title: Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.
abstract: 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.
authors.affiliation: Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 019, Houston, TX 77030, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Dejournett
authors.forename: Ryuji
authors.initials: R
authors.lastname: Kobayashi
authors.forename: Shujuan
authors.initials: S
authors.lastname: Pan
authors.forename: Chuanfen
authors.initials: C
authors.lastname: Wu
authors.forename: Laurence D
authors.initials: LD
authors.lastname: Etkin
authors.forename: Richard B
authors.initials: RB
authors.lastname: Clark
authors.forename: Oliver
authors.initials: O
authors.lastname: Bögler
authors.forename: Jian
authors.initials: J
authors.lastname: Kuang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D010450:Endopeptidases / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D006367:HeLa Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009865:Oocytes / Q000254:growth & development; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D013912:Threonine / Q000378:metabolism; D043222:Ubiquitin Thiolesterase; D029867:Xenopus Proteins / Q000378:metabolism*; D018909:src Homology Domains / Q000502:physiology
chemical_list: D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C117750:PDCD6IP protein, Xenopus; C117020:PDCD6IP protein, human; D010750:Phosphoproteins; C486696:STAMBP protein, human; C411889:Sh3kbp1 protein, rat; D029867:Xenopus Proteins; D013912:Threonine; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase