1. Identification and Characterization of a General Nuclear Translocation Signal in Signaling Proteins 
Dana Chuderland, Alexander Konson, Rony Seger 
Molecular Cell 
Volume 31, Issue 6, Pages (September 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Identification of SPS as Important Domain for ERK2's Nuclear Translocation
(A) Schematic representation of ERK2, including the TEY and the SPS domains.
(B) Role of SPS in nonregulated accumulation of ERK2 in the nucleus. CHO or COS7 cells were grown on cover slips and transfected with either WT-GFP-ERK2 (WT-ERK2) or the mutants 244-6A and Δ Twenty-four hours after transfection, the cells were serum starved (0.1% FBS, 16 hr), fixed, stained with DAPI, and visualized using a fluorescent microscope.
(C) Role of SPS in the stimulated nuclear translocation of ERK2. CHO cells were cotransfected with HA-MEK1 together with WT-ERK2, 244-6A, or Δ Twenty-four hours after transfection, the cells were serum starved and treated for 15 min with either 250 or 10 nM TPA. After treatments, the cells were fixed and stained with either αHA Ab to detect MEK1 or DAPI for nuclear staining. ERK was detected by its GFP fluorescence.
(D) Equilibration of TEY phosphorylation with different TPA concentrations. The TEY phosphorylation of GFP-ERK2 and its mutants was detected by a direct WB using αpTEY and α-general ERK Abs (αgERK).
(E) 244-6A-ERK2 binds to 6His-MEK1 and is released upon phosphorylation. Serum-starved CHO cells expressing WT-ERK2 (WT) or 244-6A-ERK2 were treated with TPA (250 nM, 15 min) followed by IP and stringently washed. The IPed constructs, as well as beads alone, were incubated with 0.5 μg recombinant 6His-MEK1 (30 min, 30°C). Thereafter, the beads were mildly washed and subjected to WB using αMEK and αgERK Abs.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Characterization of SPS Phosphorylation Using αpSPS Ab
(A) Elevation of pSPS detection upon stimulation. COS7 cells transfected with WT-GFP-ERK2 (GFP-ERK2) were serum-starved and treated with VOOH (V, 100 μM Na3VO4 and 200 μM H2O2, 15 min), TPA (T, 250 nM, 15 min), EGF (E, 50 ng/ml, 15 min), or left untreated (NT). The GFP-ERK2 was IPed with αGFP Ab, extensively washed, and subjected to WB using αpSPS and αgERK Abs.
(B) Time course of SPS phosphorylation. Serum-starved COS7 cells overexpressing GFP-ERK2 were treated with EGF (50 ng/ml) for the indicated times. The GFP-ERK2 was IPed with αGFP Ab, extensively washed, and subjected to WB with αpSPS, αpTEY and αgERK Abs.
(C) Elevation of αpSPS Ab immunoreactivity upon stimulation of endogenous ERKs. Serum-starved COS7 cells were treated with EGF (50 ng/ml) or VOOH for the indicated times. SPS phosphorylation was detected using the αpSPS Ab, and this was compared to the amount of general ERKs detected by αgERK.
(D) Specificity of the αpSPS Ab. Serum-starved COS7 cells overexpressing either GFP-ERK2 or 244-6A were treated with EGF (50 ng/ml, 15 min) following WB using αpSPS and αgERK Abs.
(E and F) Further characterization of the αpSPS Ab's specificity. GFP-ERK2 was IPed from VOOH stimulated (Act) or nonstimulated (NT), transfected COS7 cells. The IPed protein was then incubated with shrimp's alkaline phosphatase (SAP, 15 units, 30 min, 30°C) and subjected to WB with αpSPS (E), αpTEY (F), or αgERK Abs.
(G) Recognition of monophosphorylated SPS by the αpSPS Ab. Serum-starved COS7 cells overexpressing the following mutants GFP-ERK2 (WT), GFP-APS-ERK2 (AS), GFP-SPA-ERK2 (SA), and GFP-APA-ERK2 (APA) were stimulated with EGF (50 ng/ml, 10 min) or left untreated (NT). The cells were then extracted and the lysates were subjected to WB using αpSPS and αgERK Abs.
