Abstract
Connexin (Cx) 45.6, an avian counterpart of rodent Cx50, is phosphorylated in vivo, but the sites and function of the phosphorylation have not been elucidated. Our peptide mapping experiments showed that the Ser(363) site in the carboxyl (COOH) terminus of Cx45.6 was phosphorylated and that this site is within casein kinase (CK) II consensus sequence, although showing some similarity to CKI sequence. The peptide containing Ser(363) could be phosphorylated in vitro by CKII, but not by CKI. Furthermore, CKII phosphorylated Cx45.6 in embryonic lens membrane and the fusion protein containing the COOH terminus of Cx45.6. Two-dimensional peptide mapping experiments showed that one of the Cx45.6 peptides phosphorylated in vivo migrated to the same spot as one of those phosphorylated by CKII in vitro. Furthermore, CKII activity could be detected in lens lysates. To assess the function of this phosphorylation event, exogenous wild type and mutant Cx45.6 (Ser(363) --> Ala) were expressed in lens primary cultures by retroviral infection. The mutant Cx45.6 was shown to be more stable having a longer half-life compared with wild type Cx45.6. Together, the evidence suggests that CKII is likely a kinase responsible for the Ser(363) phosphorylation, leading to the destablization and degradation of Cx45.6. The connexin degradation induced by phosphorylation has a broad functional significance in the regulation of gap junctions in vivo.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Fertilized chicken eggs were obtained from SPAFAS (Roanoke, IL) and Tyson Hatchery (Gongalez, TX)
Gap junctions are channels between two adjacent cells, which allow passage of small molecules (Mr Յ 1000) such as small metabolites, ions, and second messengers
We have shown that the lens fiber Cx45.6 is phosphorylated in vivo on Ser363 at the COOH terminus and that this phosphorylation is likely to be mediated by CKII
Summary
Materials—Fertilized chicken eggs were obtained from SPAFAS (Roanoke, IL) and Tyson Hatchery (Gongalez, TX). Immunoprecipitation experiments were performed with in vitro phosphorylated lens membrane samples by Cx45.6 affinity purified antibody as described previously [33]. 32P Labeling of the Chick Embryonic Lens Organs and Two-dimensional Tryptic Phosphopeptide Analysis—Intact lenses were removed from embryonic day 9 chicken as described previously [12] and washed three times with phosphate-free Dulbecco’s modified Eagle’s medium. In vitro phosphorylated GST-Cx45.6 fusion protein and immunoprecipated samples of 32P-labeled lens organ culture were separated on 10% SDS-PAGE. Analysis of the CKII Activity in Lens Lysate—The CKII activity assay was initiated by the addition of embryonic day 16 lens lysate containing 5–10 g of proteins to CKII phosphorylation buffer (20 Ci of [␥-32P]ATP, 10 mM MgCl2, 50 mM MOPS, pH 7.0, 150 mM NaCl, 2 mM Na3VO4, 2 mM EDTA, and 25 mM -glycerolphosphate) plus or minus 2 mM peptides. The cultures were rinsed with complete medium (Dulbecco’s modified Eagle’s medium supplemented with 0.5 mM methionine and 10% fetal calf serum) and incubated in the non-radioactive medium for various chase periods
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