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

Robert E. DeJournett, Ryuji Kobayashi, Shujuan Pan, Chuanfen Wu, Laurence D. Etkin, Richard B. Clark, Oliver Bögler, Jian Kuang

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 progesterone-induced 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 (α-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 immuno-precipitates from extracts of G 2-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.

Original languageEnglish (US)
Pages (from-to)521-531
Number of pages11
JournalBiochemical Journal
Volume401
Issue number2
DOIs
StatePublished - Jan 15 2007

Fingerprint

Phosphorylation
Proline
src Homology Domains
Cell Division
Oocytes
Proteins
Xenopus
Assays
Gels
Cells
Molecules
Reentry
Electrophoretic Mobility Shift Assay
Glutathione Transferase
Astrocytes
Ionization
Progesterone
Precipitates
Desorption
Cell Cycle

Keywords

  • Apoptosis-linked-gene-2 product (ALG-2)-interacting protein X (Alix)
  • Associated molecule with the Src homology 3 domain of signal transducing adaptor molecule (AMSH)
  • M-phase phosphorylation
  • Src homology 3 domain-containing

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins. / DeJournett, Robert E.; Kobayashi, Ryuji; Pan, Shujuan; Wu, Chuanfen; Etkin, Laurence D.; Clark, Richard B.; Bögler, Oliver; Kuang, Jian.

In: Biochemical Journal, Vol. 401, No. 2, 15.01.2007, p. 521-531.

Research output: Contribution to journalArticle

DeJournett, RE, Kobayashi, R, Pan, S, Wu, C, Etkin, LD, Clark, RB, Bögler, O & Kuang, J 2007, 'Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins', Biochemical Journal, vol. 401, no. 2, pp. 521-531. https://doi.org/10.1042/BJ20061287
DeJournett, Robert E. ; Kobayashi, Ryuji ; Pan, Shujuan ; Wu, Chuanfen ; Etkin, Laurence D. ; Clark, Richard B. ; Bögler, Oliver ; Kuang, Jian. / Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins. In: Biochemical Journal. 2007 ; Vol. 401, No. 2. pp. 521-531.
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