Some aspects of the phosphorylation of alpha-crystallin A.
The cAMP-dependent phosphorylation of alpha-crystallin was investigated. The major products of in vitro phosphorylation of total bovine lens homogenate are the alpha A1 and alpha B1 polypeptides, but in addition a minor labeled spot is present which might correspond with a double phosphorylated alpha B chain. It is demonstrated that the A1 and B1 subunits of alpha-crystallin from bovine eye lenses are solely the result of phosphorylation of the primary gene products alpha A2 and alpha B2, respectively, as judged from the stoichiometry of the phosphate content of these polypeptides. Both the in vitro and in vivo phosphorylation sites of the A chain of bovine alpha-crystallin were determined and found to be the same. After in vitro incubation the majority of the 32P label was found in the tryptic peptides T17a and T16-17a, the latter being the result of incomplete tryptic cleavage between T16 and T17a. The in vivo phosphorylation site is also located in T17a, as could be concluded from the retention times on reversed-phase HPLC of T16-17a and T17a from alpha A1 as compared to those from alpha A2, and from the differences in their mobilities on high-voltage paper electrophoresis at pH 6.5. Furthermore, both T17a and T16-17a of alpha A1 contain approximately 1 mol phosphate/mol peptide. Thermolytic digestion of T16-17a of both alpha A2 and alpha A1, followed by separation on RP-HPLC, demonstrated that Ser-122 is the phosphorylation site of the A chain of bovine lens alpha-crystallin. The replacement of this phosphorylation site or the lack of basic amino acids at the N-terminal side of Ser-122 in some vertebrate species apparently results in the absence of phosphorylation of alpha-crystallin A both in vitro and in vivo.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002645:Chickens; D003459:Crystallins / Q000378:metabolism*; D004284:Dogfish; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats; D013045:Species Specificity
D000596:Amino Acids; D003459:Crystallins