Domain mapping on the human metastasis regulator protein h-Prune reveals a C-terminal dimerization domain.
The human orthologue of the Drosophila prune protein (h-Prune) is an interaction partner and regulator of the metastasis suppressor protein NM23-H1 (non-metastatic protein 23). Studies on a cellular breast-cancer model showed that inhibition of the cAMP-specific PDE (phosphodiesterase) activity of h-Prune lowered the incidence of metastasis formation, suggesting that inhibition of h-Prune could be a therapeutic approach towards metastatic tumours. H-Prune shows no sequence similarity with known mammalian PDEs, but instead appears to belong to the DHH (Asp-His-His) superfamily of phosphoesterases. In order to investigate the structure and molecular function of h-Prune, we expressed recombinant h-Prune in a bacterial system. Through sequence analysis and limited proteolysis, we identified domain boundaries and a potential coiled-coil region in a C-terminal cortexillin homology domain. We found that this C-terminal domain mediated h-Prune homodimerization, as well as its interaction with NM23-H1. The PDE catalytic domain of h-Prune was mapped to the N-terminus and shown to be active, even when present in a monomeric form. Our findings indicate that h-Prune is composed of two independent active sites and two interaction sites for the assembly of oligomeric signalling complexes.
Middelhaufe
Sabine
S
Department of Physiological Chemistry, Ruhr-University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany.
Garzia
Livia
L
Ohndorf
Uta-Maria
UM
Kachholz
Barbara
B
Zollo
Massimo
M
Steegborn
Clemens
C
D000595:Amino Acid Sequence; D001943:Breast Neoplasms; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D054778:NM23 Nucleoside Diphosphate Kinases / Q000737:chemistry* / Q000378:metabolism; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping; D010727:Phosphoric Diester Hydrolases; D010744:Phosphoric Monoester Hydrolases; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D002352:Carrier Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; D009363:Neoplasm Proteins; C517938:NME1 protein, human; C438732:PRUNE1 protein, human; D010744:Phosphoric Monoester Hydrolases; D010727:Phosphoric Diester Hydrolases