hVps15, but not Ca2+/CaM, is required for the activity and regulation of hVps34 in mammalian cells
| Autor: | Ying Yan, Haiyan Wu, Jonathan M. Backer, Rory J. Flinn, Rachel S. Schnur |
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| Rok vydání: | 2008 |
| Předmět: |
mammalian vacuolar protein sorting 34 (Vps34)
Biochemistry chemistry.chemical_compound Phosphatidylinositol 3-Kinases MEM minimal essential medium Cricetinae S6K1 S6 kinase 1 Threonine Vacuolar protein sorting 0303 health sciences CaM calmodulin HA haemagglutinin biology 030302 biochemistry & molecular biology mammalian target of rapamycin (mTOR) TOR target of rapamycin UV radiation resistance-associated gene (UVRAG) Cell biology UVRAG UV radiation resistance-associated gene beclin-1 FBS foetal bovine serum D-PBS Dulbecco's PBS PI3K phosphoinositide 3-kinase Signal Transduction Research Article autophagy Calmodulin Bif-1 Bax-interacting factor 1 hVps15 mammalian vacuolar protein sorting 15 homologue Vps vacuolar protein sorting UVRAG CHO Cells mTOR mammalian target of rapamycin Protein Serine-Threonine Kinases hVps34 mammalian vacuolar protein sorting 34 homologue Vacuolar Sorting Protein VPS15 03 medical and health sciences HEK human embryonic kidney Cricetulus BAPTA Animals Humans MLCK myosin light-chain kinase Protein kinase A Molecular Biology 030304 developmental biology Binding Sites Endosomal Sorting Complexes Required for Transport vacuolar protein sorting 15 (Vps15) Tumor Suppressor Proteins Membrane Proteins Cell Biology YFP yellow fluorescent protein EGTA BAPTA 1 2-bis-(o-aminophenoxy)ethane-N N N′ N′-tetra-acetic acid BAPTA/AM BAPTA(acetoxymethyl ester) chemistry biology.protein Apoptosis Regulatory Proteins |
| Zdroj: | Biochemical Journal |
| ISSN: | 1470-8728 |
| Popis: | The mammalian Class III PI3K (phosphoinositide 3-kinase), hVps34 [mammalian Vps (vacuolar protein sorting) 34 homologue], is an important regulator of vesicular trafficking, autophagy and nutrient sensing. In yeast, Vps34 is associated with a putative serine/threonine protein kinase, Vps15, which is required for Vps34p activity. The mammalian homologue of Vps15p, hVps15 (formerly called p150), also binds to hVps34, but its role in hVps34 signalling has not been evaluated. In the present study we have therefore compared the activity and regulation of hVps34 expressed without or with hVps15. We find that hVps34 has low specific activity when expressed alone; co-expression with hVps15 leads to a marked increase in activity. Notably, beclin-1/UVRAG (UV radiation resistance-associated gene) activation of hVps34 requires co-expression with hVps15; this may be explained by the observation that beclin-1/UVRAG expression increases hVps34/hVps15 binding. Regulation of hVps34 activity by nutrients also requires co-expression with hVps15. Finally, given a recent report that hVps34 activity requires Ca2+/CaM (calmodulin), we considered whether hVps15 might be involved in this regulation. Although hVps34 does bind CaM, we find its activity is not affected by treatment of cells with BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)] or W7. Removal of CaM by EDTA or EGTA washes has no effect on hVps34 activity, and hVps34 activity in vitro is unaffected by Ca2+ chelation. The results of the present study show that, in mammalian cells, hVps34 activity is regulated through its interactions with hVps15, but is independent of Ca2+/CaM. |
| Databáze: | OpenAIRE |
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