Molecular Cell 22, 217 230, April 21, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.molcel.2006.03.027 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, 1, * Juan Oses-Prieto, 2 Alma Burlingame, 2 Mike Fried, 1 and Frank McCormick 1 1 Cancer Research Institute and Comprehensive Cancer Center 2 Department of Pharmaceutical Chemistry University of California, San Francisco San Francisco, California 94143 Summary Ras family GTPases (RFGs) are known to share many regulatory and effector proteins. How signaling and biological specificity is achieved is poorly understood. Using a proteomics approach, we have identified a complex comprised of Shoc2/Sur-8 and the catalytic subunit of protein phosphatase 1 (PP1c) as a highly specific M-Ras effector. M-Ras targets Shoc2-PP1c to stimulate Raf activity by dephosphorylating the S259 inhibitory site of Raf proteins bound to other molecules of M-Ras or Ras. Therefore, distinct RFGs, through independent effectors, can regulate different steps in the activation of Raf kinases. Shoc2 function is essential for activation of the MAPK pathway by growth factors. Furthermore, in tumor cells with Ras gene mutations, inhibition of Shoc2 expression inhibits MAPK, but not PI3K activity. We propose that the Shoc2-PP1c holoenzyme provides an attractive therapeutic target for inhibition of the MAPK pathway in cancer. Introduction *Correspondence: pviciana@cc.ucsf.edu Ras genes code for small GTPases that act as molecular switches, cycling between an inactive GDP bound and an active GTP bound state. The exchange of GDP for GTP induces a conformational change that allows them to interact with their downstream effectors and carry out their multiple biological functions (Malumbres and Barbacid, 2003; Repasky et al., 2004). One of the best-characterized Ras effector pathways is the Raf-MEK-ERK kinase cascade, or MAPK pathway. Upon activation, Raf phosphorylates and activates MEK, which then phosphorylates and activates ERK. There are three Raf isoforms, Raf-1, A-Raf, and B-Raf, that are subject to very complex regulatory processes that are still not yet fully understood. Regulatory mechanisms include autoinhibition of the kinase domain by the N-terminal region, activation by Ras binding and recruitment to the membrane, stimulatory and inhibitory phosphorylation and dephosphorylation events, and interactions with other proteins (Wellbrock et al., 2004). Among the known proteins that interact with Raf kinases are proteins of the 14-3-3 family. These proteins bind as dimers to many target proteins through specific motifs and regulate their localization, stability, activity, and/or molecular interactions (Dougherty and Morrison, 2004). Raf proteins have two high-affinity phosphorylation-dependent 14-3-3- binding sites, with seemingly opposing functions; whereas binding to P-S621 is essential for activity, binding to P-S259 inhibits activity. In resting cells, Raf-1, the best-characterized Raf family member, is believed to exist in the cytosol in an inactive conformation by autoinhibitory interactions between the N-terminal regulatory and the C-terminal catalytic domains and by the binding of a 14-3-3 dimer that contacts the two phosphorylation sites: S259 and S621. The initiating event in Raf activation is believed to be Ras binding to the Ras binding domain (RBD) of Raf in the N-terminal regulatory region that recruits Raf to the plasma membrane, where other activating events take place. Shoc2/Sur-8 codes for a protein comprised almost entirely of leucine-rich repeats. It was independently identified in screens in C. elegans for genes that suppressed the clear phenotype of an activated form of EGL-15 FGF receptor (Selfors et al., 1998) and the multivulva phenotype caused by activated let-60 Ras (Sieburth et al., 1998). Genetic analysis indicates that it is likely to act downstream of let-60 Ras but upstream of, or parallel to, Raf to positively regulate Ras signaling. The human genome encodes a far greater number of serine/threonine (S/T) kinases than of S/T phosphatases. S/T phosphatases like PP1 and PP2A function in hetero-oligomeric complexes, interacting with a wide array of structurally unrelated regulatory subunits. Regulatory subunits confer both substrate targeting and specificity to the catalytic subunits. PP1 alone has more than 50 known regulatory proteins, and it is likely that there may be well over 100 regulatory subunits yet to be identified, allowing for the formation of holoenzymes to rival the number and function of the more abundant S/T kinases (Gallego and Virshup, 2005). Ras genes are members of a family of closely related GTPases that now includes 35 members (Colicelli, 2004). Many of these RFGs remain poorly characterized, although several do share many of the biological properties of prototypic Ras proteins (H-, K-, and N-Ras), including the ability to behave as oncogenes. There is a high degree of overlap in their sensitivity to both guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), upstream regulatory proteins that are involved in their activation (Ehrhardt et al., 2002; Quilliam et al., 2002). Therefore, some of these RFGs are likely to be activated in response to some of the same extracellular signals known to activate Ras. There is also overlap in the ability of some RFGs to interact with and regulate many of Ras 0 known effectors, with some RFGs binding to certain effectors, but not others, and doing so with variable affinities (Rodriguez-Viciana et al., 2004). Why several RFGs are simultaneously activated and how subsequent activation of their overlapping effector pathways leads to signaling and biological specificity is not understood. Tandem affinity purification (TAP) is an optimized method to purify protein complexes by the sequential use of two epitope tags (Rigaut et al., 1999). To shed more light on signal specificity by RFGs we have sought to define and identify patterns of effector interactions by
