1. Calnexin Controls the STAT3-Mediated Transcriptional Response to EGF
Asvin K.K. Lakkaraju, F. Gisou van der Goot 
Molecular Cell 
Volume 51, Issue 3, Pages (August 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1 EGF Stimulation Leads to Calnexin Cleavage by Caspase-8
(A) A431 cells were serum starved for 24 hr, pretreated with or without tyrphostin (200 nM) for 30 min, and stimulated with EGF (100 ng/ml) for 10 min. Cells were lysed and analyzed on a 15% SDS-PAGE. Western blotting was performed with antibodies against the C terminus of calnexin, EGFR, and phosphoEGFR. Please note that, for calnexin staining, the high- and low-molecular-weight parts of the SDS gel were transferred separately onto nitrocellulose (see the Experimental Procedures) but were stained with the same antibody against the C terminus of calnexin.
(B) A431 cells were serum starved for 24 hr and pretreated with or without ZVAD-fmk (10 μM) for 2 hr and with γ-secretase inhibitors and CpE (10 μM) and DAPT (10 μM) for 4 hr after being stimulated with EGF (100 ng/ml) for 10 min. Then, cells were incubated in normal medium for an additional 20 min. The cells were lysed, and proteins were migrated on a 15% SDS-PAGE, and western blotting was performed with an antibody against the C terminus of calnexin.
(C) A431 cells were serum starved for 24 hr and pretreated with the inhibitors of caspase-1, -3, and -8 (all used at 10 μM) for 2 hr prior to stimulation with EGF. The cell lysates were migrated on 15% SDS-PAGE, and western blotting was performed with an antibody against the C terminus of calnexin.
(D) A431 cells were treated with either the control RNAi or RNAi against caspase-1, -3, or -8 for 72 hr. The cells were serum starved for the last 24 hr and, before stimulation with EGF (100 ng/ml), were pretreated with or without ZVAD. The cell lysates were migrated on 15% SDS-PAGE, and western blotting was performed with an antibody against the C terminus of calnexin.
(E) A431 cells were treated with control RNAi or RNAi against FLASH for 72 hr. After serum starvation, cells were stimulated with or without EGF, and calnexin cleavage was monitored in the cell lysates with an antibody against the C terminus of calnexin.
(F) A431 cells were treated with either control RNAi or RNAi against FLASH for 72 hr after stimulation with EGF. The cells were lysed, and the cell lysate was migrated on a 15% SDS-PAGE. Western blotting was performed with an antibody against caspase-8.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Calnexin Cleavage Occurs on MAMs
(A) Amino acid sequence alignment of the cytoplasmic domains of human and mouse calnexin. Both juxtamembranous cysteines (blue) were palmitoylated. Site 1 and site 2 are the predicted caspase cleavage sites (red).
(B) A431 cells were depleted of endogenous calnexin with shRNA and recomplemented with either the WT calnexin (CalxWT) or the mutants deficient in the caspase cleavage sites (CalxS1 and CalxS2) and then stimulated with EGF. Cell lysates were migrated on 15% SDS-PAGE and were immunoblotted with an antibody against the C terminus of calnexin.
(C) A431 cells were depleted of endogenous calnexin with shRNA, and the cells were recomplemented with either the WT calnexin (CalxWT) or the calnexin mutant deficient in phosphorylation (CalxS563A). The cells were stimulated with EGF, and cell lysis was performed. The cell lysate was migrated on 15% SDS-PAGE, and western blotting was performed with an antibody against the C terminus of calnexin.
(D) A431 cells were transfected with either control RNAi or RNAi against DHHC6 and stimulated with or without EGF. The cell lysates were migrated on 15% SDS-PAGE, and western blotting was performed with an antibody against the C terminus of calnexin. Quantification was performed with FIJI software, and the error bars represent the SD.
(E) A431 cells were depleted of endogenous calnexin via shRNA, and the cells were recomplemented with either the WT calnexin (CalxWT) or the calnexin mutant-deficient in palmitoylation (CalxΔPalm). The cells were stimulated with EGF, and cell lysis was performed. An equal amount of protein was migrated on 15% SDS-PAGE, and western blotting was performed with an antibody against the C terminus of calnexin.
