Lens fiber connexin turnover and caspase-3-mediated cleavage are regulated alternately by phosphorylation.
Lens connexins are phosphorylated in vivo; however, the function and regulation of the phosphorylation remain largely unknown. We have previously identified an in vivo phosphorylation site, Ser(364), at the COOH terminus of lens connexin (Cx) Cx45.6 and phosphorylation appears to regulate connexin protein turnover. To assess the specific mechanism of Ser(364) phosphorylation in Cx45.6, exogenous wild type and Ser(364) mutant Cx45.6 were expressed in primary lens cultures through retroviral infection. Cx45.6 turnover was attenuated primarily by proteasomal inhibitors and to a lesser extent by lysosomal inhibitors. Furthermore, the level of Cx45.6 protein in ubiquitin co-expressed cells was significantly reduced as compared to the cells expressing Cx45.6 alone. Moreover, overexpression of ubiquitin led to a more significant decrease in wild type Cx45.6 than Cx45.6(S364A), a mutant deficient of phosphorylation site at Ser(364), although we did not detect any difference in the levels of ubiquitination between wild type and mutant Cx45.6. Interestingly, the mutant mimicking constitutive phosphorylation, Cx45.6(S364D), partially prevented the cleavage of Cx45.6 by caspase-3. Together, our data suggest that phosphorylation of Cx45.6 at Ser(364) appears to stimulate Cx45.6 turnover primarily through proteasome pathway and this phosphorylation inhibits the cleavage of Cx45.6 by caspase-3. These findings provide further insights into regulatory mechanism of the specific phosphorylation of connexins in the lens.
Yin
Xinye
X
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, USA.
Liu
Jialu
J
Jiang
Jean X
JX
D000818:Animals; D053148:Caspase 3 / Q000502:physiology*; D002460:Cell Line; D002642:Chick Embryo; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D007908:Lens, Crystalline / Q000502:physiology*; D009924:Organ Culture Techniques; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000502:physiology; D012694:Serine / Q000235:genetics / Q000378:metabolism
D017630:Connexins; C082920:connexin 45; D012694:Serine; D053148:Caspase 3; D046988:Proteasome Endopeptidase Complex