Amino Acid Residue Val362 Plays a Critical Role in Maintaining the Structure of C Terminus of Connexin 50 and in Lens Epithelial-fiber Differentiation

Abstract

We have previously shown that connexin (Cx) 50, unlike the other two lens connexins, Cx43 and Cx46, promotes chicken lens epithelial-fiber differentiation in a channel-independent manner. Here, we show that deletion of the PEST motif at the C terminus (CT) domain of Cx50 attenuates the stimulatory effect of Cx50 on lens fiber differentiation. Valine 362, a residue located within the PEST domain, is functionally involved. The structure of the Cx50 CT predicted by molecular modeling revealed four ␣-helices and Val 362 was found to be located in the middle of the 3rd helix. Replacement of Val 362 with amino acid residues that disrupt the ␣-helical structure predicted by molecular modeling, such as arginine, glutamate, or phenylalanine, attenuated the stimulatory effects of Cx50 on lens differentiation, whereas replacement with threonine, isoleucine, leucine, or proline, which maintain the structure preserved the function of Cx50. Circular dichroism (CD) studies supported the structural predictions and showed that the substitution with Glu, but not Thr or Pro, disrupted the ␣-helix, which appears to be the structural feature important for lens epithelial-fiber differentiation. Together, our results suggest that Val 362 is important for maintaining the helical structure and is crucial for the role of Cx50 in promoting lens epithelial-fiber differentiation.

Connexin Controls Cell-Cycle Exit and Cell Differentiation by Directly Promoting Cytosolic Localization and Degradation of E3 Ligase Skp2

“…Co-expressing Cx50 and either Skp2 shRNA further enhanced the primary lens cell differentiation. We have previously shown that unlike WT Cx50, the Cx50(V362E) mutant has a minimal effect on lens differentiation (Shi et al, 2010). Expression of the Cx50(V362E) mutant, unlike WT Cx50, failed to augment lens differentiation induced by either Skp2 shRNA (Figure 2B).…”

“…The hypothetical structure of Cx50-CT contains four hypothetical helical loops (Shi et al, 2010). The fragment, Skp2 (aa 70–120), was predicted to be located in a helical loop (PDB: 1FQV).…”

“…Previous studies show that analogous to full-length Cx43, the CT domain of Cx43 by itself that does not form functional gap junction can inhibit tumor cell growth (Zhang et al, 2003a). We have previously shown that the role of Cx50 in promoting lens cell differentiation is independent of its function in forming gap junctions or hemichannels (Banks et al, 2007, 2009; Gu et al, 2003), and the CT domain and Val-362 residue are primarily responsible for this function (Banks et al, 2007; Shi et al, 2010). Unveiling the sequential similarity between Cx50 and cyclin A inspired us to examine the relationship between Cx50 and the cyclin A-binding partner, Skp2.…”

“…Although Cx50 and cyclin A appear to bind to a similar region on Skp2; however, this is unlikely a competitive binding since unlike Cx50, the interaction with cyclin A primarily occurs in the nucleus. We have previously shown that the V362E mutation compromises the ability of Cx50 in promoting lens cell differentiation (Shi et al, 2010). Here, we show that Val-362 is located in the interaction interface of Cx50 and Skp2.…”

“…The intracellular C terminus (CT) of Cx50 is sufficient to promote lens fiber cell differentiation. Moreover, the Val-362 (V362) residue within the CT domain, by maintaining an α-helical structure, functionally participates in lens epithelial-fiber differentiation (Shi et al, 2010). …”