(F) A431 cells were transfected with control RNAi or RNAi against PACS-2, Rab32, and Bap31. The cells were stimulated with or without EGF, and cell lysates were migrated on 15% SDS-PAGE before western blotting was performed with an antibody against the C terminus of calnexin. The cleaved cytoplasmic domain of calnexin was quantified with FIJI software, and histograms were plotted. Error bars represent the SD. ∗∗∗p <
See also Figures S2C–S2F.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 The Cleaved Calnexin Tail Modulates STAT3 Signaling
(A–D) A431 cells were starved for 24 hr prior to stimulation with EGF (100 ng/ml). Total RNA was extracted from the samples, and real-time qPCR was performed in order to analyze the expression of early genes that are activated within 30 min of stimulation with EGF. RNA expression profiles were analyzed in the cells depleted of calnexin by shRNA (A) and complemented with either the WT calnexin (CalxWT), calnexin with mutated caspase cleavage site (CalxS1), or the GFP-tagged calnexin expressing the cytoplasmic tail (CalxCT) (B) or calnexin lacking the palmitoylation site (CalxΔPalm) (C). RNA was also analyzed in the cells depleted of FLASH (D) by RNAi. Error bars represent the SD.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Full-Length Calnexin, but Not the CTD, Interacts with Caspase-8 and FLASH
(A–D) A431 cells were stimulated with or without EGF, cell lysis and immunoprecipitation were performed with either of the N- and C terminus-binding calnexin antibodies (A and B), FLASH (C), and caspase-8 (D). The immunoprecipitates were migrated on 15% SDS-PAGE, and western blotting was performed with antibodies against the C and N termini of calnexin, caspase-8, PIAS3, STAT3, and FLASH. The western blot analysis of the corresponding total cell extracts is shown in Figure S5A.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 PIAS3 Is Inhibited by Calnexin Cytoplasmic Domain
(A and B) A431 cells were treated with control shRNA or shRNA against calnexin for 7 days. Cells were stimulated with or without EGF, and the cell lysates were immunoprecipitated with either anti-PIAS3 (A) or anti-STAT3 (B). The western blot analysis of the corresponding total cell extracts is shown in Figure S5B. The immunoprecipitates were migrated on 15% SDS-PAGE, and western blotting was performed with antibodies against PIAS3, STAT3, and the C terminus of calnexin. The amount of STAT3 coprecipitated in the PIAS3 immunoprecipitation, and the amount of PIAS3 in STAT3 immunoprecipitation was quantified and plotted as histograms. For total cell extracts, see Figure S5B. Error bars represent the SD. ∗∗∗p <
(C) A431 cells grown in 10 cm plates were stimulated with or without EGF, digitonin permeabilization was performed in order to extract the cytoplasmic fraction, and centrifugation was performed in order to separate the nuclei and membrane fraction (see the Experimental Procedures). The protein content in each fraction was quantified with BCA reagent, and equal amounts of protein samples (20 μg) were loaded on the 15% gels. After western blotting, the membranes were blotted for full-length calnexin with an antibody that recognizes its N terminus (CalxFL[N]) or the calnexin antibody that recognizes the C terminus (CalxCTD), anti-PIAS3, anti-GAPDH, which was a marker for the cytoplasmic fraction, and anti-NUP, a marker for the nuclear fraction. In the right panel, PIAS3 was immunoprecipitated from the nuclear (N) and cytoplasmic fraction (C), and the immunoprecipitates were migrated on 15% SDS-PAGE and blotted against PIAS3 and calnexin antibody.
(D) A431 cells were treated with control shRNA or shRNA against calnexin for 7 days. On day 5, the cells were treated with either control RNAi or RNAi against PIAS3. After 24 hr serum starvation, the cells were stimulated with EGF for 10 min. RNA was extracted from these cells, and qPCR was performed in order to monitor the expression levels of early genes that were identified previously. Error bars represent the SD.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 ER Stress Prevents Calnexin Cleavage upon EGF Stimulus
(A) A431 cells were depleted of endogenous calnexin with shRNA and complemented with either the WT calnexin (CalxWT) or calnexin bearing a truncated lumenal domain (CalxΔB). The cells were pretreated with or without thapsigargin for 6 hr and then stimulated with or without EGF. The cell lysates were migrated on 15% SDS-PAGE, and western blot was performed with an antibody against the C terminus of calnexin.
(B) Representation of the lumenal domain of calnexin with all the four mutated residues highlighted in color.
(C) A431 cells were depleted of endogenous calnexin with shRNA and complemented with the calnexin mutants (CalxY165A-K167A and CalxY186A-M189A) deficient in binding to the substrates. Serum-starved cells were pretreated with or without thapsigargin for 6 hr and were stimulated with or without EGF. The lysates were migrated on 15% SDS-PAGE, and western blot was performed with an antibody against the C terminus of calnexin.
(D) Experiments were performed as in (A) except that RNA was isolated, and the expression of early genes identified previously was monitored by qPCR. Error bars represent the SD. ∗∗∗p <
(E) Experiments were performed as in (C), the RNA was extracted, and the transcriptional response of early genes was monitored by qPCR. Error bars represent the SD. ∗∗p < 0.01.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

8. Figure 7
Schematic Model of the Role of Calnexin in STAT3-Mediated EGF Signaling
The binding of EGF to EGFR leads to the activation of a multitude of signal-transduction pathways, among which is the STAT3 signaling pathway. STAT3 is activated by EGFR-activated kinases, which phosphorylate STAT3, promoting its dimerization. Activated STAT3 translocates into the nucleus, where it binds to the DNA and modulates transcription. PIAS3 are natural inhibitors of STAT3, which prevents the binding of activated STAT3 to DNA. The activation of the EFGR pathway leads to FLASH-mediated caspase-8 activation and its recruitment to the calnexin cytosolic domain in MAMs. The cleaved calnexin cytoplasmic domain moves into the nucleus where it binds to PIAS3, thereby preventing PIAS3 from binding to STAT3 and, thus, promoting EGF-mediated STAT3 signaling.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions