The prostate is a relatively homogeneous tissue that is highly specialized in synthetic and secretory functions. The frequency of malignant growth explains its great clinical significance. We used here a combination of subcellular fractionation, 1-DE (one-dimensional gel electrophoresis) protein separation and mass spectrometry, to establish a prostate protein expression profile in mice. Analysis of proteins present in cytosolic (C) and membrane (P) prostate fractions led to the identification of 619 distinct proteins. A majority of abundant proteins were found to compose the metabolism and protein synthesis machinery. Those identified also correspond to known endoplasmic reticulum and Golgi residents, chaperones and anterograde cargos. They included a series of proteins involved in exocytic/endocytic trafficking. Among the signaling proteins, we identified the ubiquitin-like peptides smt3. We showed that both free small ubiquitin-related modifier SUMO-2/3 and SUMO-1 levels are subject to tight control by the androgen 5α-dihydrotestosterone (DHT). By contrast with SUMO-2/3, free SUMO-1 peptides are particularly abundant in the prostate when compared with other tissues. Therefore, we report prostate protein expression profiles of cytosolic and membrane fractions in mice. Our data suggest that the identified free SUMO peptides play an important role in this secretory tissue.
Mouse prostate membrane-associated proteins of the annexin family showed changes in SUMOylation during androgen treatment. Among these the calcium-binding annexin A1 protein (ANXA1) was chosen for further characterization given its role in protein secretion and cancer. SUMOylation of ANXA1 was confirmed by overexpressing SUMO-1 in LNCaP cells. Site-directed mutagenesis indicated that K257 located in a SUMOylation consensus motif in the C-terminal calcium-binding DA3 repeat domain is SUMOylated. Mutation of the N-terminal Y21 decreased markedly the SUMOylation signal while EGF stimulation increased ANXA1 SUMOylation. A structural analysis of ANXA1 revealed that K257 is located in a hot spot where Ca2 + and SUMO-1 bind and where a nuclear export signal and a polyubiquitination site are also present. Also, Y21 is buried inside an α-helix structure in the Ca2 +-free conformation implying that Ca2 + binding, and the subsequent expelling of the N-terminal α-helix in a disordered conformation, is permissive for its phosphorylation. These results show for the first time that SUMOylation can be regulated by an external signal (EGF) and indicate the presence of a cross-talk between the N-terminal and C-terminal domains of ANXA1 through post-translational modifications.
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