Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.


Autoria(s): Dejournett, Robert E; Kobayashi, Ryuji; Pan, Shujuan; Wu, Chuanfen; Etkin, Laurence D; Clark, Richard B; Bögler, Oliver; Kuang, Jian
Data(s)

15/01/2007

Resumo

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

Identificador

http://digitalcommons.library.tmc.edu/uthmed_docs/342

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1820820/?tool=pmcentrez

Publicador

DigitalCommons@The Texas Medical Center

Fonte

UT Medical School Journal Articles

Palavras-Chave #Amino Acid Sequence #Animals #Calcium-Binding Proteins #Carrier Proteins #Cell Cycle Proteins #Cell Division #Electrophoretic Mobility Shift Assay #Endopeptidases #Endosomal Sorting Complexes Required for Transport #Hela Cells #Humans #Neoplasm Proteins #Nerve Tissue Proteins #Oocytes #Phosphoproteins #Phosphorylation #Rats #Threonine #Ubiquitin Thiolesterase #Xenopus Proteins #src Homology Domains #Medicine and Health Sciences
Tipo

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