(H) TEY phosphorylation is not required for SPS phosphorylation. COS7 cells overexpressing WT-ERK2 or TEY-AAA were treated and analyzed as described in (D).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
SPS Phosphorylation Is Required for the Nuclear Translocation of ERK2
(A) Replacing the Ser residues in the SPS with Glu induces the nuclear accumulation of ERK2. Serum-starved CHO cells overexpressing WT-GFP-ERK2 (WT), APA-GFP-ERK2 (APA), or EPE-GFP-ERK2 (EPE), were fixed, stained with DAPI, and visualized (as in Figure 1).
(B) Subcellular fractionation confirms the fluorescence results. Serum-starved COS7 cells overexpressing the ERK2 constructs (as in [A]) were grown in 10 cm plates and then subjected to cellular fractionation. WB of aliquots of the fractions was performed with αGFP Ab to detect ERKs, and with αtubulin or αc-Jun Abs as markers for cytoplasmic and nuclear fractions respectively.
(C) EPE-ERK2 translocates to the nucleus faster than WT-ERK2. CHO cells were transfected with either WT-ERK2 or EPE-ERK2 constructs. Eight or 12 hr later the cells were either stained with DAPI and visualized or lysed and subjected to WB with αGFP Ab to confirm equal expression.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
The SPS domain, within a 19 Amino Acid Stretch, Is an Autonomous NTS
(A) Schematic representation of the β-Gal constructs.
(B) Equal expression of the β-Gal constructs. The constructs were overexpressed in COS7 cells, which were grown for 24 hr, serum starved, extracted, and subjected to WB with αGFP Ab.
(C) SPS, APA, and EPE peptides fused to the β-Gal construct modify its subcellular localization. Serum-starved CHO and COS7 cells overexpressing the β-Gal constructs were fixed, stained with DAPI, and visualized.
(D) Subcellular fractionation confirms the fluorescence results. Subcellular fractionation was performed on COS7 cells transfected with the β-Gal constructs. WB of aliquots of the fractions was performed with αGFP Ab to detect the β-Gal constructs, and with αtubulin and α-histone-H1 Abs as cytoplasmic and nuclear markers respectively.
(E) TPA stimulation enhances nuclear localization of the β-Gal-SPS. Serum-starved CHO and COS7 cells, overexpressing β-Gal-GFP, β-Gal-SPS, and β-Gal-APA were stimulated with TPA (250 nM, 15 min) or left untreated, and then fixed, stained with DAPI and visualized.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 The Nuclear Translocation of ERKs Is Mediated by Imp7
(A) EGF-induced ERKs translocation is prevented by SiRNA of Imp7. HeLa cells were transfected with SiRNA of Imp7 or scrambled control SiRNA using oligofectamin. Seventy-two hours after transfection, the cells were serum starved and then either treated with EGF (50 ng/ml, 15 min) or left untreated. Then, the cells were washed, fixed, and stained with αgERK Ab and DAPI.
(B) LMB-induced ERKs translocation is partially prevented by SiRNA of Imp7. HeLa cells were transfected with control or Imp7 SiRNAs as above. After serum starvation, the cells were either left untreated (basal) or were treated with LMB (5 ng/ml, 30 min), washed, fixed, and stained with αgERK and DAPI.
(C) Nuclear accumulation of WT-GFP-ERK2 is partially prevented by SiRNA of Imp7. HeLa cells overexpressing the SiRNA of Imp7 or the control SiRNA (72 hr after transfection) were further cotransfected with WT-GFP-ERK2 (GFP-ERK2). Forty-eight hours after this transfection the cells were serum-starved and then fixed and stained with DAPI.
(D) SiRNA abolishes Imp7 expression. HeLa cells overexpressing the SiRNA of Imp7 or control SiRNA were harvested and subjected to WB using αImp7 and αgERK Abs.
