1. Volume 43, Issue 1, Pages 45-56 (July 2011)
SHP2 Tyrosine Phosphatase Converts Parafibromin/Cdc73 from a Tumor Suppressor to an Oncogenic Driver 
Atsushi Takahashi, Ryouhei Tsutsumi, Ippei Kikuchi, Chikashi Obuse, Yasuhiro Saito, Azadeh Seidi, Robert Karisch, Minerva Fernandez, Taewoo Cho, Naomi Ohnishi, Orit Rozenblatt-Rosen, Matthew Meyerson, Benjamin G. Neel, Masanori Hatakeyama 
Molecular Cell 
Volume 43, Issue 1, Pages (July 2011)
DOI: /j.molcel
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2. Molecular Cell 2011 43, 45-56DOI: (10.1016/j.molcel.2011.05.014)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 Interaction of the PAF Complex with Substrate-Trapping SHP2
(A) NIH 3T3 cells were transiently transfected with the indicated vectors. Total cell lysates prepared were immunoprecipitated with an anti-Myc antibody and the immunoprecipitates (IPs) were immunoblotted with the indicated antibodies. FAK, a known SHP2 substrate, was used as a control. ST, substrate-trapping SHP2; ST/T507K, substrate-trapping SHP2-T507K; pY, phosphotyrosine.
(B) lacC9 cells with or without induction of SHP2-specific shRNA by IPTG treatment were subjected to immunostaining (top) or immunoblotting (bottom) with the indicated antibodies. Nuclei were visualized by DAPI staining. Scale bar represents 20 μm.
(C–E) AGS (C and D) or lacC9 cells with or without induction of SHP2-shRNA (E) were transiently transfected with the indicated vectors. Total cell lysates (TCLs) prepared were immunoprecipitated with the respective antibodies and the IPs were immunoblotted with the indicated antibodies. PF, parafibromin.
(F) AGS (top) or HEK293A (bottom) cell lysates were immunoprecipitated with an anti-parafibromin (PF) antibody or control IgG and the IPs were immunoblotted with an anti-PF antibody or anti-phosphotyrosine (pY) antibody.
(G) Ptpn11fl/KO mice-derived MEFs expressing tamoxifen (4-OHT)-inducible Cre-ER were treated with or without 4-OHT. TCLs prepared were immunoprecipitated with an anti-PF antibody. The IPs were immunoblotted with the indicated antibodies. See also Figure S1.
Molecular Cell  , 45-56DOI: ( /j.molcel )
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4. Figure 2 Tyrosine Dephosphorylation of Parafibromin by SHP2
(A) AGS cells were transiently transfected with the indicated parafibromin (PF) vector or control empty vector. Total cell lysates (TCLs) prepared were immunoprecipitated with the respective antibodies and the immunoprecipitates (IPs) were immunoblotted with the indicated antibodies.
(B and C) AGS cells were cotransfected with SHP2-ST-Myc (B) or SHP2-ST/T507K-Myc (C) vector and Flag-parafibromin (wild-type or Y290/293/315F) vector. TCLs prepared were immunoprecipitated with an anti-Myc (B) or anti-Flag (C) antibody, and the IPs were immunoblotted with the indicated antibodies.
(D) AGS cells were triple-transfected with Flag-parafibromin (PF), c-Abl, and SHP2-Myc vectors. TCLs prepared were immunoprecipitated with an anti-Flag antibody, and the IPs were immunoblotted with the indicated antibodies.
(E) AGS cells were cotransfected with Myc-tagged SHP2 (wild-type, T507K, D61G, or E76K) and Flag-parafibromin (PF) vectors. TCLs prepared were immunoprecipitated with an anti-Flag antibody and the immunoprecipitates were immunoblotted with the indicated antibodies. Values indicate levels of parafibromin tyrosine phosphorylation relative to that in the absence of ectopic SHP2 expression. See also Figure S2.
Molecular Cell  , 45-56DOI: ( /j.molcel )
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5. Figure 3 Regulation of Parafibromin/β-Catenin Interaction by SHP2
(A–C) HEK293A (A) or AGS (B) cells were transiently transfected with the indicated vectors. The lacC9 cells (C) were induced to express SHP2-shRNA by IPTG treatment. Total cell lysates (TCLs) were immunoprecipitated with the respective antibodies and the immunoprecipitates (IPs) were immunoblotted with the indicated antibodies. Error bars indicate + standard deviation (SD); n = 3, ∗∗∗p < (A). Values indicate relative amounts of β-catenin that bound to parafibromin (PF) (B and C). See also Figure S3.
