Characterization of the Novel Protein SH3BP4 Death Domain during Oxidant-induced Apoptosis in Human Pigment Epithelial (ARPE-19) Cells

Abstract

Abstract: : Purpose: SH3BP4 is a 107 kD protein with a C-terminus death domain (DD) whose protein expression has been shown to be elevated early upon induction of apoptotic stress both in vitro in RPE cultures and in vivo in the rat neural retina. The purpose of this study was to determine the role of the death domain in SH3BP4 in oxidant-induced apoptosis. Methods: SH3BP4 fusion proteins were constructed with a c-myc epitope tag in the pCMV-Tag 3 vector (Stratagene). Tag 1: SH3BP4 DD-814 bp encodes the tyrosine phosphorylation site and the putative death domain. Tag 2: SH3BP4 DD-1520 bp encodes the bipartite nuclear target site, a tyrosine phosphorylation site and death domain. Tag 3: SH3BP4-4297 bp encodes for the open reading frame starting at amino acid 6. Cos-7 and ARPE-19 were transfected with the 3 Tag vectors using TransIT LT-1 (Mirus Corp.). Cells were treated with the pro-oxidant tert-butylhydroperoxide (tBH) for various lengths of time, lysed in 2% SDS lysis buffer and SH3BP4 levels were measured by Western blot analysis using c-myc and SH3BP4 specific antibodies. Coverslips were fixed in 100% methanol and Hoechst 33258 nuclear stain was used to measure nuclear condensation as an indicator of apoptosis. Results: Data from transfected cells indicated that fusion protein expression was maximal at 48 hours for Cos-7 and 24 hours for ARPE-19. Immunofluorescence staining of the c-myc fusion proteins was positive for all 3 Tags. Tag 1 showed punctate cytoplasmic staining although no protein was detectable on Western blots. Tag 2 staining was visible in the cytoplasm with a small amount in the nucleus. Tag 3 staining showed punctate cytoplasmic staining and diffuse nuclear staining with exclusion from nucleoli. Preliminary results indicate a change in general cell morphology with Tag 2 and Tag 3 overexpression compared to the transfection reagent (LT-1) only control in ARPE-19. It appears that overexpression of SH3BP4 primes the RPE cells for apoptosis and this effect appears enhanced when the truncated DD-containing fusion protein is expressed. Conclusions: Preliminary data suggests that SH3BP4 may play an active role in apoptosis. ARPE-19 cells transfected with SH3BP4 fusion proteins containing the C-terminus death domain may prime the cells to progress through apoptosis at a faster rate.

Keywords: apoptosis/cell death • retinal pigment epithelium • molecular biology