(E) GFP-ERK interaction with Imp7 is stimulation dependent. Serum-starved HeLa cells overexpressing GFP-ERK2 or GFP control were incubated with or without EGF (50 ng/ml, 15 min). This was followed by Co-IP with αGFP Ab and mild washes. The amount of Co-IPed Imp7 and the amount of IPed GFP or GFP-ERK2 were determined by WB using αImp7 and αGFP Abs.
(F) Interaction of endogenous Imp7 and ERK2 is elevated by EGF. Serum-starved HeLa cells were treated with EGF (50 ng/ml) for 0, 5, and 15 min and then subjected to Co-IP with αgERK2 Ab or beads alone. The amount of Co-IPed Imp7, the amount of the IPed ERKs, and a loading marker (L) were detected by WB using αImp7 and αgERK Abs.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
pSPS Interacts with Imp7 and Releases Nup153c Binding without Involvement of Imp-β
(A) Interaction of ERK2 with Imp7, but not Imp-β, is enhanced by EGF stimulation and EPE mutation. Serum-starved HeLa cells overexpressing WT-ERK (WT), ERK-APA (APA), ERK-EPE (EPE), or GFP were treated with EGF (50 ng/ml, 10 min). Their extracts were then subjected to Co-IP with α-GFP Ab and mild washes. The Co-IPed Imp7 and Imp-β, as well as their loading amounts, were determined by WB using αImp7 and αImp-β Abs. The equal amount of IPed ERKs was determined with αGFP Ab.
(B) The interaction of ERK2 and Imp7 in vitro is affected by the SPS domain. Reduced glutathione-released GST-ERK2 or GST constructs were incubated with IPed Imp7. This was followed by a mild wash, boiling, and WB using αgERK and αImp7 Abs.
(C) Interaction of 2GFP-SPS and its mutants with Imp7. HeLa cells overexpressing 2GFP, 2GFP-SPS, 2GFP-APA, and 2GFP-EPE constructs were subjected to Co-IP as described in (A) (without EGF stimulation).
(D) Interaction with Nup153c is elevated in APA-ERK2 but reduced in EPE-ERK2. IPed and stringently washed WT-ERK2 (WT), APA-ERK2 (APA), or EPE-ERK2 from COS7 cells pretreated with or without TPA (250 nM, 15 min) were incubated with 0.5 μg recombinant His-Nup153c. This was followed by mild washes, and the beads were then subjected to WB using αHis and αGFP Abs.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

8. Figure 7
The NTS Motif Mediates Nuclear Translocation of SMAD3 and MEK1
(A) Elevation of pSPS detection upon stimulation by SMAD3. Serum-starved COS7 cells overexpressing Flag-SMAD3 were stimulated with VOOH, TPA (T, 250 nM, 15 min), TGF-β (TG, 1 ng/ml, 15 min), or left untreated (NT). Flag-SMAD3 was IPed with αFlag Ab, extensively washed, and subjected to WB with αpSPS or αFlag Abs.
(B) Replacement of SPS with alanines prevents the nuclear translocation of full-length SMAD3. CHO cells overexpressing either Flag-SMAD3 or its AAA mutant were fixed and stained with both αFlag Ab (Flag) and DAPI.
(C) Mutants of the SPS domain modulate the subcellular localization of the MH2 region of SMAD3. CHO cells overexpressing GFP-MH2 domain of SMAD3 (WT-MH2) or the AAA and DPD mutants were fixed and visualized.
(D) Subcellular fractionation confirms the fluorescence results. Subcellular fractionation was performed on COS7 cells transfected with the MH2 constructs. WB of aliquots of the fractions was performed with αGFP Ab to detect the SMAD3 constructs and with αtubulin or αc-Jun Abs as cytoplasmic and nuclear markers respectively.
(E) The NTS motif regulates MEK1 shuttling into the nucleus. Serum-starved CHO cells overexpresing WT-GFP-MEK1, AAA-GFP-MEK1, and EPE-GFP-MEK1 were treated with LMB (5 ng/ml, 1 hr), LMB + TPA (5 ng/ml, 45 min and then 250 nM TPA for an additional 15 min), or left untreated (NT). Subsequently the cells were fixed, stained with DAPI, and visualized.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions