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‐α and ‐γ, 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‐α 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 α isoform.