FRAP/mTOR Localization in the ER and the Golgi Apparatus Ryan M. Drenan1, Xiangyu Liu2 ...

36 Pages·2003·0.49 MB·English

JBC Papers in Press. Published on October 24, 2003 as Manuscript M305912200
FRAP/mTOR Localization in the ER and the Golgi Apparatus
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Ryan M. Drenan1, Xiangyu Liu2, Paula G. Bertram2, and X.F. Steven Zheng2,3 ttp://w
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1Molecular Cell Biology Graduate Program and 2Department of Pathology and gu
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Immunology, Washington University School of Medicine, 660 South Euclid Ave, p
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St. Louis, MO 63110. 0
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Running Title: Subcellular localization of FRAP/mTOR
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Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
3To whom correspondence should be addressed:
Tel: [phone], Fax: [phone], E-mail: [email protected]
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Summary
FKBP12-rapamycin-associated protein (FRAP) or mammalian target of rapamycin
(mTOR) and its effector proteins form a critical signaling pathway that regulates
eukaryotic cell growth and proliferation. While the protein components in this pathway
have begun to be identified, little is known about their subcellular localization or the
physiological significance of their localization. By immunofluorescence, we find that
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both endogenous and recombinant FRAP/mTOR proteins show localization o
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predominantly in the endoplasmic reticulum (ER) and the Golgi apparatus. Consistent ed
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with this finding, FRAP/mTOR is co-fractionated with calnexin, an ER marker protein. ttp
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Biochemical characterization suggests that FRAP/mTOR is a peripheral ER/Golgi protein .jb
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with tight membrane-association. Finally, we have identified domains of FRAP/mTOR b/
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that may mediate its association with the ER and the Golgi apparatus. t on
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Introduction
Rapamycin is a macrolide isolated from the Easter Island soil bacterium Streptomyces
Hygroscopicus, and is currently used as an immunosuppressant in kidney transplantation
(1) and in drug-eluting stents to prevent restenosis after angioplasty (2). In addition,
rapamycin ester derivatives are in clinical trials for cancer treatment (3). The FKBP12-
rapamycin associated protein (FRAP), also known as mammalian target of rapamycin
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(mTOR), is a 289kDa phosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) (4,5). o
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The PIKK family also includes ATM, ATR, DNA-PK, and SMG-1. FKBP12- ed
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rapamycin binds to and inhibits FRAP in mammalian cells and its homolog Tor1/2 in ttp
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yeast via the conserved FKBP12-rapamycin-binding (FRB) domain (6-9). The C- .jb
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terminal kinase domain is homologous to PI3K catalytic domains, although it only b/
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appears to have protein kinase activity (4,5). At the N-terminus, there is an extensive t on
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array of tandem HEAT repeats (10,11) that mediate protein-protein interactions with 0
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CLIP-170, Gephyrin and Raptor (12-15). Two other domains conserved in the PIKK
family, FAT and FATC, flank the kinase domain to the N and C-terminus, respectively
(16).
FRAP is linked to translational control through its two main effector proteins, the
ribosomal S6 kinases (S6Ks) and the translation regulator eIF4E-binding protein 1 (4E-
BP1) (17-21). S6Ks promotes protein synthesis by phosphorylation of ribosomal protein
S6, allowing the translation of 5’ oligopyrimidine tract-containing mRNAs that encode
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for ribosomal proteins and translational regulators (17-21). 4E-BP1 binds to eIF4E and
inhibits CAP-dependent translation under unfavorable growth conditions. 4E-BP1
becomes phosphorylated by FRAP and is released from eIF4E when growth conditions
become favorable (17-21). FRAP also controls protein synthesis by regulating ribosome
biogenesis (22-24). FRAP signaling is regulated both by nutrients, including amino acids
and glucose, and by mitogens (17-21).
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Both the endoplasmic reticulum (ER) and the Golgi apparatus are internal o
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membrane structures constituting over one-half of the totalmembranes in a cell. The ER d fro
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is known to have two essentialfunctions. First, proteins destined for transport to other ://w
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organelles and the cell surface or for secretion are synthesized inthe ER. During .org
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translation, they are translocated intothe ER lumen through a pore in the ER membrane, st o
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and are folded and become glycosylated inside the ER lumen. Secondly, synthesis of ril 10
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lipids and cholesterol takes place on the cytoplasmic side ofthe ER membrane. The ER is
also an important participant in cell signaling, including the unfolded protein response
(UPR), the ER overload response (EOR) and the ER-to-nuclear sterol signaling by
SREBP (25). Several recent studies suggest that the ER is also involved in signal
transduction pathways traditionally thought to occur solely through the plasma
membrane. For example, the small GTPase Ras that is restricted to the ER and the Golgi
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apparatus can actively engage in signal transduction to MAP kinases (26). These studies
indicate that the ER and the Golgi apparatus can serve as anchors for diverse cell
signaling events.
A large body of evidence shows that proper localization plays a crucial role in the
specificity and/or regulation of cell signaling events. Despite the importance of FRAP in
cell growth and therapeutic intervention, little is known about its precise subcellular
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localization. We have examined common mammalian cell lines for FRAP localization. o
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We find that FRAP is predominantly localized to the ER and the Golgi apparatus. ed
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Biochemical analyses suggest that FRAP is a peripheral ER membrane protein but tightly ttp
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anchored to the ER/Golgi membranes. Finally, we found two discrete domains in the N- .jb
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terminus that are crucial for targeting to the ER and the Golgi apparatus, respectively. b/
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Like the sterol-SREBP pathway, FRAP may use the ER/Golgi as anchors for nutrient t on
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sensing and signaling. 0
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Experimental Procedures
Plasmids, cell lines, antibodies and chemicals
FLAG-FRAP plasmids have been described previously (12). Mammalian cell lines were
obtained from American Type Culture Center (ATCC) and were maintained as
recommended by ATCC. FRAP antibodies were produced (Harlan) by immunizing
rabbits with polypeptides corresponding to the amino acids 1-120 of human FRAP. The
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rabbit FLAG polyclonal antibody was purchased from Sigma, Calnexin antibodies were o
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purchased from BD Transduction Laboratories, Golgin-97 antibodies and Mitotracker ed
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were purchased from Molecular Probes, phospho-Thr389 antibody was purchased from ttp
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Cell Signaling, FRAP H-266 FRB-specific antibody was purchased from Santa Cruz .jb
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Biotechnology, and HRP-conjugated secondary antibodies were purchased from Pierce. b/
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Leptomycin B, sodium azide, and trypsin and chymotrypsin were purchased from Sigma. t on
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Affinity-purification of FRAP antibodies
The N-terminal FRAP antigenic polypeptide was conjugated to CNBR-activated
Sepharose beads (Pharmacia) according to manufacturer’s instruction. The N-terminal
FRAP antiserum (150 mg IgG) was equilibrated in Binding Buffer (supplied from Biorad
cat. No. 153-6160) and was loaded twice onto the N-terminal FRAP-Sepharose affinity
column. After washing with Binding Buffer, FRAP-specific IgG was eluted with Elution
Buffer (Biorad). Fractions were collected, analyzed, and subsequently dialyzed against
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10mM HEPES pH7.5, 150mM NaCl, 50% Glycerol until further use. Purification and
dialysis were performed at 4°C.