Molecular Cell  , 45-56DOI: ( /j.molcel )
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6. Figure 4 Effect of β-Catenin or SUV39H1 on Parafibromin Function
(A–D) AGS cells were transiently transfected with luciferase reporters together with the indicated expression vector and were subjected to the dual luciferase reporter assay. β-catenin S33Y is a degradation-resistant β-catenin mutant. The levels of transfected proteins are shown (representative images from triplicate experiments). In (B), AGS cells were cotransfected with Paf1-specific shRNA and wild-type parafibromin vector. Error bars represent ± SD; n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001.
(E) AGS cells transfected with the parafibromin (PF) vector were immunoblotted with the respective antibodies. Values indicate amounts of c-Myc or cyclin D1 relative to Actin.
(F and G) AGS cells transfected with the c-myc reporter together with the indicated vectors were subjected to the dual luciferase assay. Error bars indicate mean ± SD; n = 3 (F), n = 6 (G), ∗∗∗p <
(H and I) HEK293T cells were transfected with the indicated vectors. Total cell lysates (TCLs) were immunoprecipitated with an anti-Flag (H and I) or anti-Myc (H) antibody, and the immunoprecipitates (IPs) were immunoblotted with the respective antibodies. See also Figure S4.
Molecular Cell  , 45-56DOI: ( /j.molcel )
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7. Figure 5 Nuclear Accumulation of β-Catenin by Parafibromin or SHP2
(A) AGS cells transfected with the Flag-parafibromin (PF) vectors were immunostained with respective antibodies. The signal intensity of β-catenin was quantified. Error bars indicate ± SD; n = 10, Scale bar represents 20 μm.
(B) AGS cells transfected with the PF vectors were subjected to immunoblotting with respective antibodies.
(C) AGS cells transfected with the SHP2-Myc vectors were immunostained with respective antibodies. The signal intensity of β-catenin was quantified. Error bars indicate ± SD; n = 10, Scale bar represents 20 μm.
(D) AGS cells transfected with the parafibromin-shRNA and SHP2-T507K-Myc vectors were immunostained with an anti-parafibromin antibody (top left) or anti-Myc plus anti-β-catenin antibodies (bottom left) or were subjected to immunoblotting with the indicated antibodies (top right). The signal intensity of β-catenin was quantified (bottom right). Error bars indicate ± SD, n = 10, scale bars represent 20 μm.
(E) Anti-β-catenin or anti-c-Myc immunostaining of myeloid cells infiltrating enlarged spleens of Ptpn11D61G/+ mice. Spleens from littermate mice were used as a control. Scale bars represent 20 μm. See also Figure S5.
Molecular Cell  , 45-56DOI: ( /j.molcel )
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8. Figure 6 RAS Signal-Dependent Translocalization of SHP2
(A) AGS cells, either proliferating (left) or confluent (middle), were immunostained with an anti-SHP2 antibody (green). The confluent cells were re-seeded at a reduced cell density and stained with an anti-SHP2 antibody (right). Scale bar represents 20 μm.
(B and C) Confluent HEK293T cells were transiently transfected with K-RASV12 vector (B) or SOS1-M269R-Flag vector (C). Transfected cells were then stained with anti-SHP2 antibody (green) and anti-RAS or anti-Flag antibody (red). Scale bar represents 20 μm. Box and whisker plots on the right show the ratio of the fluorescence intensity of the nuclear SHP2 to that of the cytoplasmic SHP2. Boundaries of the box: 25th and 75th percentiles, A line within the box: median; whiskers: 10th and 90th percentiles; filled circles: outliers. n = 34, Student's t test.
(D) Nuclear extracts prepared from proliferating or confluent AGS cells were immunoprecipitated with an anti-parafibromin (PF) antibody. The immunoprecipitates (IPs) were immunoblotted with an anti-phosphotyrosine (pY) or anti-parafibromin antibody.
Molecular Cell  , 45-56DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions