Transcript 424_2013_1361_MOESM1_ESMx

Pflügers Archiv - European Journal of Physiology
Stromal interaction molecule 1 (STIM1) regulates sarcoplasmic/endoplasmic
reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle
Keon Jin Lee1, Changdo Hyun1, Jin Seok Woo1, Chang Sik Park2, Do Han Kim2, Eun Hui Lee1,*
1Department
2School
of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea,
of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, 500-712, Korea
*Correspondence: [email protected]
List of supplementary materials
Supplemental Fig. 1. PCR primers for the cloning of GST-fused STIM1-UI in pGEX-4T-1 or STIM1-SBR in pMO91.
Supplemental Fig. 2. Materials and methods.
Supplemental Fig. 3. Sequences of siRNA used for knocking down STIM1 and qPCR results.
Supplemental Fig. 4. MS spectrums for bands 1 to 4 in Fig. 1d.
Supplemental Fig. 5. Releasable Ca2+ from the SR in myotubes expressing STIM1-SBR.
Supplemental Fig. 6. Prediction of possible phosphorylation sites on mouse STIM1-SBR.
Supplemental Fig. 7. Sequence comparison of mouse STIM1-SBR and other proteins.
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Supplemental Figure 1
PCR primers for the cloning of GST-fused STIM1-UI in pGEX-4T-1 or STIM1-SBR in
pMO91.
PCR primers
enzyme sites
forward
5’ – CGGAATTCATGTCACTGGTGGCTGCCCTCAAC –3’
backward
5’– CGTCGACTCACCGGCCTGGGCTGGAGTCTGTTTC –3’
Sal I
forward
5’ – CCGCTCGAGATGTCACTGGTGGCTGCCCTCAAC –3’
Xho I
backward
5’ – GGGGATCCGTCCGGCCTGGGCTGGAGTCTGTTTC –3’
EcoR I
GST-STIM1-UI
STIM1-SBR
2
BamH I
Supplemental Figure 2
Materials and methods.
(A) Binding assay of GST-fused STIM1-UI protein with triad proteins.
Briefly, affinity beads were prepared by immobilizing GST-fused STIM1-UI proteins on GST beads
(Amersham Biosciences, Pittsburgh, PA, USA). The affinity beads were then incubated with 150 μg of the
solubilized triad vesicle sample from rabbit skeletal muscle for 8 h at 4 °C. The proteins that were bound to
the affinity beads were separated on a 10% SDS-PAGE gel, and the gel was stained with Coomassie Blue in
order to obtain the protein bands that were specifically bound to the GST-fused STIM1-UI protein for qTOF
MS and database searches.
(B) Protein identifications by qTOF MS and database searches.
The digested peptide solution by the in-gel digestion with trypsin was desalted and concentrated, and was
eluted using a homemade C18 nano-column (100-300 nl with trypsin of POROS reverse-phase R2 material
(20–30 μm in bead size, PerSeptive Biosystems, Foster City, CA, USA)) and 1.5 ul of 50% MeOH, 49% H2O,
and 1% HCO2H. qTOF MS of the eluted peptides was performed by Hybrid Quadrupole-TOF LC/MS/MS
Mass Spectrometer (AB Sciex Instruments, Framingham, MA, USA) equipped with an electrospray
ionization (ESI) source. Source temperature, room temperature; potential, 1 kV; flow rate, 10–30 nl/min;
cone voltage, 40 V; collision gas and energy, Ar at a pressure of 6 -7 x 10-5 mbar and 25–40 V. A quadrupole
analyzer was used to select precursor ions for the fragmentation in the hexapole collision cell. The produced
ions were analyzed using an orthogonal TOF analyzer and fitted with a reflector, a micro-channel plate
detector, and a time-to-digital converter. Mode, ESI+; MS parameter and scan type, positive TOF MS;
intensity threshold, 1 count; MCA No., GS1: 2.00 and GS2: 0.00; CUR, 30.00; IS, 1000.00; CID, 50~55;
scan range and rate, m/z 50-2000 and 2 sec/scan. Database searches were conducted using the Mascot server
(www.matrixscience.com). Type of search, MS/MS ion search; enzyme, trypsin; fixed modifications,
carbamidomethyl (C); variable modifications, oxidation (M); mass values, monoisotopic; protein mass,
unrestricted; peptide mass tolerance, ±1 Da; fragment mass tolerance, ±0.6 Da; max missed cleavages, 1;
instrument type, default; number of queries, 1.
3
Supplemental Figure 2 (continued)
(C) Co-immunoprecipitation assay, immunoblot assay, and immunocytochemistry.
For the co-immunoprecipitation assay, the solubilized triad vesicle sample from rabbit skeletal muscle (800
µg of total protein) was incubated with anti-STIM1 (10 μg/ml, abcam, Cambridge, MA, USA) or antiSERCA1a antibody (10 μg/ml, abcam, Cambridge, MA, USA) overnight at 4 °C, followed by incubation
with Protein G–Sepharose beads (Amersham Biosciences, Pittsburgh, PA, USA) for 4 h at 4 °C. Beads were
washed five times with a buffer (10 mM Tris-HCl, 1 mM Na3VO4, 10% glycerol, 150 mM NaCl, 5 mM
EDTA, and Protease Inhibitor Cocktail Tablets (Roche, Switzerland), and pH 7.4). Bound proteins were
eluted by boiling in a SDS sample buffer and were subjected to SDS-PAGE (10% gel) followed by
immunoblot assay with anti-STIM1 (1:1,000) or anti-SERCA1a antibody (1:1,000). For the
immunocytochemistry, the myotubes were fixed in cold methanol (-20 °C) for 15 min and permeabilized
with 0.05% Tween 20 in phosphate-buffered saline (PBS) for 1 min. After blocking with 2% normal goat
serum (NGS) in PBS, the myoblasts were incubated with anti-STIM1 (1:500), anti-SERCA1a (1:200, Cell
Signaling Technology, Danvers, MA, USA), or anti-CFP antibodies (1:500, Cell Signaling Technology,
Danvers, MA, USA) for 3 h, washed three times with 2% NGS in PBS for 10 min, incubated with a Cy-3conjugated anti-mouse (1:2,000, Jackson ImmunoResearch, West Grove, PA, USA) or FITC-conjugated
anti-rabbit secondary antibody (1:200, Sigma-Aldrich, St. Louis, MO, USA) for 45 min at room temperature,
and visualized with an inverted fluorescence microscope using a 40x objective lens (ECLIPSE Ti, Nikon
Instruments, Melville, NY, USA) equipped with a monochrome camera (ProgRes MF, JENOPTIK Optical
Systems, Jupiter, FL, USA).
4
Supplemental Figure 3
Sequences of siRNA used for knocking down STIM1 and qPCR results.
#2 siRNA knocked down STIM1 more efficiently, and was used for the subsequent experiments. The results were
adopted from our previous study (Lee KJ, Woo JS, Hwang JH, Hyun C, Cho CH, Kim DH, Lee EH (2013) STIM1
negatively regulates Ca2+ release from the sarcoplasmic reticulum in skeletal myotubes. Biochem J 453(2):187-200).
siRNA
Sense
Antisense
Scrambled siRNA
5’ CUGCCGUCCAAAGUUGUAAUU 3’
5’ UUACAACUUUGGACGGCAGUU 3’
#1 siRNA
5’ GGGAAGACCUCAAUUACCAUU 3’
5’ UGGUAAUUGAGGUCUUCCCUU 3’
#2 siRNA
5’ GCAGAGAAGGAGCUGGAAUUU 3’
5’ AUUCCAGCUCCUUCUCUGCUU 3’
qPCR primers
Forward
Backward
5’ AGAATGAGAGGAGCCGTCAA 3’
5’ GCCTCTCTGCATTTTGCTTC 3’
(expected size of PCR product: 192 bp)
5
Supplemental Figure 4
MS spectrums for bands 1 to 4 in Fig. 1d.
(A) MS spectrum of band 1.
Max. 23.5 counts.
+TOF MS: 0.201 to 0.617 min from Sample 6 (1) of 12ms90.wiff
a=3.56737113718504650e-004, t0=-3.59317734214128000e+001 (Ion Spray)
536.29(1)
23.0
22.0
21.0
20.0
19.0
18.0
17.0
No matching signal
I n t e n s it y , c o u n t s
16.0
15.0
14.0
508.27
13.0
12.0
11.0
529.36(1)
637.31(1)
10.0
9.0
8.0
573.39(1)
7.0
6.0
507.33
5.0
503.30(1)
4.0
523.29
681.24(1)
571.32(2)
1.0
0.0
500
530.21
755.26(1)
581.25(2) 597.40(1) 705.48(1)
539.16
663.20(3)
550
767.41(3)
661.43(1)
3.0
2.0
737.70(3)
617.42(1)
551.34
600
675.42(2)
650
700
739.61(1)
729.34(3)
795.48(1)
748.31(1)
750
856.42(1)
800
871.34(1)
901.39 954.39 964.39(1)
900
m/z, Da
850
6
950
1000
1112.40
1050
1100
1150
1200
1250
1300
(B) MS spectrum of band 2.
+TOF MS: 0.167 to 0.333 min from Sample 9 (2) of 12ms90.wiff
a=3.56737113718504810e-004, t0=-3.59317734214128000e+001 (Ion Spray)
Max. 61.2 counts.
60
55
50
595.40(1)
505.26(1)
I n t e n s it y , c o u n t s
45
40
35
30
507.30(1)
25
536.29
20
545.30(2)
15 515.28
5
637.32(1)
537.29(1)
555.29(2)
10
Identified (SERCA1a from rabbit)
571.29(1)
639.37(1) 653.36
562.27(5) 603.32(2)
554.18(3)
592.49
738.37(2) 781.41(2)
657.37(1)
667.37(1)
623.16(5) 701.22(5) 715.37(2) 756.29(1)
823.37(2)
868.16(2) 945.55(2) 958.46(2)
0
500
550
600
650
700
750
800
850
900
m/z, Da
7
950
1000
1041.82
1050
1103.65(1)
1100
1181.14 1192.30 1230.23
1150
1200
1250
1300
(C) MS spectrum of band 3.
+TOF MS: 0.117 to 0.300 min from Sample 11 (3) of 12ms90.wiff
a=3.56737113718504810e-004, t0=-3.59317734214128000e+001 (Ion Spray)
Max. 63.6 counts.
536.28(1)
64
508.26(1)
60
55
50
45
I n t e n s it y , c o u n t s
637.31(1)
40
35
529.36(1)
30
25
573.38(1)
507.32
20
15
617.42(1)
Identified
634.26(1)
551.33(1)
10
502.36
5
(Chain A, chaperonin groel from E.coli)
737.68(3)
661.45(1)
571.30(2)
589.27(2)
659.30(1) 705.48(1)
581.30(2) 625.32(1)
767.40(3)
804.29(1)
681.26(1)749.49(1)
793.38(1)
730.48(2)
0
500
550
600
650
700
750
800
829.24 870.36(3) 911.45(2)
850
900
m/z, Da
8
969.16(2) 980.44(2) 1029.34(2) 1080.82 1146.46(1)
950
1000
1050
1100
1150
1206.19 1216.77(1)
1200
1250
1300
(D) MS spectrum of band 4.
+TOF MS: 0.167 to 0.234 min from Sample 12 (4) of 12ms90.wiff
a=3.56737113718504760e-004, t0=-3.59317734214128000e+001 (Ion Spray)
Max. 195.4 counts.
637.30(1)
195
190
180
170
507.32(1)
160
150
140
634.30(1)
I n t e n s it y , c o u n t s
130
536.28(1)
120
110
100
551.34(1)
90
595.38(1)
80
70
529.35(1)
804.29(1)
60
525.32
50
606.25(1)
571.30(1) 617.38(1)
40
567.32
10
0
500
599.31(1)
581.30(2)
522.27
557.18(5)
550
683.41(1)
655.37
603.31(1) 647.37
639.83(3)
600
687.68(2)
650
737.70(3)
723.37
749.48(1)
793.50
716.96(3)
700
Identified
Identified
735.03(3)
625.31(2)
30 519.26
20
(Chain A, Ompf porin mutant D74a from E.coli)
573.37(1)
870.45(2)
886.38
750
800
850
925.11(3)
881.42(2)
900
m/z, Da
9
976.44(1)
950
1000
1102.05(2)
1185.48(3)
1036.17
1050
1100
1150
1200
1285.61(2)
1250
1300
Supplemental Figure 5
Releasable Ca2+ from the SR in myotubes expressing STIM1-SBR.
To measure the releasable Ca2+ from the SR to the cytosol, the myotubes loaded with fluo-5N (which detects higher
levels of Ca2+ ranging from μM to mM) were incubated in the imaging solution with zero Ca 2+ for 5 min and then
were treated with caffeine. The releasable Ca2+ is summarized as histograms (the area under the curve was normalized
to the mean value of those from the control vectors). The releasable Ca 2+ from SR to the cytosol was increased by
STIM1-SBR. *Significant difference compared with the control vector (p < 0.05).
10
Supplemental Figure 6
Prediction of possible
phosphorylation sites on
mouse STIM1-SBR.
Phosphorylation sites for mouse
STIM1-SBR were predicted using
NetPhos (Blom N, Gammeltoft S,
Brunak S (1999) Sequence and
structure-based prediction of
eukaryotic protein
phosphorylation sites. J Mol Biol
294(5): 1351-1362). Twenty-five
serines and three threonines were
predicted as possible
phosphorylation sites. Two
phosphorable amino acids in helix
I in Fig. 7a are indicated by red
lines. A higher score means a
higher possibility for
phosphorylation. Numbers
indicate the amino acid sequences
of mouse STIM1-SBR. The first
and the last amino acids of the
STIM1-SBR are presented as
numbers 1 and 223, respectively.
Pos, position; Pred (V in Context),
predicted phosphorylation site.
11
Supplemental Figure 7
Sequence comparison of mouse STIM1-SBR and other proteins.
Using RaptorX (Kallberg M, Wang H, Wang S, Peng J, Wang Z, Lu H, Xu J (2012) Template-based protein structure
modeling using the RaptorX web server. Nat Protoc 7(8):1511-1522), the amino acid sequence of mouse STIM1-SBR
was compared with those of
proteins with 3D structures that
were previously known. 1gg9,
1gge, 1ggj, 1p80, 1p81 or 1qws is
the PDB ID of heme-dependent
catalase HPII or one of its five
mutants (H128N, wild-type HPII,
N201A, D181Q, D181E, or D181N,
respectively). Red letter, small,
hydrophobic, or aromatic amino
acid, but not Y; blue letter, acidic
amino acid; magenta, basic amino
acid; green, hydroxyl, amine, or
amide amino acid; gray, other
amino acids; *, identical amino
acid; :, conserved substitution (the
same color group); ∙, semiconserved substitution (similar
shape). The homology for amino
acid sequences between mouse
STIM1-SBR and HPII or one of its
five mutants was approximately
29%.
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