Molecular Cell 218 individual RFGs in an unbiased proteomics approach by using TAP methodology. Here, we report the identification of a complex comprised of Shoc2/Sur-8 and the catalytic subunit of protein phosphatase 1 (PP1c) as a highly specific effector of M-Ras that plays an essential role in MAPK pathway activation, both in response to growth factors and in tumor cell lines with Ras mutations. Shoc2 functions as a regulatory subunit of PP1c that upon M-Ras activation targets PP1c to specifically dephosphorylate the P-S259 inhibitory site of Raf kinases and stimulate Raf activity at specialized signaling complexes. Several effectors regulated by different GTPases of the Ras family can therefore simultaneously contribute to the activation of the same effector pathway, regulating different steps in the activation process of Raf kinases and contributing to modulate signal intensity. Results Identification of Shoc2 and PP1c as Effectors of M-Ras To identify M-Ras effectors, we stably expressed by retroviral infection a TAP-tagged M-Ras L81-activated mutant in HCT116 human colon carcinoma and HEK293 cell lines. After two affinity columns, protein complexes copurifying with the M-Ras bait were resolved by gel electrophoresis and specific protein bands were excised and analyzed by mass spectrometry. Several known and novel interacting proteins were identified (Figure 1A). Raf-1 and a PI3K complex comprised of the p110a catalytic subunit with p85a or p85b regulatory subunits are already known to interact with M-Ras (Rodriguez-Viciana et al., 2004). Hsp70 isoforms always appear on our TAP purifications with all the baits we have used. Hsp70 isoforms 5 and 8 (HSPA5 and HSPA8) were identified in complexes with M-Ras as was Rap1GDS1/SmgGDS, a protein known to interact in a GTP-independent manner with other Ras and Rho family GTPases (Takai et al., 1993). Interestingly, the a, b, and g isoforms of the catalytic subunit of PP1c also copurify with M-Ras. However, the most prominent band was identified as Shoc2/Sur-8. To validate the interactions and study the specificity of binding to other RFGs we performed immunoprecipitations (IP)-Western blot (WB) experiments in a variety of cell lines. There are currently no good antibodies to immunoprecipitate many of the endogenous RFGs. We therefore transfected tagged versions of the RFGs and detected interactions with endogenous proteins by WB after IPs. As shown in Figure 1B, both Shoc2 and PP1c bind specifically to M-Ras, but not to any of the other RFGs we tested. The interactions are GTP dependent, as shown by the preferred binding to the constitutively active L81 mutant than to the wild-type protein. Even under conditions of Shoc2 overexpression, M- Ras is the only RFG able to interact with Shoc2 (data not shown). Raf-1 and B-Raf, on the other hand, not only interact in a GTP-dependent manner with M-Ras but also interact with other RFGs, with Ras proteins (H-, N-, and K-Ras) showing the strongest interaction. Effectors of RFGs are defined biochemically as proteins that bind preferentially to the RFG when in its active, GTP bound conformation and whose binding is disrupted by mutations in the core effector domain. As shown in Figure 1C, the A48 core effector domain mutation of M-Ras dramatically ablates the ability of M-Ras to interact with both Shoc2 and PP1c. Thus, the activationdependent and effector domain-dependent binding is consistent with both Shoc2 and PP1c behaving as M- Ras effectors. Shoc2/Sur8 has been reported to interact directly with both Ras and Raf, allowing for formation of a ternary complex (Li et al., 2000). This is in clear contrast to our results, where we see binding to M-Ras, but not to Ras proteins (H-, N-, or K-Ras) or any other RFGs (Figure 1B). Furthermore, when we have used H-, N-, or K-Ras proteins as baits in TAP experiments, we have never identified Shoc2 in the purified complexes, whereas we readily detect Raf isoforms (data not shown). To further explore this discrepancy, we performed cotransfection experiments to compare binding of overexpressed M- Ras, N-Ras, and Raf-1 proteins to Gst-Shoc2. As shown in Figure 1D, M-Ras, but not N-Ras nor Raf-1, binds in a GTP-dependent manner to Shoc2. Conversely, when we cotransfect Gst-Raf-1 with myc-shoc2, M-Ras, and N-Ras, we detect strong binding to Raf-1 of both N- Ras and M-Ras, but not Shoc2 (data not shown). Thus, it appears that M-Ras-Shoc2 complexes do not contain Raf, and this is consistent with binding of different effectors to the same RFG being mutually exclusive. The interaction between the endogenous Shoc2, PP1c, and M-Ras can