Cataract EPHA2 SAM Domain Mutations Alter Receptor Stability and Function

Abstract

Purpose: : Mutations in EPHA2 receptor tyrosine kinase have been shown to cause cataracts in human populations. However, the molecular mechanisms underlying cataractogenesis by these mutations are currently unknown. Here we examine the functional deficits of four EPHA2 cataract mutants with mutations specifically in the Sterile Alpha Motif (SAM) domain.

Methods: : Human EPHA2 cataract mutants were constructed and expressed in HEK293T human embryonic kidney and αTN4-1 mouse lens epithelial cell lines. The EPHA2 protein levels and their ability to activate downstream signals were examined with Western blot analysis. Alterations in their biological functions were analyzed using the wound-healing assay.

Results: : The SAM domain cataract mutations reduce EPHA2 protein stability, as levels of the mutant proteins are significantly lower in comparison to wild-type EPHA2. This protein instability is due to increased ubiquitin-mediated proteasomal degradation, since the proteasome inhibitor MG132 markedly enhanced mutant EPHA2 protein levels. The SAM domain mutants also have decreased ability to activate AKT (Ser473) and ERK1/2 signaling pathways and fail to promote cell migration.

Conclusions: : This study shows that the human EPHA2 SAM domain mutations cause protein degradation along with AKT and ERK down regulation. As a consequence, this may lead to defects in lens fiber cell migration and result in cataract development.

Keywords: cataract • proteolysis • wound healing