Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.
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PMID:Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens. 298 31

cAMP-dependent protein kinase, derived from either calf lens or bovine heart, promotes the phosphorylation of three lens plasma membrane proteins of molecular mass 28 kDa, 26 kDa and 18 kDa. Correlation of the maximal level of phosphorylation of these components with the Coomassie blue staining intensity of fractionated lens membranes suggests that the phosphorylation of the 28 kDa and 18 kDa components may be approximately stoichiometric. The protein kinase substrates could be dephosphorylated by a cardiac sarcoplasmic-reticulum-bound protein phosphatase activity. The 26 k Da component comigrated with MP26, the major lens membrane component that has been localized to the lens fiber cell junction. Treatment of phosphorylated lens membranes with chymotrypsin did not suggest that any of the three major phosphorylated components was derived from the partial proteolysis of a larger phosphoprotein. After electrophoretic separation of phosphorylated proteins, treatment with N-chlorosuccinimide confirmed that there was little similarity in the structure of the three phosphoproteins. Chymotrypsin did, however, reveal a cryptic phosphorylation site in a 22 kDa fragment that appeared to be derived from MP26. Treatment of phosphorylated membranes with reducing agents resulted in the disappearance of the 28 kDa phosphorylated component and the appearance of a new phosphorylated component of 18 kDa; neither MP26 nor the original 18 kDa component was affected by such treatment. It is not clear whether the original 18 kDa phosphoprotein, present in unreduced samples, is the same as that generated with reducing agents from the 28 kDa phosphorylated lens membrane component.
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PMID:Characterization of the bovine lens plasma membrane substrates for cAMP-dependent protein kinase. 299 Sep 30

A major protein with a molecular weight of 17,000, designated as MP17, has been identified in mammalian eye lens plasma membranes. Hydrophobic photolabeling experiments revealed that MP17 is a genuine intrinsic membrane protein. By using monoclonal antibodies we demonstrated that MP17 is not detectable in liver, heart, muscle, spleen and kidney, and thus can be considered, like MP26, as a lens-specific membrane protein. Furthermore, we showed that MP17 is a substrate for cAMP-dependent protein kinase and that it is a calmodulin-binding protein.
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PMID:MP17, a fiber-specific intrinsic membrane protein from mammalian eye lens. 337 Oct 69

The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at Mr 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at Mr 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at Mr 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated Mr 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within approximately 20 to 40 residues from the COOH-terminus of MP26. Published work indicates that the phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10% threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein.
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PMID:A lens intercellular junction protein, MP26, is a phosphoprotein. 395 48

Purification of the lens fiber cell membrane proteins MP20 and MP26, and the partial co-purification of the lens connexin-related proteins MP70 and connexin 46 has been achieved using anion- and cation-exchange chromatography of lens fiber cell membrane proteins solubilized in n-octyl-beta-D-glucopyranoside (octyl glucoside). The apparent molecular weights of the solubilized protein-detergent complexes were significantly greater than that expected for the monomeric proteins. The purified proteins retained their ability to be phosphorylated by cAMP-dependent protein kinase, and to bind calmodulin in a calcium and magnesium dependent manner. The heterobifunctional covalent chemical crosslinking agent N-5-azido-2-nitro-benzoyloxysuccinimide (ANB-NOS), and the thiol oxidant cupric phenanthroline were used to identify the oligomeric states of these proteins. Crosslinking of either the purified proteins or native lens membranes generated a ladder of crosslinked MP20 or MP26 homo-oligomers. The largest detectable crosslinked homo-oligomer of MP20 was at least a hexamer, while for MP26 the largest crosslinked homo-oligomer was at least a tetramer. The possible oligomeric states of MP70 and connexin 46 could not be determined with the crosslinking reagents used in this study. The procedure described here for the purification of detergent-solubilized major lens proteins should provide a valuable approach in future studies aimed at clarifying the roles of these different lens membrane proteins.
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PMID:Purification and oligomeric state of the major lens fiber cell membrane proteins. 852 19

The cAMP-dependent protein-kinase-catalyzed phosphorylation of the two major intrinsic lens fiber cell plasma membrane proteins, MP20 and MP26, is likely restricted to the inner cortical and nuclear regions of the lens in vivo. The ovine-lens-specific connexin, MP70, that has been identified as Cx50 in mice and Cx45.6 in the chick, is also a protein kinase substrate although it does not appear to be phosphorylated by a number of protein kinases including cAMP-dependent protein kinase, calmodulin-dependent protein kinase or protein kinase C. Rather, an extrinsic lens membrane fraction was isolated which contained protein kinase activity that catalyzed the phosphorylation of MP70; this protein kinase activity was cAMP-independent, Ca(2+)-independent, Mg(2+)-dependent, phosphorylated MP70 on a serine residue(s) and migrated with a molecular mass of 35 kDa on a gel filtration column. Both MP70 phosphorylation and the endogenous protein kinase activity were restricted to the lens outer cortical region. This membrane-associated protein kinase activity represents the first reported partial characterization of an endogenous lens fiber cell protein kinase activity that catalyzes the phosphorylation of a lens connexin protein. The phosphatase-induced shift in the electrophoretic mobility of MP70 is not reversed by this protein kinase, indicating that MP70 is likely phosphorylated on different residues by two or more protein kinases.
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PMID:Characterization of the ovine-lens plasma-membrane protein-kinase substrates. 853 18

Salivary secretion occurs in response to stimulation by neurotransmitters released from autonomic nerve endings. The molecular mechanisms underlying the secretion of water, a main component of saliva, from salivary glands are not known; the plasma membrane is a major barrier to water transport. A 28-kDa integral membrane protein, distributed in highly water-permeable tissues, was identified as a water channel protein, aquaporin (AQP). Thirteen AQPs (AQP0 - AQP12) have been identified in mammals. AQP5 is localized in lipid rafts under unstimulated conditions and translocates to the apical plasma membrane in rat parotid glands upon stimulation by muscarinic agonists. The importance of increases in intracellular calcium concentration [Ca(2+)](i) and the nitric oxide synthase and protein kinase G signaling pathway in the translocation of AQP5 is reviewed in section I. Signals generated by the activation of Ca(2+) mobilizing receptors simultaneously trigger and regulate exocytosis. Zymogen granule exocytosis occurs under the control of essential process, stimulus-secretion coupling, in salivary glands. Ca(2+) signaling is a principal signal in both protein and water secretion from salivary glands induced by cholinergic stimulation. On the other hand, the cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase system has a major role in zymogen granule exocytosis without significant increases in [Ca(2+)](i). In section II, the mechanisms underlying the control of salivary protein secretion and its dysfunction are reviewed.
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PMID:Water channels and zymogen granules in salivary glands. 1679 62