also be detected in vivo. Both PP1c and M-Ras can be detected in Shoc2 immunoprecipitates from serum-starved cells, and EGF stimulation (that leads to M-Ras activation, see later) further increases complex formation (Figure 1E). To further explore the relationship between the binding of Shoc2, PP1c, and Raf to M-Ras, we analyzed the ability of M-Ras to interact with PP1c and Raf-1 after inhibiting Shoc2 expression by RNA interference (RNAi). As shown in Figure 1F, three different Shoc2 sirnas, which strongly decrease Shoc2 expression levels, dramatically inhibit the ability of active M-Ras to interact with PP1c while having little effect on the interaction of Raf-1 with M-Ras or N-Ras. This is consistent with Shoc2 being necessary for M-Ras to interact with PP1c, but not with Raf. When using purified proteins in an in vitro binding assay, M-Ras can interact with immobilized Shoc2 only when in its GTP bound state. However, the interaction only takes place in the presence of purified PP1c (Figure 1G). Similarly, the interaction between PP1c and Shoc2 is strongly enhanced in the presence of M- Ras-GTP. When immobilized Gst-PP1c was used, similar results were obtained, with GTP bound M-Ras interacting with PP1c only in the presence of purified Shoc2 (data not shown). These data (see also Figure 3) strongly suggest that M-Ras, when in its active, GTP bound state, interacts in a ternary complex with Shoc2 and PP1c. Shoc2 Enhances M-Ras Activation of the Raf-ERK Pathway Shoc2 was identified in C. elegans as a gene playing a positive role in MAPK pathway activation. To address its effect on the ability of M-Ras to regulate the MAPK pathway, Shoc2 was cotransfected with active M-Ras in a variety of cell lines, and MAPK pathway activation
M-Ras-Shoc2-PP1c Complex Modulates Raf Activity 219 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 (2) or stimulated with 20 ng/ml EGF for 5 min. Lysates were immnunoprecipitated with a Shoc2 antibody (2 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 220 was assayed and compared to PI3K pathway activation by measuring ERK and Akt phosphorylation, respectively, with phospho-specific antibodies. M-Ras is considerably weaker than Ras proteins at activating the MAPK pathway, and its ability to induce detectable ERK activation varies depending on the type of assay used (Rodriguez-Viciana et al., 2004), the cell line, and the level of sensitivity of the assay (e.g., length of WB exposure) (Figure 2A). Shoc2 expression has no effect by itself but strongly stimulates the ability of M-Ras to activate ERK. In contrast, Shoc2 has no effect on the ability of M-Ras to activate the PI3K pathway, as measured by Akt phosphorylation. An M-Ras effector mutant is defective in activating both MAPK and PI3K pathways (Figure 2A). To assess at what level Shoc2 affects M-Ras activation of the MAPK pathway, we analyzed activation of Raf, the most upstream kinase of the MAPK cascade, by performing Raf kinase assays. M-Ras activates Raf-1, although not as strongly as N-Ras (Figure 2B). Shoc2 expression increases the ability of M-Ras to stimulate Raf-1 activity to levels comparable to those achieved by N-Ras. Raf activation by N-Ras is not affected by Shoc2 in this assay (see below). The ability of Shoc2 to stimulate M-Ras activation of ERK was also measured in the presence of the different Raf family members A-Raf, Raf-1, and B-Raf (Figure 2C). Coexpression of Shoc2 with A-Raf and Raf-1 had no detectable effect on ERK phosphorylation but, when coexpressed with active M-Ras, cooperated to give stronger ERK activation than M-Ras with either Raf or Shoc2 alone (see also panel for lysates in Figure 3A). In the case of B-Raf, which is known to have higher basal activity, overexpression of B-Raf alone is sufficient to lead to ERK phosphorylation that was further increased by either Shoc2 or M-Ras expression. Expression of all three proteins had no further detectable effect in this assay, suggesting that maximal activation had already been achieved. These data thus suggest that Shoc2 enhances ERK activation by M-Ras by stimulating the ability of M-Ras to activate all three Raf kinase isoforms. Binding of Shoc2 and PP1c to M-Ras Leads to Raf P-S259 Dephosphorylation We next investigated the mechanism by which Shoc2 stimulates Raf activation. M-Ras was cotransfected with tagged Raf kinases in the presence or absence of Shoc2, and interactions with M-Ras were analyzed. It has previously been reported that Shoc2 can function as a scaffold-type protein by facilitating an interaction between Ras and Raf (Li et al., 2000). As shown in Figure 1, however, we did not see any interaction between Shoc2 and N-Ras or Raf-1. Furthermore, GTP-dependent binding of M-Ras to Raf kinases was unaffected by overexpression of Shoc2. Likewise, Shoc2 binding to M-Ras was not affected by Raf expression. This, however, is in sharp contrast to the interaction of PP1c with M-Ras, which is greatly increased by Shoc2 expression (Figure 3A). Analysis of the phosphorylation state of Raf with phospho-specific antibodies revealed that although the amount of Raf bound to M-Ras was not changed, Shoc2 expression (and the corresponding increase in PP1c binding) correlated with a dramatic decrease in 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 (2) 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 (2) 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. the phosphorylation state of the S259 inhibitory site in Raf (Figure 3A). Remarkably, the decrease in phosphorylation appears to be specific to the P-S259 site, as Shoc2 overexpression has no effect on the phosphorylation state of the S338 site (Figures 3A and 3B). The dephosphorylation of the P-S259 site on Raf by Shoc2 expression is seen on the Raf-1 bound to M- Ras, but not the Raf bound to N-Ras, and correlates with a decrease of the 14-3-3 bound to the M-Ras bound Raf (Figure 3B). Strikingly, this effect is much more prominent on the population of Raf-1 molecules bound to
