Endogenous casein kinase I catalyzes the phosphorylation of the lens fiber cell connexin49.
The lens fiber cell-specific gap junction protein connexin49 is a substrate for a membrane-associated Ser/Thr protein kinase that can be extracted from lens cell membranes by 0.6 M KCl. However, the identity of this protein kinase has not been defined. In this report, evidence is presented indicating that it is casein kinase I. Thus, connexin49 was shown to be a substrate for purified casein kinase I but not for casein kinase II; the endogenous connexin49 protein kinase activity extracted from lens membranes with KCl was inhibited by the casein kinase I-specific inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7); the connexin49 protein kinase activity in the lens membrane KCl extract, which could be partially purified by gel filtration and affinity purification with a casein-Sepharose 4B column, copurified with casein kinase activity; phosphopeptide analysis showed that casein kinase I and the connexin49 protein kinase activity in the lens membrane KCl extract probably share the same phosphorylation sites in connexin49. Reverse transcription-PCR using total ovine lens RNA and casein kinase I isoform-specific oligonucleotide primers resulted in the amplification of cDNAs encoding casein kinase I-alpha and -gamma, while an in-gel casein kinase assay indicated casein kinase activity in the lens membrane KCl extract was associated with a major 39.2-kDa species, which is consistent with the 36 to 40-kDa size of casein kinase I-alpha in other animal species. These results demonstrate that the protein kinase activity present in the lens membrane 0.6 M KCl extract that catalyzes the phosphorylation of connexin49 is casein kinase I, probably the alpha isoform.
Cheng
H L
HL
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
Louis
C F
CF
D000818:Animals; D001483:Base Sequence; D047390:Casein Kinase II; D047388:Casein Kinases; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D017630:Connexins / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007904:Lens Cortex, Crystalline / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012756:Sheep; D013379:Substrate Specificity
D017630:Connexins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C109089:connexin 49 protein, Ovis aries; D011494:Protein Kinases; D047390:Casein Kinase II; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases