S-nitrosylation of ERK inhibits ERK phosphorylation and
induces apoptosis
Xiujing FENG, Tingzhe SUN, Yuncheng BEI, Sen DING, Wei ZHENG,
Yan LU * and Pingping SHEN *
State Key Laboratory of Pharmaceutical Biotechnology and Model Animal Research Center
(MARC), Nanjing University, Nanjing 210093, China
Tel: 86-25-83686635
Fax: 86-25-83594060
*Author for correspondence: Dr. Yan LU and Dr. Pingping SHEN
State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University, and Model Animal
Research Center (MARC) of Nanjing University, Nanjing University, Nanjing 210093, China.
Tel: 86-25-83686635;
fax: 86-25-83594060,
E-mail: luyan@nju.edu.cn , ppshen@nju.edu.cn
Xiujing FENG and Tingzhe SUN share the first authorship.
Supplementary data
Name of primers
Sequence 5’-3’
positi
on
VGAI (forward)
233
VGAI (reversed)
TYAQ(forward)
5'CTC AGC CAG AAT TGC GCC TAC AGA CCA GAT GT3'
82
TYAQ(reversed)
HIAY(forward)
144
183
5'GAC CTT AAG ATT GCT GAT TTC GGC CCG G3'
5'CCG GGC CGA AAT CAG CAA TCT TAA GGT C3'
178
TTAD(reversed)
NAII(forward)
5' AGC AAT GAC CAT ATC GCC TAC TTC CTC TAC3'
5'GTA GAG GAA GTA GGC GAT ATG GTC ATT GCT3'
KIAD(reversed)
TTAD(forward)
5'TTC GAA CAT CAG ACC TAC GCC CAG CGC ACG3'
5'CGT GCG CTG GGC GTA GGT CTG ATG TTC GAA3'
HIAY(reversed)
KIAD(forward)
5'ACA TCT GGT CTG TAG GCG CAA TTC TGG CTG AG3'
5'CTC ATC AAC ACC ACC GCC GAC CTT AAG ATT TG 3'
5'CAA ATC TTA AGG TCG GCG GTG GTG TTG ATG AG3'
271
5'CGA CAT CTG GTC TGT GGG CGC CAT TCT GGC TGA G3'
NAII(reversed)
5'CTC AGC CAG AAT GGC GCC CAC AGA CCA GAT GTC G3'
p-ERK1(forward)
5'CACACCGGCTTCCTGGAGGAGGATGTGGCTACGCGCTGGTAC3'
p-ERK1(reversed)
5'GTACCAGCGCGTAGCCACATCCTCCTCCAGGAAGCCGGTGTG3'
Table1. The primers used in the paper
Figure.S1: S-nitrosylation of ERK1 inhibits ERK phosphorylation and promotes
apoptosis in HeLa cell. (A) HeLa Cells were treated with different doses of SNP and
phosphorylation level of ERK1/2 was quantified at 6h after treatment by western
blotting. (B) S-nitrosylation of ERK1 level was determined by BST under different
doses of SNP. (C) Apoptotic effects of SNP in HeLa cells transfected with either wild
type or C183A mutant ERK1 were determined by flow cytometry.
Figure.S2. Sequence of S-nitrosylation site of ERK1 was analyzed. There exist six
potential cysteine residues (Cys) in ERK1 sequence. A software, GPS-SNO 1.0, was
applied to predict the S-nitrosylation sites [1]. In order to make sure which site is most
possible S-nitrosylation site, we analyzed the sequence of the protein and found that
KICD is predicted to be the most possible S-nitrosylation site [2]. (A) Sequence
analysis of the ERK1 protein and scoring of S-nitrosylation site using GSP-SNO 1.0
software. (B) The BLAST result of the conserved cysteine in ERK1.
Figure.S3. Effect of ERK (C183A) on ERK1/2 activity and cell apoptosis under
H2O2 or TNF- treatment for 12 hour, respectively. (A) 293T cells were transfected
with ERK mutant form (C183A) and treated with SNP for 12 hours. Then the
phosphorylation of ERK was determined by immunoblots. (B) 293T cells were
transfected with C183A mutant ERK and the process of procaspase-9, PARP-1 were
quantified by immunoblots under 1mM SNP stress for 12 hours.(C) 293T cells were
transfected with wild type ERK and treated with SNP for 12 hours. Then the
phosphorylation of ERK was determined by immunoblots. (D) 293T cells were
transfected with wild type ERK and the process of procaspase-9,PARP-1 were
quantified by immunoblots under 1mM SNP stress for 12 hours.
Figure.S4. Quantification of NO levels using DAF fluorescence staining under H2O2
treatment in MCF-7 cells.
Figure S5. S-nitrosylation of ERK inhibits ERK phosphorylation under TNF- stress
system and promotes apoptosis. (A)MCF-7 cells were transfected with HA-ERK and
ERK mutant form (C183A) and phosphorylation of ERK was determined by
immunoblots. (B). PPARγ, a phosphorylation target of ERK was examined for
phosphorylation status. (C). MCF-7 cells were transfected with either wild type or
C183A mutant ERK and the process of procaspase-8 was quantified by immunoblots.
(D). Apoptotic effects of TNF in MCF-7 cells transfected with either wild type or
C183A mutant ERK were determined by flow cytometry. *: p<0.05, **: p<0.01, ***:
p<0.001.
Supplemental reference:
[1] Marino, S. M. and V. N. Gladyshev (2010). Structural analysis of cysteine S-nitrosylation: a modified
acid-based motif and the emerging role of trans-nitrosylation. J Mol Biol, 395 (4): 844-859.
[2] Xue, Y. and Z. Liu, et al. (2010). GPS-SNO: computational prediction of protein S-nitrosylation sites with a
modified GPS algorithm. PLoS One, 5 (6): e11290.
Full-length gels and blots
Fig.1c: Elevated NO levels induce apoptosis in MCF-7 cells. SNP accelerates the process of
procaspase-9
Figure2A: Cells were treated with 1mM SNP and the phosphorylation of ERK was examined by
immunoblots.
Figure5A: MCF-7 cells were transfected with ERK1-HA or ERK1 mutant form (C183A), the
phosphorylation of ERK was determined by immunoblots.