M-Ras-Shoc2-PP1c Complex Modulates Raf Activity 221 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 (2) 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 14-3-3 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 222 M-Ras. Only a significantly smaller decrease in P-S259 was observed when the phosphorylation state of the total Raf population was analyzed in Raf IPs (Figure 3B, compare lanes 1 and 2 of the left panel and lanes 3 and 4 of the right panel, see also Figure 3D). This decrease in the total Raf phosphorylation likely represents the percent of the total Raf that is bound to M-Ras under our experimental conditions. In clear contrast, both M-Ras and N-Ras induce a clear increase in S338 phosphorylation on the total Raf population that was unaffected by Shoc2 overexpression (Figure 3B, right). The genetic screens in C. elegans that originally identified Shoc2 also identified loss of function mutations in the protein (Selfors et al., 1998; Sieburth et al., 1998). When the corresponding mutations C260Y, D175N, E457K, and P510L were engineered in the human Shoc2, those mutants that were defective in M-Ras interaction (D175N and E457K) showed a concomitant decrease in their ability to stimulate PP1c binding to M-Ras and induce P-S259 dephosphorylation of M-Ras bound Raf (Figure 3C). The lack of any detectable effect of the C260Y and P510L mutations in our assay may be accounted for by differences between the C. elegans and human Shoc2 proteins or by the nature of our assay, where more subtle defects may be masked under the conditions of overexpression used. In support of the latter possibility, even the strongly reduced levels of Shoc2 E457K binding to M-Ras are able to stimulate ERK activation by M-Ras in our assay. All these effects are seen upon Shoc2 expression, but not when other molecules involved in MAPK pathway activation, such as Ksr or CNK1, are overexpressed (Figure 3D). S259A Mutation on Raf Isoforms Mimics Stimulatory Effect of Shoc2 The above data suggest that binding of a Shoc2-PP1c complex to active M-Ras leads to dephosphorylation of P-S259 on molecules of Raf recruited by nearby molecules of M-Ras. To further address the role of P-S259 dephosphorylation, we generated Raf mutants that can no longer be phosphorylated at that site. S259A mutation in Raf-1 is known to confer higher basal activity (Michaud et al., 1995). This mutant is further activated by M-Ras as well as N-Ras (Figure 4A and References). In fact, whereas M-Ras is weaker than N-Ras at activating the kinase activity of the wild-type Raf protein, it stimulates S259A Raf to the same levels as N-Ras. This stimulation, however, is no longer enhanced by Shoc2 expression (Figure 4A). The S259A mutation does not affect the ability of Raf-1 to interact with M-Ras. This is in clear contrast to the 89L mutation in the RBD of Raf-1, which strongly blocks binding to M-Ras (Figure 4B). Similarly, the equivalent mutation to S259A in B-Raf, S365A, does not affect interaction with M-Ras, whereas the 189L mutation in the B-Raf RBD domain strongly blocks it (Figure 4B, right). Mutation of other regulatory phosphorylation sites does not affect the ability of Shoc2 to induce P-S259 dephosphorylation (Figure 4B). As previously shown (Dhillon et al., 2003; Light et al., 2002), S259A Raf leads to increased ERK activation. This higher basal ERK activity can be further increased by active M-Ras but is no longer stimulated by Shoc2 expression (Figure 4C). The same effect was observed with the corresponding S214A mutation in A-Raf (Figure 4C). B-Raf has a higher basal kinase activity and upon overexpression induces detectable levels of ERK phosphorylation (Figure 4C, see also Figure 2C). In the case of B-Raf, expression of Shoc2 alone (or M- Ras) further increases the ability of B-Raf to induce ERK phosphorylation. S365A B-Raf has higher basal activity that is no longer stimulated by Shoc2. The data from Figure 4 are consistent with a model where M- Ras can activate Raf activity through several cooperating mechanisms, one of them being Shoc2 acting on the S259 inhibitory site. Mutation of that site makes the protein insensitive to the stimulatory effect of Shoc2 but still able to be activated by M-Ras, likely through direct interaction with the RBD domain. The loss of the inhibitory function of the P-S259 site (and subsequent activation) of Raf activity that is achieved by S259A mutation can cooperate with other mutations that mimic activating phosphorylation sites to further increase Raf activity (Figure 4D). For example, Y341D substitution in Raf-1 