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Endogenous casein kinase I catalyzes the phosphorylation of the lens fiber cell connexin49
- Source :
- European Journal of Biochemistry. 263:276-286
- Publication Year :
- 1999
- Publisher :
- Wiley, 1999.
-
Abstract
- 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.
- Subjects :
- animal structures
In Vitro Techniques
Protein Serine-Threonine Kinases
Mitogen-activated protein kinase kinase
Biology
Biochemistry
Connexins
Substrate Specificity
MAP2K7
Casein Kinase I
Casein kinase 2, alpha 1
Animals
Humans
Enzyme Inhibitors
Phosphorylation
Casein Kinase II
Protein kinase A
DNA Primers
Sheep
Base Sequence
Cell Membrane
Lens Cortex, Crystalline
Molecular biology
Rats
Isoenzymes
Cattle
Rabbits
Casein kinase 1
Casein kinase 2
Casein kinases
Casein Kinases
Protein Kinases
Subjects
Details
- ISSN :
- 14321033 and 00142956
- Volume :
- 263
- Database :
- OpenAIRE
- Journal :
- European Journal of Biochemistry
- Accession number :
- edsair.doi.dedup.....96593a225c8e22cf2ba066091d833599