Immunoprecipitation and Western Blot
Cell lysates for immunoprecipitation and Western blot were prepared using ice-cold lysis
buffer (LB) containing 50 mM HEPES-KOH (pH 7.4), 40mM NaCl, 1 mM EDTA, 0.1%
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Triton X-100, 5% glycerol, 10 mM sodium pyrophosphate, 10 mM ²-glycerophosphate, o
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1.5 mM Na3VO4, 50 mM NaF, and 1x protease inhibitor cocktail (Roche Diagnostics, d fro
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GmbH). Immunoprecipitation was performed by incubating the cell lysates with 10 ¼l ttp://w
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FRAP antibody per 1 mg protein extract at 4ºC for 90 min. The immune complexes were .jb
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bound to Protein A-agarose beads (Pharmacia) for 1 hr at 4ºC, which were washed three y g
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times with wash buffer (WB) containing 50 mM HEPES-KOH, pH 7.4, 1 M LiCl, 0.1% A
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Triton X-100, 5% glycerol, 10 mM sodium pyrophosphate, 10 mM ²-glycerophosphate, , 2
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1.5 mM Na3VO4, 50 mM NaF, and 1x protease inhibitor cocktail. Protein A-bound
materials were eluted by boiling in SDS protein sample buffer. For Western blot, protein
samples were separated on SDS polyacrylamide gels and transferred onto an Immobilon-
P membrane. After blocking with 5% dry milk in TBST, the membrane was incubated
with primary antibodies for 1 hr to overnight, followed by incubation with HRP-
conjugated secondary antibodies (1:10,000) for 30 min and with ECL (Amersham Life
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Sciences).
FRAP kinase assay
Following immunoprecipitation, FRAP or control immunocomplexes were incubated at
30ÚC with the FRAP kinase reaction mix (50 mM HEPES-KOH pH 7.4, 10 mM MgCl2,
1 mM DTT, 2 mM ATP, 0.5 ¼g GST-p70 per 20 ¼l KB). Aliquots of samples were
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withdrawn at different times and the kinase reaction was stopped by boiling in SDS w
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sample buffer. Phosphorylation of GST-p70S6K1 at Thr-389 was analyzed by Western d fro
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blot with a phospho-Thr389 antibody. ttp://w
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Transfection and Immunofluorescence Microscopy y g
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HeLa cells were transfected with plasmids using the calcium phosphate method. For A
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indirect immunofluorescence, Cells were fixed in 3% paraformaldehyde, 2% sucrose in , 2
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H2O for 10 min at 37ÚC, permeabilized with ice-cold Triton buffer (0.5% Triton X-100 in
20 mM HEPES, pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose) for 5 min on ice,
blocked with 0.1% BSA in PBS for 10 min on ice, and incubated with primary antibodies
for 20 min (rabbit N-terminal FRAP antibody at 1:500, rabbit C-terminal FRAP
antibody at 1:50, FRAP FRAP H-266 FRB-specific antibody at 1:50, mouse anti-
Calnexin at 1:50, mouse anti-Golgin-97 at 1:100, rabbit anti-FLAG at 1:1000) at 37ÚC
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in a moisture chamber. Unbound antibodies were removed by washing ten times with
TBST. Secondary antibodies labeled with Texas Red-X or Alexa Fluor 488 (Molecular
Probes) were applied for 15 min at room temperature and washed as with the primary
antibodies. Glass cover slips carrying treated cells were mounted with Cytoseal mounting
medium onto glass slides and analyzed using a Zeiss laser scanning confocal microscope
(Zeiss Axiovert 200 m microscope and LSM 5 Pascal laser scanning confocal), or an
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Olympus BX51 fluorescence microscope equipped with a Qimaging Retiga EXi digital o
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camera. No significant difference in IF staining was observed with the N-terminal FRAP ed
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antiserum or affinity-purified N-terminal FRAP antibody. Mitotracker Red was used to ttp
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stain HeLa cells according to manufacturer’s instructions. .jb
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Subcellular fractionation and related biochemistry t on
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HeLa cells were washed with ice-cold HME buffer (10 mM HEPES, 250 mM Mannitol, 0
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0.5 mM EDTA, pH 7.4), resuspended in 5 volumes of ice-cold HME buffer containing
0.1 mM PMSF and dounce homogenized with 10 gentle strokes. Nuclei and unbroken
cells were pelleted at 1,500g, followed by a spin at 10,000g (10 min, 4ÚC). The pellets
(P10) were resuspended in HME buffer. The S10 supernatant (after saving an aliquot)
was overlaid on a 20% sucrose cushion and further centrifuged at 100,000g (60 min,
4ÚC). The pellets (P100) were resuspended in HME. The S100 was also saved for
western blot analysis. To determine the nature of FRAP membrane association, the P100
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