase phosphorylation of Na,K-ATPase subunits are still not well understood. There are 36 sites that could qualify as protein kinase C motifs in rat alpha 1. Here we have used protein fragmentation with trypsin to localize the site of phosphorylation of the rat Na,K-ATPase alpha 1 subunit to within the first 32 amino acids of the N terminus and then used direct sequencing of the phosphorylated protein to determine which of two candidate serine residues was modified. The result was that at most 25% of the 32P was found on Ser-11, a site that is well conserved in Na,K-ATPase alpha 1 subunits. The remaining 75% or more of the 32P was found on Ser-18, a site that is absent in many Na,K-ATPase alpha subunit sequences. This accounts for the observation that dog and pig alpha 1 subunits can be phosphorylated by protein kinase C only to much lower levels than can rat alpha 1. It is also likely to be relevant to other known species-specific effects of protein kinase C on Na,K-ATPase.
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PMID:Structural basis for species-specific differences in the phosphorylation of Na,K-ATPase by protein kinase C. 777 68

The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves.
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PMID:In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators. 801 40

Alveolar hypoxia occurs during ascent to high altitude and is also observed in patients with ARDS and acute hypoxemic respiratory failure, in which alveolar flooding is associated with a decrease in edema fluid clearance and increased mortality. The mechanisms that lead to the impairment of alveolar fluid clearance are not completely understood. Alveolar fluid reabsorption is accomplished mostly by active Na+ transport across the alveolar epithelium which creates an osmotic gradient responsible for the clearance of lung edema from the alveolar spaces. In vivo and in vitro hypoxia inhibits both the epithelial sodium channels, responsible for the apical sodium entry, and the basolateral Na,K-ATPase, responsible for Na+ extrusion. We have shown that acute hypoxia inhibits Na,K-ATPase function by promoting its endocytosis from the plasma membrane to intracellular compartments. This process is mediated by the generation of mitochondrial reactive oxygen species (ROS) as shown by pharmacological and genetic approaches. Hypoxia and ROS promote the PKC-zeta dependent phosphorylation of the Na,K-ATPase alpha subunit triggering its endocytosis in a clathrin-AP2 dependent process. The phosphorylation occurs at the Ser-18 in the alpha subunit N-terminus, and mutation of this serine prevents both the decrease in function and the endocytosis. More prolonged hypoxia causes the ubiquitination and degradation of Na,K-ATPase. Thus, methods that counterbalance the inhibition of edema clearance during hypoxia and improve the lung's ability to clear pulmonary edema are needed. As such, a better understanding of the mechanisms that increase Na,K-ATPase function, (i.e., activation of dopaminergic or adrenergic receptors, gene transfer) may lead to the development of therapeutic approaches to upregulate the Na-K-ATPase function and increase edema clearance.
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PMID:Hypoxic inhibition of alveolar fluid reabsorption. 1826 95

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped alpha-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase alpha and beta subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na(+)/K(+) ion binding site of the Na,K-ATPase alpha subunit.
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PMID:Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1. 1976 58