The Subcellular Localization of SH3BP4 in Human Retinal Pigment Epithelial and COS-7 Cell Lines

Abstract

Abstract: : Purpose: SH3BP4 is a novel gene, encoding a putative death domain, whose protein expression has been shown to be elevated early upon induction of apoptosis stress both in vitro in RPE cell cultures and in vivo in the rat neural retina. The propose of this study is to examine the subcellular localization of SH3BP4 and determine if the localization is altered during increased SH3BP4 expression as well as during apoptosis. Methods: GFP- SH3BP4 in pEGFP-C2 or ss- SH3BP4-GFP in pEGFP-N2 vectors were transfected into ARPE-19 or COS-7 cells using lipofectamine 2000 (Gibco) or TransIT LT1 (Mirus Corp.). Apoptosis was induced by treating cells with the pro-oxidant t-Butyl Hydroperoxide (tBH). Subcellular fractionation was performed using differential centrifugation. SDS-PAGE followed by Western blotting was used to determine SH3BP4 localization in each subcellular fraction. Likewise, subcellular markers were used as positive controls. Results: Specificity of subcellular markers localization indicated a high purity of each fraction. Preliminary results showed that in ARPE-19 cells, SH3BP4 was found mainly in the membrane fraction. Treatment of ARPE-19 with tBH causes an early increase in SH3BP4 concentrations well before apoptosis is evident by cellular morphology. Subcellular fractionation indicates there is an increase in membrane associated SH3BP4 after 4-8 hrs of tBH treatment; this increase is particularly evident when compared to the membrane control marker TGN46. In COS-7 cells, endogenous SH3BP4 was also found mainly in the membrane fraction with a smaller portion in the nucleus. However, both transfected SH3BP4-GFP constructs were localized mainly to the nucleus, while some also localized to the membrane and cytoplasmic fractions. These results may indicate an alteration from steady state localization when SH3BP4 is over-expressed. This is supported by the finding that in COS-7 cells transfected with ss- SH3BP4-GFP, the majority of endogenous SH3BP4 was re-routed from a membrane to a nuclear localization. Conclusions:These results show that SH3BP4 is most likely a membrane protein with the ability to also translocate into the nucleus and that overexpression of SH3BP4 may alter this steady state distribution. In ARPE-19 treated with tBH, the subcellular fractionation studies indicate an accumulation of SH3BP4 primarily in the membrane fraction.

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