mimics Y341 phosphorylation by Src family kinases and leads to higher Erk activity. A S259A-Y341D double mutant leads to higher Erk activity than either of the single mutations by themselves. Similarly, substitutions of T491 and S494 in the activation segment for acidic residues (ED) also lead to an activated Raf kinase that cooperates with either S259A or S338D-Y341D (DD) substitutions to induce ERK phosphorylation. This is consistent with P-S259 dephosphorylation being one of many activating signals that Raf kinases are able to integrate to tightly modulate their level of activation and the corresponding signal output strength of the MAPK pathway. Shoc2 Plays a Critical Role in MAPK Pathway Activation by Growth Factors To address the contribution of Shoc2 to MAPK pathway activation by extracellular signals, we have taken advantage of sirna technology to inhibit Shoc2 function. Three different Shoc2 sirnas strongly inhibit the ability of EGF and bfgf to activate the MAPK pathway in 293T cells when compared to a control Lamin sirna. (Figure 5A). This inhibition is more pronounced at submaximal doses of growth factor, takes place in a sirna dosedependent manner (data not shown), and is observed in multiple cell lines (Figure 5B and data not shown). In contrast, Shoc2 knockdown has no effect on EGF-induced Akt phosphorylation (Figure 5B). Shoc2 therefore plays an essential contribution to ERK pathway activation by growth factors. M-Ras is coactivated with Ras proteins after EGF treatment and can be pulled down from lysates by using an RBD-activation assay (Figure 5C). Activation of exogenously expressed M-Ras by extracellular signals has been described before (Ehrhardt et al., 2004; Kimmelman et al., 2002), but to our knowledge, this is the first report of activation of the endogenous protein. The EGF-induced activation of endogenous M-Ras correlates with the increased binding to Shoc2 and PP1c shown in Figure 1E. Shoc2 Can Stimulate Raf-ERK Activation by Ras To address any possible contribution of Shoc2 to Erk activation by Ras, the three Ras isoforms were
M-Ras-Shoc2-PP1c Complex Modulates Raf Activity 223 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 (2) 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 (2) 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. expressed at varying levels in 293T cells in the presence or absence of overexpressed Shoc2. Ras proteins induce ERK phosphorylation in a dose-dependent manner that plateaus, after a defined level of Ras expression, when maximal ERK activation is achieved (Figure 6A). Importantly, there is a narrow window at very low levels of Ras where Shoc2 expression synergizes with the three Ras isoforms at activating ERK (Figure 6A). In our constructs, the three Ras isoforms express at different levels. Because our Ras genes do not carry the same 5 0 and 3 0 UTR sequences as their full-length mrnas, the physiological relevance of this observation is not clear. However, in all three cases, the pattern is the same and synergy with overexpressed Shoc2 is observed at the lowest levels of exogenous Ras, before Ras overexpression leads to maximal ERK activation. This result is consistent with the observations of Li et al. (2000). Shoc2 expression also strongly stimulates the much
Molecular Cell 224 and M-Ras indeed cooperated to stimulate the specific activity of the Raf bound to N-Ras. Thus, active M-Ras can target the Shoc2-PP1c complex to act not only on the Raf bound to M-Ras but also on the Raf bound to active Ras (and other RFGs) to further stimulate Raf specific activity. To study whether similar regulation of the Ras bound Raf by Shoc2 can be seen in vivo in the context of growth factor stimulation, we analyzed the Raf associated with Ras proteins after EGF stimulation by performing Ras IPs. The amount of Raf that associates with endogenous Ras after EGF treatment is not affected by Shoc2 sir- NAs (Figure 6D). However, when the phosphorylation state of the Ras bound Raf-1 is measured, inhibition of Shoc2 expression leads to a marked increase in S259 phosphorylation of the Ras bound Raf (Figure 6D). 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 (2 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. weaker ability of other RFGs, such as R-Ras, TC21, Rit, and Rin, to activate the MAPK pathway (data not shown). To explore how Shoc2 cooperates with Ras at stimulating ERK activity, we performed experiments to study the effect of active M-Ras on the ability of Shoc2 to regulate Raf activation by Ras. As previously shown, expression of Shoc2 leads to dephosphorylation in P- S259 of the Raf bound to M-Ras but has only a very small effect on the Raf bound to N-Ras. Expression of active M-Ras leads to more pronounced P-S259 dephosphorylation on N-Ras bound Raf, presumably by interacting with endogenous Shoc2. Coexpression of both active M-Ras and Shoc2 leads to a dramatic decrease in P- S259 dephosphorylation on the N-Ras bound Raf (Figure 6B). This is accompanied by a pronounced decrease in the amount of 14-3-3 proteins bound to Raf. Dephosphorylation is specific to the S259 site, as Shoc2 and M-Ras have no effect on the phosphorylation state of S338 on the N-Ras bound Raf. To study whether the effect seen on P-S259 dephosphorylation and 14-3-3 