Cataract-linked serine mutations in the gap junction protein connexin50 expose a sorting signal that promotes its lysosomal degradation.

Many human connexin50 (Cx50) mutants have been linked to cataracts including two carboxyl terminus serine mutants that are known phosphorylation sites in the lens (Cx50S258F and Cx50S259Y). To examine the behavior of these mutants and the role of phosphorylation at these positions, we stably transfected HeLa cells with cataract-linked and phosphorylation-mimicking (Cx50S258D and Cx50S259D) Cx50 mutants. We observed that gap junctional plaques were rarely detected in Cx50S258F-expressing and Cx50S259Y-expressing cells compared with wild-type cells. In contrast, gap junction abundance and size were greatly increased for Cx50S258D and Cx50S259D mutants. Cx50S258F and Cx50S259Y supported very low levels of gap junctional coupling, whereas Cx50S258D and Cx50S259D supported extensive intercellular communication. Furthermore, Cx50 levels as detected by immunoblotting were lower in Cx50S258F and Cx50S259Y mutants than in the wild-type or the aspartate substitution mutants, and chloroquine or ammonium chloride treatment significantly increased Cx50S258F and Cx50S259Y protein levels, implying participation of the lysosome in their increased degradation. Alanine substitution of amino acids within a predicted tyrosine-based sorting signal in Cx50S258F and Cx50S259Y increased levels of gap junctional plaques and intercellular transfer of neurobiotin. These results suggest that the absence of phosphorylatable serines at these positions exposes a sorting signal leading to lysosomal degradation of Cx50, whereas phosphorylation at these sites conceals this signal and allows targeting of Cx50 to the plasma membrane and stabilization of gap junction plaques. We propose that in the lens, degradation of Cx50S258F and Cx50S259Y decreases Cx50 levels at the plasma membrane and consequently Cx50 function, leading to cataracts.

p62/Sequestosome 1 levels increase and phosphorylation is altered in Cx50D47A lenses, but deletion of p62/sequestosome 1 does not improve transparency.

Purpose: p62/Sequestosome 1 (p62) is a stress-induced protein that is involved in several different intracellular pathways, including regulation of aspects of protein degradation. p62 levels are elevated in several types of cataracts. We investigated whether levels of p62 and its phosphorylation were altered in the lenses of Cx50D47A mice, which express a mutant of connexin50 (Cx50) that leads to cataracts and impaired lens differentiation. To evaluate the importance of p62 in the lens defects caused by a connexin50 mutant, we also examined the effect of deleting p62 in homozygous Cx50D47A mice. Methods: Protein levels were determined with immunoblotting. Mouse lenses were examined with dark-field illumination microscopy. Intensities of the opacities and lens equatorial diameters were quantified using ImageJ. Nuclei and nuclear remnants were detected with fluorescence microscopy of lens sections stained with 4',6-diamino-2-phenylindole dihydrochloride (DAPI). Results: Levels of total p62 were increased in the lenses of homozygous Cx50D47A mice compared to those of the wild-type animals. The ratio of p62 phosphorylated at threonine-269/serine-272 (T269/S272) to total p62 was significantly decreased, whereas the ratio of p62 phosphorylated at serine-349 (S349) to total p62 was significantly increased in lenses of homozygous Cx50D47A mice. However, deletion of p62 did not affect the sizes of the lenses or the severity of their cataracts in homozygous Cx50D47A mice. Deletion of p62 did not improve connexin50 or connexin46 levels. Moreover, deletion of p62 did not change the levels of crystallins, histone H3, the mitochondrial import receptor subunit TOM20 homolog, or the abundance of nuclei and nuclear fragments in the lenses of homozygous Cx50D47A mice. Homozygous deletion of p62 led to an 84% increase in the levels of ubiquilin 2, but did not significantly affect the levels of ubiquilin 1 or ubiquilin 4. Conclusions: Although homozygous Cx50D47A lenses have increased levels of p62, a specific reduction in p62 phosphorylation at T269/S272, and a specific increase in p62 phosphorylation at S349, this protein is not a critical determinant of the severity of the abnormalities of these lenses (reduced growth or differentiation and cataracts). The lens may utilize redundant or compensatory systems (such as changes in levels of ubiquilin 2) to compensate for the lack of p62 in homozygous Cx50D47A lenses.