PMID- 21738781
OWN - NLM
STAT- MEDLINE
DCOM- 20111201
LR  - 20211203
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 6
IP  - 6
DP  - 2011
TI  - Phosphoproteomic profiling of in vivo signaling in liver by the mammalian target 
      of rapamycin complex 1 (mTORC1).
PG  - e21729
LID - 10.1371/journal.pone.0021729 [doi]
LID - e21729
AB  - BACKGROUND: Our understanding of signal transduction networks in the 
      physiological context of an organism remains limited, partly due to the technical 
      challenge of identifying serine/threonine phosphorylated peptides from complex 
      tissue samples. In the present study, we focused on signaling through the 
      mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is at the center 
      of a nutrient- and growth factor-responsive cell signaling network. Though 
      studied extensively, the mechanisms involved in many mTORC1 biological functions 
      remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We developed a 
      phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat 
      liver homogenates. Using the anticancer drug rapamycin, the only known target of 
      which is mTORC1, we characterized signaling in liver from rats in which the 
      complex was maximally activated by refeeding following 48 hr of starvation. Using 
      protein and peptide fractionation methods, TiO(2) affinity purification of 
      phosphopeptides and mass spectrometry, we reproducibly identified and quantified 
      over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive 
      phosphorylation events, we identified 62 new rapamycin-responsive candidate 
      phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We 
      observed similar proportions of increased and reduced phosphorylation in response 
      to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway 
      components among rapamycin-sensitive phosphopeptide candidates. 
      CONCLUSIONS/SIGNIFICANCE: In addition to identifying potential new 
      mTORC1-mediated phosphorylation events, and providing information relevant to the 
      biology of this signaling network, our experimental and analytical approaches 
      indicate the feasibility of large-scale phosphoproteomic profiling of tissue 
      samples to study physiological signaling events in vivo.
FAU - Demirkan, Gokhan
AU  - Demirkan G
AD  - Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, 
      Rhode Island, United States of America.
FAU - Yu, Kebing
AU  - Yu K
FAU - Boylan, Joan M
AU  - Boylan JM
FAU - Salomon, Arthur R
AU  - Salomon AR
FAU - Gruppuso, Philip A
AU  - Gruppuso PA
LA  - eng
GR  - R21AI083908/AI/NIAID NIH HHS/United States
GR  - R01 HD024455/HD/NICHD NIH HHS/United States
GR  - P20 RR015578/RR/NCRR NIH HHS/United States
GR  - 1R01AI083636/AI/NIAID NIH HHS/United States
GR  - R21 AI083908/AI/NIAID NIH HHS/United States
GR  - R01 AI083636/AI/NIAID NIH HHS/United States
GR  - R01HD024455/HD/NICHD NIH HHS/United States
GR  - 2P20RR015578/RR/NCRR NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20110628
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Phosphoproteins)
RN  - EC 2.7.11.1 (TOR Serine-Threonine Kinases)
SB  - IM
MH  - Animals
MH  - Liver/*metabolism
MH  - Male
MH  - Mass Spectrometry
MH  - Phosphoproteins/*metabolism
MH  - Proteomics/*methods
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Signal Transduction
MH  - TOR Serine-Threonine Kinases/*metabolism
PMC - PMC3125343
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2011/07/09 06:00
MHDA- 2011/12/13 00:00
PMCR- 2011/06/28
CRDT- 2011/07/09 06:00
PHST- 2011/04/21 00:00 [received]
PHST- 2011/06/07 00:00 [accepted]
PHST- 2011/07/09 06:00 [entrez]
PHST- 2011/07/09 06:00 [pubmed]
PHST- 2011/12/13 00:00 [medline]
PHST- 2011/06/28 00:00 [pmc-release]
AID - PONE-D-11-07325 [pii]
AID - 10.1371/journal.pone.0021729 [doi]
PST - ppublish
SO  - PLoS One. 2011;6(6):e21729. doi: 10.1371/journal.pone.0021729. Epub 2011 Jun 28.