Clusterin interacts with SCLIP (SCG10-like protein), a microtubule destabilizing protein

초록

Clusterin has been known that it may play critical roles in tissue remodeling, differentiation and apoptosis. In the brain, clusterin is up-regulated in reactive astrocytes, following experimental brain injury and in the state of neurodegenerative diseases or neuroinflamation processes. Therefore, it has been known as an important molecule in these processes, although regulation and molecular mechanism of clusterin have not been identified. Clusterin has two coiled-coil motifs, providing chance to bind other molecules. We isolated several clusterin binding proteins (CLUB1-CLUB4) using yeast two hybrid screening in rat brain cDNA library. DNA sequence analysis revealed CLUB1 to be SCG10-like protein (SCLIP), known as a neuronal growth associated protein and microtubule destabilizing phosphoprotein. Systematic yeast two hybrid assay using alpha/beta-galactosidase assay showed that clusterin binds with SCLIP in yeast. For the purpose of further confirmation of the interaction between two molecules, we performed GST-pull down assay in vitro and co-immunoprecipitation in vivo. In vitro GST pull down assay showed that GST-clusterin fusion protein binds with His-SCLIP fusion protein expressed in PC12 cells. Co-immunoprecipitation studies in PC12 cells expressing SCLIP revealed that His-SCLIP fusion protein co-precipitated with clusterin in vivo. At the same time, both clusterin and SCLIP were co-localized in cytoplasm as shown by confocal microscopic analysis. SCLIP has two coiled-coil motifs in its stathmin (STMN) domain commonly shown in STMN family proteins. These findings showed that STMN family proteins including SCLIP may be as a binding partner of coiled-coil domain of clusterin and provide first evidence for regulatory effects of clusterin on microtubule stabilizing/destabilizing processes including cell proliferation, differentiation, remodeling and apoptosis.