1. Volume 22, Issue 2, Pages 217-230 (April 2006)
A Phosphatase Holoenzyme Comprised of Shoc2/Sur8 and the Catalytic Subunit of PP1 Functions as an M-Ras Effector to Modulate Raf Activity 
Pablo Rodriguez-Viciana, Juan Oses-Prieto, Alma Burlingame, Mike Fried, Frank McCormick 
Molecular Cell 
Volume 22, Issue 2, Pages (April 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Identification of Shoc2 and PP1c as M-Ras Effectors
(A) TAP purification of M-Ras-interacting proteins in HEK 293 cells. Protein complexes copurifying with TAP-tagged M-Ras were resolved by PAGE and proteins identified by mass spectrometry. TAP-tagged Noey2, another Ras family GTPase, was used in parallel purifications as a specificity control.
(B) M-Ras, but not other RFGs, interacts with endogenous Shoc2 and PP1c in a GTP-dependent manner. Gst-tagged versions of wild-type (wt) and activated mutants of RFGs were transfected into 293T cells and purified with glutathione beads. Interacting endogenous proteins were detected by Western blot (WB) with the specific antibodies indicated on the right-hand side. 1/100 of the lysate was also loaded as a reference.
(C) Interaction is effector domain dependent. Interactions of transfected Gst-M-Ras-activated mutant (L81) or an effector domain mutant in an activated background (L81, A48) with endogenous Shoc2 and PP1c were determined as in (B). Gst-GFP was used as a control.
(D) Shoc2 interacts with active M-Ras, but not Ras or Raf. Gst-Shoc2 was cotransfected in 293T cells with activated myc-M-Ras or myc-N-Ras mutants and either myc-Raf-1 or empty vector, and myc-tagged proteins interacting with purified Gst-Shoc2 were detected by WB with myc antibody.
(E) In vivo interaction between endogenous proteins. HeLa cells were serum starved overnight and either left untreated (−) or stimulated with 20 ng/ml EGF for 5 min. Lysates were immnunoprecipitated with a Shoc2 antibody (− and + lanes) or a GFP control antibody (c lane). Immunoprecipitates and 1/100 of the lysate were probed with the indicated antibodies.
(F) Shoc2 is required for M-Ras to interact with PP1c. Gst-M-Ras or Gst-N-Ras (wild-type and activated mutants) were transfected into 293T cells a day after transfection with either lamin sRNA (L) or three different siRNAs against Shoc2 (1, 2, and 3). After purification of Gst proteins on glutathione beads, interactions with endogenous Shoc2, PP1c, or Raf-1 were detected with specific antibodies as above.
(G) In vitro interactions: trimeric complex formation between Shoc2 and PP1c is dependent on M-Ras-GTP. Purified M-Ras was bound to either GDP or GMP-PMP and added to immobilized Gst-Shoc2 in the presence or absence of PP1c. After washing, bound M-Ras and PP1c were detected by WB. 1/200 and 1/66 of the amount of proteins used in the assay were run alongside as a reference.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Shoc2 Stimulates M-Ras Activation of Raf-ERK Pathway
(A) Shoc2 stimulates M-Ras activation of ERK, but not Akt, in different cell lines. GFP-myc, a myc-M-Ras-activated mutant (L81), or a myc-M-Ras effector domain mutant in an activated background (L81, A48) was transfected into different cell lines together with empty vector (−) or myc-hoc2 (+). P-Akt and P-ERK were detected on whole-cell lysates by WB with phospho-specific antibodies.
(B) Shoc2 increases M-Ras stimulation of Raf kinase activity. Flag-Raf-1 was transfected in 293T cells with empty vector or active mutants of M-Ras or N-Ras in the presence or absence of Shoc2. Raf activity was measured on Flag immunoprecipitates in a coupled MEK-ERK kinase assay with MBP as the final substrate. Error bars represent the standard deviation of duplicates, representative of at least three different experiments.
