Endogenous casein kinase I catalyzes the phosphorylation of the lens fibercell 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 fr om 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 indicati ng 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 w as inhibited by the casein kinase I-specific inhibitor, N-(2-aminoethyl)-5- chloroisoquinoline-8-sulfonamide (CKI-7); the connexin49 protein kinase act ivity in the lens membrane KCI extract, which could be partially purified b y gel filtration and affinity purification with a casein-Sepharose 4B colum n. copurified with casein kinase activity; phosphopeptide analysis showed t hat casein kinase I and the connexin49 protein kinase activity in the lens membrane KCl extract probably share the same phosphorylation sites in conne xin49. Reverse transcription-PCR using total ovine lens RNA and casein kina se 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 ext ract 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. Th ese results demonstrate that the protein kinase activity present in the len s membrane 0.6 M KCl extract that catalyzes the phosphorylation of connexin 49 is casein kinase I, probably the alpha isoform.