Abstract
Purpose::
The ubiquitin proteolytic pathway (UPP) is involved in the regulation of a wide variety of cellular processes, such as cell cycle, responses to stress, and some receptor-mediated signal transduction. In the UPP ubiquitin is activated by the ubiquitin activating enzyme, E1. It is then passed to one of many ubiquitin conjugating enzymes (E2s). Then ubiquitin is transferred to either the substrate or an E3. There are hundreds of E3s. Earlier we showed that levels of UbcH7 vary during cell cycle. Roles have also been indicated for UbcH7 in oncogenic transformation, embryonic morphogenesis and neurodegeneration through its interaction with different E3s. The purpose of this study was to investigate the expression and the function of UbcH7 and related E3s in ocular tissues.
Methods::
Human lens epithelial cells (HLEC), ARPE19, and HeLa cells were cultured in complete DMEM medium supplemented with 10% FBS. Immunofluorescence microscopy was performed using an Axiovert200 microscope (Zeiss, Oberkochen, Germany). For western blotting, the membranes were immunoblotted with anti-UbcH7 and antibodies to E3s that are known to interact with UbcH7, specifically, Cbl and E6AP. Wild type (wt) and mutant (mut) GST-UbcH7 constructs were over-expressed in bacteria and the fusion proteins were purified using Glutathione Sepharose 4B. A thiolester assay was performed to assure that the wt GST-UbcH7 was active and the mut GST-UbcH7 was not. Microinjection into HeLa cells was performed in buffer containing 0.1 M KCl, 1.7 mM NaCl, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4.
Results::
Immunoblotting detected UbcH7 in HLEC, ARPE19, and HeLa cells. Immunofluorescent staining showed predominantly cytoplasmic localization of UbcH7 throughout the cell cycle. A small amount of UbcH7 was also localized in the nucleus. The subcellular localization of Cbl and E6AP was similar to that of UbcH7. Microinjection of GST-UbcH7 fusion protein into HeLa cells showed a defect in cell cycle progression in mitosis.
Conclusions::
UbcH7 and its associated E3s are present in HLEC and ARPE19 cells. The involvement of UbcH7 in timed destruction and turnover of regulators responsible for cellular proliferation and normal growth is indicated by the block of cell cycle that is induced when UbcH7 is introduced. The data further corroborate roles for the UPP in regulation of lens and retina epithelial cell cycle.
Keywords: proteolysis • proliferation • microscopy: light/fluorescence/immunohistochemistry