| Research Abstract |
Ran binding protein in microtubule organising centre (RanBPM) was originally isolated as a protein that binds to the small GTPase Ran. RanBPM also associates with the HGF receptor MET, p75 the neurotrophin receptor, and integrin LFA-1, and modifies the signaling of some of these molecules. Therefore, RanBPM may function as a scaffolding protein that coordinates the signaling input of cell surface receptors with intracellular signaling pathways. Previously, we identified RanBPM as a component of a 20S large protein complex. Here we purified the complex from soluble extract of HEK293 cells by an antibody against RanBPM, and identified Muskelin, ARMC8α, p48EMLP, ARMC8β, and p44CTLH as complex components by tandem mass spectrometry. ARMC8α and ARMC8βare novel armadillo-repeat proteins. Since the N-terminal 364 amino acids of ARMC8a and ARMC8βwere completely conserved, these proteins are probably alternatively spliced products from the same gene. Interestingly, RanBPM, Muskelin, p48EMLP, and p44CTLH possess LisH/ CTLH motifs, which are present in proteins involved in microtubule dynamics, cell migration, nucleokinesis, and chromosome segregation. Immunoblot analysis showed dominant expression of ARMC8α, ARMC8β, p48EMLP, and p44CTLH mRNAs in skeletal muscle and testis. We next confirmed the in vivo association of each complex component by co-immunoprecipitation assays using the Cos-7 cells in which these components were exogenously overexpressed. Pull-down assay using bacterially-expressed Twa1 revealed that each In vitro-translated component could bind to the Twa1. Finally, we confirmed the cellular co-localization of these proteins. Taken together, we revealed that RanBPM, Muskelin, p48EMLP, Twa1, p44CTLH, and ARMC8 form complexes in cells.
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