displacement is accompanied by an increase in Raf activity, Raf kinase activity was assayed in Ras pull downs from similar transfections as above to specifically analyze the activity of the Raf bound to active Ras. As shown in Figure 6C, Shoc2 Shoc2 Function Is Necessary for Basal ERK Activity in Tumor Cell Lines with Ras Mutations Shoc2 can stimulate the specific activity of Raf bound to Ras and can synergize with low levels of active Ras to activate ERK activity. We set to address any possible contribution of Shoc2 function to ERK activation in the context of endogenous active Ras in tumor cells with Ras gene mutations. We also addressed the contribution of the individual Ras isoforms to activation of both the MAPK and PI3K pathways in tumor cell lines that had either K-Ras or N-Ras mutations (Figure 7). Efficient inhibition of individual N-, K-, and H-Ras protein levels was achieved with sirnas against the respective Ras isoforms. Knockdown of K-Ras expression, but not N-Ras or H-Ras, strongly inhibits the basal levels of ERK phosphorylation in cell lines with K-Ras mutations such as MiaPaca2, ASPC1, A427, A549, and PANC1 cells (Figure 7). Similar results were seen in HPAF, SW480, and MDA-MB-231 cell lines (Figure S1 available in the Supplemental Data with this article online). Similarly, knockdown of K-Ras, but not N-Ras or H-Ras, inhibits the basal PI3K pathway activity detected in cells with mutant K-Ras, as seen by a decrease in the phosphorylation state of Akt in MiaPaca2, ASPC1, A427 (Figure 7), SW480, and HPAF cells (Figure S1). In cells with mutations in the N-Ras gene, such as SKMEL-2 melanoma cells (Figure 7F), inhibition of N- Ras expression, but not K-Ras nor H-Ras, inhibits basal ERK activation. Similar results were observed in other cell lines with mutant N-Ras, such as HepG2, HT1080, and RD cells where K-Ras knockdown had no effect (Figure S1). In a few cell lines, such as HT1080 and RD (Figure S1), H-Ras sirna also inhibited ERK activation. We do not know the biological relevance of this observation. Inhibition of Shoc2 expression decreases the level of ERK, but not Akt activity, in tumor cell lines with both K-Ras and N-Ras mutations (Figure 7 and Figure S1). The degree of inhibition varied between different cell lines. In some, such as ASPC1, A549, and PANC1 (Figure 7), Shoc2 knockdown inhibited ERK activity to the same extent as knockdown of the mutant Ras gene. In contrast to Shoc2, knockdown of Raf-1, B-Raf, or even in some instances Raf-1 and B-Raf had little effect on ERK activity.
M-Ras-Shoc2-PP1c Complex Modulates Raf Activity 225 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 (2). 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 (2) 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. The effect of Shoc2 sirnas on ERK activity in Ras mutant cell lines suggests that Shoc2 activity is needed to cooperate with mutant Ras in the activation of the MAPK pathway. To address this possibility, the phosphorylation state of the Raf associated with mutant Ras was analyzed in Ras IPs after transfections with control or Shoc2 sirnas. Inhibition of Shoc2 expression leads to an increase in S259 phosphorylation of the Raf associated with Ras that correlates with decreased ERK activity (Figure S2). This suggests that, even under conditions of serum starvation, there is some basal Shoc2 activity able to regulate the specific activity of the Raf associated with mutant Ras. It is worth noting that even under the resting conditions used some M-Ras can still be detected in complex with Shoc2 and PP1c (see Figure 1E), consistent with the existence of basal Shoc2 activity. In summary, in at least some tumor cells with Ras gene mutations, mutant Ras is involved in activating both the MAPK and PI3K pathways. Importantly, Shoc2 is required to cooperate with mutant Ras in the activation of the MAPK pathway. Discussion Using a proteomics approach, we have identified Shoc2 and PP1c as M-Ras-interacting proteins. Our data suggest that Shoc2 is a regulatory subunit of PP1c and that M-Ras interacts with a Shoc2-PP1c dimer to form a ternary complex. The interaction is extremely specific to M- Ras and not to any other RFGs. M-Ras and Shoc2 have a targeting function as well as a substrate specifying function for PP1c. M-Ras targets the Shoc2-PP1c complex to act on the Raf that is recruited to specific signaling complexes by other molecules of M-Ras, Ras, or other RFGs. In addition, M-Ras and Shoc2 have a substrate specifying function in that they lead to specific dephosphorylation of P-S259, but not P-S338, on Raf. Furthermore, Shoc2-PP1c recruitment does not affect stimulation of Akt phosphorylation by M-Ras, consistent
Molecular Cell 226 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). with the phosphatase activity of the complex acting to regulate Raf activation (but not other effectors) by targeting specifically the S259 inhibitory site. The crystal structure of PP1c bound to the myosin phosphatase targeting subunit 1 (MYPT1) illustrates how the binding of a regulatory subunit can regulate phosphatase specificity (Terrak et al., 2004). We predict that as shown for the myosin phosphatase holoenzyme, Shoc2 and M-Ras binding to PP1c will lead to a remodeling of the PP1 catalytic site, specifically reshaping it to target P-S259 in Raf. Shoc2-PP1c-induced S259 dephosphorylation leads to increased Raf activation by M-Ras and to increased activation of ERK. S259A Raf, a mutant that can no longer be phosphorylated, has an increased ability to activate ERK that is, however, no longer sensitive to the