(C) Shoc2 stimulates activation of ERK through all three Raf kinase isoforms. Shoc2 (+) or empty vector (−) was cotransfected in 293T cells with activated M-Ras and wild-type versions of A-Raf, Raf-1, or B-Raf or empty vector as indicated and Erk phosphorylation analyzed on cell lysates.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Mechanism of Action of Shoc2
(A) Shoc2 expression increases PP1c binding to M-Ras and leads to P-S259 dephosphorylation of Raf. Wt or L81-activated mutant of Gst-M-Ras was cotransfected in 293T cells with Flag-Raf isoforms and either empty vector (−) or myc-Shoc2 (+). Gst-M-Ras was purified with glutathione beads and complexes resolved by PAGE. Interacting Raf and Shoc2 were detected with Flag and myc antibodies, respectively, and endogenous PP1c with a PP1c antibody. Filters were reprobed with phospho-specific antibodies against P-S259 and P-S338 on Raf-1. Erk phosphorylation was measured on cell lysates by WB.
(B) Shoc2 induces selective dephosphorylation of P-S259 on the pool of Raf bound to M-Ras and leads to displacement from Raf. 293T cells were transfected with Flag-Raf-1 and either Gst-M-Ras or Gst-N-Ras in the presence or absence of Shoc2. Two days later, 3/4 of the lysate was purified with glutathione beads to look at proteins associated with either active Gst-N-Ras or Gst-M-Ras (left). The remaining 1/4 of the lysate was immunoprecipitated with Flag beads to measure the total Raf population (right). Proteins and phosphorylation sites were detected with the antibodies indicated on the right-hand side as in (A).
(C) The ability of Shoc2 mutants to interact with M-Ras correlates with M-Ras binding to PP1c and dephosphorylation of bound Raf on P-S259. Myc-tagged versions of Shoc2 wt or Shoc2 point mutants C260Y, D175N, E457K, and P510L were cotransfected with Flag-Raf and activated Gst-M-Ras and bound proteins purified and detected as above. Expression levels of tagged proteins were also measured on lysates. ERK activation was measured with a phospho-specific antibody in lysates of parallel transfections without Flag-Raf.
(D) Increase in M-Ras binding to PP1c and P-S259 dephosphorylation of bound Raf is specific to Shoc2, but not Ksr or CNK1. Gst-M-Ras was cotransfected in 293T cells with Flag-Raf-1 and either empty vector or myc-tagged Ksr, CNK1, or Shoc2. Proteins bound to M-Ras and phosphorylation status of either M-Ras bound or total Raf were measured as in (B). Expression of myc-tagged proteins was detected on lysates with myc antibody.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
“S259A” Mutation on Raf Isoforms Mimics the Stimulatory Effect of Shoc2 and Cooperates with M-Ras Binding and Other Phosphorylation Sites to Stimulate Raf Activity
(A) S259A Raf-1 has higher kinase activity that is no longer stimulated by Shoc2. Wt or S259A Flag-Raf versions were cotransfected in 293T cells with M-Ras L81 or N-Ras V12 and either Shoc2 or empty vector and Raf activity measured on flag-immunoprecipitates. Error bars represent the standard deviation of duplicates, representative of at least three different experiments.
(B) Interaction of Raf-1 and B-Raf mutants with active M-Ras and sensitivity to Shoc2 function. Flag-tagged Raf-1 and B-Raf wt and mutant versions were cotransfected with Gst-M-Ras L81 and either empty vector (−) or Shoc2 (+). Gst-M-Ras bound Raf was measured on glutathione pull downs and total Raf on cell lysates. Raf-1 mutants used were 259A, 89L, 338A-341F (AF), 338D-341D (DD), 491A-494A (AA), and 491E-494D (ED). B-Raf mutants used were 365A, 189L, 598A-601A (AA), and 598E-601D (ED). Note that the anti-P-S259 antibody also recognizes the equivalent P-S365 site in B-Raf.
(C) Effect of S259A mutation on Raf isoforms' ability to activate ERK. Wt or S214A A-Raf or S259A Raf-1 or S365A B-Raf was cotransfected with activated M-Ras-L81 and either empty vector (−) or Shoc2 (+). ERK phosphorylation was measured on cell lysates with a phospho-specific antibody.
(D) Cooperation of S259A with other phosphorylation site mutations on Raf-1 activity. Flag-tagged Raf-1 mutants were transfected in 293T cells and ERK activation measured on lysates as above. Raf-1-AF represents S338A-Y341F, DD represents S338D-Y341D, and ED is T491E-S494D.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Shoc2 Plays a Critical Role in the Activation of ERK Activity by Growth Factors
(A) Shoc2 RNAi inhibits ERK activation by growth factors. 293T cells were transfected with siRNAs against lamin or three different target sequences of Shoc2. Two days later cells were serum starved overnight. On day 3, cells were stimulated with 0.2 ng/ml EGF (a), 1 ng/ml bFGF (b), or 5 ng/ml bFGF (c) for 8 min. Total ERK, phospho-ERK, and Shoc2 levels on cell lysates were analyzed by WB with specific antibodies.
(B) Shoc2 siRNAs inhibit EGF-induced ERK activation, but not Akt activation, in multiple cell lines. MCF10A, BT549, and HBL-100 cells were transfected with siRNAs against Lamin (L), two different Shoc2 siRNAs (1 and 2), or a control nonsilencing siRNA (N) at 80 nM as above. Cells were stimulated with 1 ng/ml EGF (except the first lane) and lysates analyzed by WB with the indicated antibodies.
(C) Activation of endogenous M-Ras by EGF. Lysates from untreated or EGF-stimulated Hela cells (− and +, respectively) were incubated with immobilized GST- RBD of RalGDS and bound M-Ras and Ras proteins (H-, N-, and K-Ras) detected with M-Ras and Pan-Ras antibodies, respectively.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Relationship between Shoc2 and Ras in the Activation of Raf-ERK Pathway
(A) Shoc2 stimulates Ras activation of ERK at low Ras levels. Myc-tagged GFP or serial dilutions of myc-tagged V12 K-Ras, N-Ras, and H-Ras plasmids were transfected into 293T cells with Shoc2 (+) or empty vector (−). ERK phosphorylation and myc-tagged proteins were detected on lysates with phospho-ERK and myc antibodies, respectively.
(B) M-Ras and Shoc2 cooperate to dephosphorylate P-S259 on Raf bound to active Ras. Flag-Raf was cotransfected in 293T cells with Gst-N-Ras V12 or Gst-M-Ras L81 and myc-tagged V12 N-Ras, L81 M-Ras, and Shoc2 as indicated. Gst-Ras and Gst-M-Ras were purified on glutathione beads and bound proteins analyzed by WB with the indicated antibodies.
(C) M-Ras and Shoc2 increase the specific activity of Raf bound to active Ras. The kinase activity of Raf-1 associated with Gst-Ras V12 proteins or empty vector (−) was measured by performing Raf kinase assays on Gst-pull downs from transfections as in (B). Error bars represent the standard deviation of duplicates, representative of at least three different experiments.
(D) Shoc2 is required for growth factor-induced S259 dephosphorylation of endogenous Ras bound Raf. HeLa cells were transfected with siRNAs against Lamin (L) or two different siRNAs against Shoc2 (1 and 2). Cells were stimulated with 10 ng/ml EGF for 5 min, and endogenous Ras (H- and K-Ras) were immunoprecipitated with the 238 anti-Ras antibody. In lane N, lysates from a nonsilencing siRNA transfection were immunoprecipitated with a control HA antibody. Immunoprecipitates and cell lysates were probed with the indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
RNAi of Mutant Ras Isoform Inhibits ERK and AKT Activity in Cell Lines with Ras Mutations, whereas RNAi of Shoc2 only Inhibits ERK
siRNAs were transfected into various cell lines as indicated at 80 nM. Shoc2-1, -2, and -3 represent three different Shoc2 siRNAs. 1 + B represents combined Raf-1 and B-Raf siRNAs at 80 nM each. Two days after transfection, cells were serum starved overnight. P-ERK, P-Akt, and targeted proteins were detected in whole-cell lysates with the indicated antibodies. Cell line name, tumor type origin, and Ras gene mutation are as follows: Mia Paca2, pancreatic carcinoma, and K-Ras (A); ASPC1, pancreatic carcinoma, and K-Ras (B); A427, nonsmall cell lung carcinoma (NSCLC), and K-Ras (C); A549, NSCLC, and K-Ras (D); PANC1, pancreatic carcinoma, and K-Ras (E); and SKMEL2, melanoma, and N-Ras (F).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions