HVps15, but not Ca2+/CaM, is required for the activity and regulation of hVps34 in mammalian cells

Ying Yan, Rory J. Flinn, Haiyan Wu, Rachel S. Schnur, Jonathan M. Backer

Research output: Contribution to journalArticle

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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; coexpression 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 showthat, inmammalian cells, hVps34 activity is regulated through its interactions with hVps15, but is independent of Ca2+/CaM.

Original languageEnglish (US)
Pages (from-to)747-755
Number of pages9
JournalBiochemical Journal
Volume417
Issue number3
DOIs
StatePublished - Feb 1 2009

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Nutrients
Cells
Food
Ethane
1-Phosphatidylinositol 4-Kinase
Protein-Serine-Threonine Kinases
Egtazic Acid
Autophagy
Protein Transport
Calmodulin
Chelation
Phosphatidylinositols
Sorting
Edetic Acid
Ultraviolet radiation
Acetic Acid
Yeast
Transcriptional Activation
Esters
Phosphotransferases

Keywords

  • Autophagy
  • Beclin-1
  • Mammalian target of rapamycin (mTOR)
  • Mammalian vacuolar protein sorting 34 (Vps34)
  • UV radiation resistance-associated gene (UVRAG)
  • Vacuolar protein sorting 15 (Vps15)

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

HVps15, but not Ca2+/CaM, is required for the activity and regulation of hVps34 in mammalian cells. / Yan, Ying; Flinn, Rory J.; Wu, Haiyan; Schnur, Rachel S.; Backer, Jonathan M.

In: Biochemical Journal, Vol. 417, No. 3, 01.02.2009, p. 747-755.

Research output: Contribution to journalArticle

Yan, Ying ; Flinn, Rory J. ; Wu, Haiyan ; Schnur, Rachel S. ; Backer, Jonathan M. / HVps15, but not Ca2+/CaM, is required for the activity and regulation of hVps34 in mammalian cells. In: Biochemical Journal. 2009 ; Vol. 417, No. 3. pp. 747-755.
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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; coexpression 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 showthat, inmammalian cells, hVps34 activity is regulated through its interactions with hVps15, but is independent of Ca2+/CaM.",
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AU - Flinn, Rory J.

AU - Wu, Haiyan

AU - Schnur, Rachel S.

AU - Backer, Jonathan M.

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N2 - 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; coexpression 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 showthat, inmammalian cells, hVps34 activity is regulated through its interactions with hVps15, but is independent of Ca2+/CaM.

AB - 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; coexpression 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 showthat, inmammalian cells, hVps34 activity is regulated through its interactions with hVps15, but is independent of Ca2+/CaM.

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