Characterization of the bovine lens plasma membrane substrates for cAMP-dependent protein kinase.
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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D000818:Animals; D020346:Aquaporins; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D002918:Chymotrypsin / Q000494:pharmacology; D003459:Crystallins / Q000032:analysis*; D005136:Eye Proteins / Q000378:metabolism; D007908:Lens, Crystalline / Q000032:analysis*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000032:analysis*; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis*; D012394:Rosaniline Dyes
D020346:Aquaporins; D003459:Crystallins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D012394:Rosaniline Dyes; C030328:aquaporin 0; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D002918:Chymotrypsin; C004692:coomassie Brilliant Blue