期刊
BIOCHEMICAL JOURNAL
卷 431, 期 -, 页码 93-102出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20100314
关键词
apoptosis-linked gene 2-interacting protein X (Alix); haemopoietic cell kinase (Hck); protein-protein interaction; Src homology 3 domain (SH3 domain); Src family kinase (SFK)
资金
- Centre National de la Recherche Scientifique
- Institut National de la Sante et de la Recherche Medicate (Inserm)
- Agence Nationale de Recherche sur le SIDA et les hepatites virales (ANRS)
- Ministry of Science and Technology, China [2007CB914304, 2006AA02A313]
- National Natural Science Foundation of China (NSFC) [30800181, 30625011]
- Research Foundation Flanders (FWO)
- Katholieke Universiteit Leuven
- National Institutes of Health [CA016672]
SFKs (Src family kinases) are central regulators of many signalling pathways. Their functions are tightly regulated through SH (Src homology) domain-mediated protein protein interactions. A yeast two-hybrid screen using SH3 domains as bait identified Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] as a novel lick (haemopoietic cell kinase) SH3 domain interactor. The Alix-Hck-SH3 interaction was confirmed in vitro by a GST (glutathione transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cells. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 and determined a dissociation constant of 34.5 mu M. Heteronuclear NMR spectroscopy experiments were used to map the Hck-SH3 residues that interact with an ALIX construct containing the V and PRR domains or with the minimum identified interacting motif. Finally, SAXS (small-angle X-ray scattering) analysis showed that the N-terminal PRR of Alix is unfolded, at least before Hck-SH3 recognition. These results indicate that residues outside the canonical PxxP motif of Alix enhance its affinity and selectivity towards Hck-SH3. The structural framework of the Hck-Alix interaction will help to clarify how Hck and Alix assist during virus budding and cell-surface receptor regulation.
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