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

Authors

Robert E Dejournett, Program in Genes and Development, University of Texas Graduate School of Biomedical Sciences, Houston, TX 77030, U.S.A.; University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 019, Houston, TX 77030, U.S.A.
Ryuji Kobayashi, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 019, Houston, TX 77030, U.S.A.
Shujuan Pan, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 019, Houston, TX 77030, U.S.A.
Chuanfen Wu, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 019, Houston, TX 77030, U.S.A.
Laurence D Etkin, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 019, Houston, TX 77030, U.S.A.
Richard B Clark, Department of Integrative Biology and Pharmacology, The University of Texas Medical School, Houston, TX 77225, U.S.A.
Oliver Bögler, Program in Genes and Development, University of Texas Graduate School of Biomedical Sciences, Houston, TX 77030, U.S.A.
Jian Kuang, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 019, Houston, TX 77030, U.S.A.Follow

Publication Date

1-15-2007

Journal

Biochem Journal

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.

Keywords

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

Comments

PMC1820820