M-Ras-Shoc2-PP1c Complex Modulates Raf Activity 227 stimulatory effect of Shoc2. This is seen for all three Raf kinases, indicating that this regulatory mechanism is conserved among the three family members and is consistent with P-S259 being the only Shoc2-PP1c target on Raf. M-Ras can contribute to Raf activation by several independent mechanisms. It recruits a Shoc2-PP1c complex to specialized signaling complexes to stimulate Raf activity by dephosphorylating S259. In addition, through direct binding to the RBD, M-Ras also can recruit Raf to the membrane where other Shoc2-independent activating events take place. The stimulation of S338 phosphorylation on total Raf, for example, is not affected by Shoc2-PP1c, and M-Ras still induces S338 phosphorylation of S259A Raf (data not shown). Furthermore, M-Ras can still stimulate the activity of S259A Raf and in fact does so more efficiently than wild-type Raf (see later). Therefore, M-Ras can utilize two different direct effectors, Raf and Shoc2-PP1c, to regulate the same effector pathway. Ras, through interactions with Raf and IMP (Matheny et al., 2004), can also use several effectors to regulate the MAPK pathway. It will be interesting to determine if this property of using multiple effectors to regulate the same pathway is shared by other RFGs and/or other effector pathways. PP2A is known to play a positive role in Raf-ERK activation, can interact with Raf-1, and has been proposed to dephosphorylate P-S259 (Abraham et al., 2000; Ory et al., 2003; Sieburth et al., 1999). However, its role in P-259 dephosphorylation was mainly based on the use of okadaic acid, a phosphatase inhibitor that also inhibits PP1. A C. elegans LIN-45 Raf mutant equivalent to S259A still requires the SUR-6 PR55/B regulatory subunit of PP2A, suggesting that SUR-6 must positively regulate Raf activity by a mechanism other than S259 dephosphorylation (Kao et al., 2004). PP2A may be preferentially involved in Raf activation by dephosphorylation of the five sites that are targeted by ERK as part of a negative feedback loop (Dougherty et al., 2005). PP2A also plays a positive role in ERK activation by regulation of Ksr function (Ory et al., 2003). All of the above are consistent with different phosphatase holoenzymes acting at different levels in the activation of the MAPK pathway, with Shoc2-PP1c being the main phosphatase targeting the Raf P-S259 inhibitory site. However, we cannot rule out that PP2A may also target P-S259 under some circumstances. P-S259 is part of a 14-3-3 binding site, and dephosphorylation correlates with loss of 14-3-3 binding to Raf. 14-3-3 binding, rather than phosphorylation, seems to be responsible for inhibition of Raf activity (Light et al., 2002), implying that dephosphorylation is part of the mechanism to displace bound 14-3-3. However, the relationship between S259 dephosphorylation and 14-3-3 displacement is not straightforward. Dephosphorylation correlates with loss of 14-3-3 from Raf bound to M-Ras and Ras, consistent with dephosphorylation leading to 14-3-3 displacement. However, the phosphate on the serine of peptides bound to 14-3-3 is deeply buried and would be inaccessible to phosphatases (Yaffe et al., 1997), suggesting that 14-3-3 displacement must precede dephosphorylation. In support of this, when looking at the total Raf population, Ras has a modest effect on 14-3-3 displacement without affecting S259 phosphorylation (Light et al. [2002] and our data). However, 14-3-3 can still be readily detected in the Raf bound to Ras (see Figures 3B and 6B). Expression of active M-Ras and Shoc2, on the other hand, leads to S259 dephosphorylation and very pronounced displacement of 14-3-3 from the Ras bound Raf (Figure 6B). Both Ras and 14-3-3 have secondary binding sites within the cysteine-rich domain (CRD) of Raf and thus may compete with each other for binding to the CRD (Daub et al., 1998; Rommel et al., 1996). This competition may be partly responsible but is not sufficient for efficient 14-3-3 displacement. We propose a model where upon activation, Ras (or other RFGs) will bind to the high-affinity RBD in Raf and then compete with 14-3-3 for binding to the secondary site on the CRD. Equilibrium will be reached, with 14-3-3 molecules getting displaced and binding back. A second signal, via Shoc2- PP1c, will lead to P-S259 dephosphorylation when the bound 14-3-3 is displaced and the buried phosphate exposed. This will prevent 14-3-3 from binding back and therefore strongly shift the equilibrium toward 14-3-3 displacement and Ras (or other RFGs) binding to the CRD. This in turn may fully relieve intramolecular inhibition, stabilize Raf interaction at the plasma membrane, and/or facilitate other activating events. The differential abilities of RFGs to interact with and activate Raf may be explained by their different abilities to compete with 14-3-3 for binding to the CRD. This would help explain the observation that M-Ras (and other RFGs) can activate S259A Raf more