Characterization of nucleomorphin, a novel nuclear breast cancer carboxy-terminal-domain containing calmodulin-binding protein from Dictyostelium discoideum.
Published: 2005
Language: eng
Number of pages: 186
Description: Probing of Dictyostelium discoideum cell extracts using  35S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca2+-dependent calmodulin binding proteins (CaMBPs). Here, we report the molecular and cellular characterization of a gene encoding a novel CaMBP we have called nucleomorphin. Initially, nucleomorphin (NumA) was isolated as a cDNA (1119bp) encoding a 38.7kDa protein. Subsequently two other isoforms, NumB and NumC were found to be expressed differentially from NumA. The CaM-binding of NumA was subsequently verified and, using deletion constructs, a loose IQ motif that mediates binding (182LQKQQKIYKDLERF 195) in both the presence and absence of calcium ions was identified. GFP-NumA localizes as bright patches of fluorescence in the nucleoplasm, adjacent to the nuclear envelope. NumA and B contain an extensive acidic domain rich in aspartic and glutamic acid residues (DEED) that when deleted results in the formation of highly multinucleate cells however the larger NumB isoform contains an amino-terminal breast cancer carboxy-terminal-domain (BRCT) common to cell cycle checkpoint proteins. Using the green fluorescent protein (GFP) fused to NumA we have identified a nuclear localization sequence (NLS;  48KKSYQDPEIIAHSRPRK64) that contains both a bipartite and classical NLS. Using the yeast two-hybrid system we show that nucleomorphin interacts with the calcium binding protein 4a in a Ca2+-dependent manner, and the D. discoideum puromycin-sensitive aminopeptidase requiring the carboxy-terminal residues 161--340. The expression of NumA is repressed during starvation of shaking cultures or in the presence of either exogenous ammonia (NH4+/NH3) or cAMP. However, this repression can be inhibited by the Dictyostelium developmental morphogen differentiation-inducible factor (DIF-1). Our work suggests that NumA is involved in some fundamental cell cycle-dependent event in the nucleus of Dictyostelium discoideum.
Notes: Thesis (Ph.D.)--University of Toronto, 2005.

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Source: Dissertation Abstracts International, Volume: 66-06, Section: B, page: 3002.