1. hVps15, but not Ca2+/CaM, is required for the activity and regulation of hVps34 in mammalian cells
- Author
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Ying Yan, Haiyan Wu, Jonathan M. Backer, Rory J. Flinn, and Rachel S. Schnur
- Subjects
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 - Abstract
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.
- Published
- 2008