Lens fiber connexin turnover and caspase-3-mediated cleavage are regulated alternately by phosphorylation

Abstract

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.

Figure 1

Cx45.6 degradation is primarily through proteasomal pathway. The lens primary cultures were treated with 20 µg/ml of cycloheximide alone (A and B, control), cycloheximide plus lysosomal inhibitor, 100 µM leupeptin (A), proteasomal inhibitor, 10 µM clasto-lactacystin β-lactone (A), lysosomal inhibitor 10 mM NH₄Cl (B), or proteasomal inhibitor, 0.5 µM epoxomicin (B) for 0 h (lane 1), 4 h (lane 2), 10 h (lane 3), and 24 h (lane 4). The Cx45.6 protein bands from three separate Western blot analyses were quantified by densitometry (C). The data are presented as the mean ± s.e.m. (n = 3).

Figure 2

Co-expression of ubiquitin and connexin constructs in N2A cells. N2A cells were transiently transfected with wild type and mutant Cx45.6 constructs with or without ubiquitin. Cells were lysed 24 h after transfection. (A) Same amount of proteins from wild type Cx45.6 (lanes 1 and 3) and Cx45.6(S364A) (lanes 2 and 4) transfected cells co-expressing ubiquitin (lanes 3 and 4) or without ubiquitin (lanes 1 and 2) were immunoblotted by anti-flag antibody and anti-β actin antibody. The protein band intensity was measured by densitometry and the intensity ratio of Cx45.6 versus β-actin was calculated (right panel). (n=3). (B) The transfected N2A cell lysates were immunoprecipitated by anti-Cx45.6 antibody. The immunoprecipitates from ubiquitin only (lane 1), wild type Cx45.6 (lanes 2 and 4) and Cx45.6(S364A) (lanes 3 and 5) transfected cells co-expressing ubiquitin (lanes 4 and 5) or without ubiquitin (lanes 1–3) were analyzed on 7.5% SDS-PAGE and immunoblotted with anti-myc and anti-flag antibodies, respectively. (n = 3).

Figure 3

Constitutively phosphorylated mutant, Cx45.6(S364D), was resistant to the cleavage by caspase-3 as compared to wild type Cx45.6. Exogenous wild type (WT) Cx45.6 (lanes 1 and 2) and Cx45.6 single phosphorylation site mutants, Cx45.6(S364D) (lanes 3 and 4) were expressed in CEF cells through retroviral infection. Crude membranes isolated from those infected cultures were treated in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of caspase-3 in vitro at 37°C for 6 h. Arrows indicate the cleaved fragments. The protein band intensity was measured by densitometry and the intensity ratio of truncated versus full-length Cx45.6 was calculated (lower panel). (n = 3).

Figure 4

A model for the regulation of proteolysis of Cx45.6 in the lens by phosphorylation at Ser³⁶⁴. (A) The phosphorylation at Ser³⁶⁴ renders Cx45.6 unstable and undergoes the degradation mediated by proteasome. (B) If Ser³⁶⁴ is not phosphorylated, Cx45.6 becomes a substrate for caspase-3, which cleaves behind Glu³⁶⁸. The truncated Cx45.6 mainly accumulates in the mature fibers at the center core region of the lens (Yin et al. 2001).