efficiently than wt Raf (and to a similar extent as Ras) (Light et al. [2002] and our data); their weaker ability to initiate 14-3-3 displacement is no longer required in S259A Raf. Regulation of Raf activity is clearly a very complex process, and P-S259 dephosphorylation is just one of many regulatory signals that Raf kinases are able to integrate to finely modulate their activity. For example, growth factor (GF) receptors can simultaneously activate multiple independent signals that regulate Raf activity, such as Ras-GTP binding, Shoc2-PP1c-mediated dephosphorylation of S259, Y341 phosphorylation by Src family kinases, and likely others. Different combinations of regulatory events will lead to different levels of Raf activity and spatially modulate MAPK pathway output at sites of receptor activation. Inhibition of Shoc2 expression by RNAi strongly inhibits ERK activation by growth factors, indicating that Shoc2 function plays a critical contribution to Raf activation. The inhibition observed is more pronounced at submaximal doses of growth factors. This is consistent with a model where synergy between combinations of Raf-activating signals is more pronounced at low levels of receptor activation, where a higher specific activity of the fewer Raf molecules recruited to sites of receptor activation is necessary to reach a determined threshold of pathway activity. It is worth noting that under physiological conditions, growth factors are much more likely to act at submaximal doses than at the saturating concentrations used in tissue culture. Shoc2 can also synergize with Ras to stimulate ERK activation. This, however, is only seen at low levels of Ras. Above a certain threshold of active protein, Ras can lead to maximal MAPK pathway activation. The same considerations of protein concentration versus
Molecular Cell 228 specific activity likely apply: at high levels of Ras-GTP at the membrane, higher amounts of recruited Raf with a lower specific activity may lead to the same level of signaling output as lower levels of Raf with higher specific activity. It is likely that these high levels of active Ras, although easily and routinely achieved in most experimental systems, may be supraphysiological. On the contrary, we speculate that the low levels of exogenous Ras that can synergize with Shoc2 more accurately reflect physiological levels of active Ras. The requirement for Shoc2 for ERK activation seen in tumor cells with Ras mutations supports this possibility (see below). M-Ras can target Shoc2-PP1c to dephosphorylate the Raf bound to Ras-GTP and stimulate its specific activity. Ras proteins and M-Ras, closely related RFGs, can therefore act in parallel, simultaneously contributing to the activation of the same effector pathway. It will be interesting to determine if similar crosstalk between other RFGs is found in the regulation of other effector pathways. M-Ras shares broadly overlapping sensitivities to the GEFs and GAPs that regulate Ras activation. It is likely that, depending on the cell type and the type and intensity of the extracellular signal, different sets of GEFs and GAPs will lead to the activation of different sets of RFGs at different membrane microdomains (Bivona et al., 2003; Ehrhardt et al., 2004; Perez de Castro et al., 2004). This, in turn, will result in different combinations of Raf-activating signals cooperating to modulate MAPK pathway intensity at these membrane microdomains (as well as the activation of different sets of effector pathways). This type of cooperation between closely related family members may, at least partly, account for the evolutionary conserved diversity in RFGs. It will be interesting to see if Shoc2 is found overexpressed or mutated in human tumors, providing yet another mechanism of MAPK pathway upregulation in cancer. Similarly, M-Ras, with its ability to activate both the MAPK and PI3K pathways, is an excellent candidate gene to be found mutated in human cancer. In cell lines derived from a wide variety of tumor types with activating K-Ras or N-Ras mutations, inhibition of the mutant Ras isoform strongly inhibits ERK activity. Importantly, in many cases where basal AKT phosphorylation was detected, inhibition of mutant Ras also leads to inhibition of AKT activity. Ras activation of MAPK and PI3K pathways has been observed in many systems. However, they most often involved ectopic Ras overexpression. There is also indirect evidence that the ability of Ras to activate both MAPK and PI3K pathways is critical in human tumorigenesis (Tsao et al., 2004). On the other hand, expression at physiological levels of knockin mutant K-Ras in MEFs did not detectably induce ERK or AKT activation (Tuveson et al., 2004). To our knowledge, our results provide the first report where endogenous mutant Ras in human tumor cells has been shown to be involved in the activation of both the MAPK and PI3K pathways. Strikingly, inhibition of Shoc2 expression also inhibits ERK activity in cells with mutant Ras. This suggests that mutant Ras, at the low protein levels that are likely found under physiological conditions, critically relies on Shoc2-PP1c function to cooperate in the activation of Raf. This is consistent with the ability of Shoc2 to synergize with low levels of ectopic active Ras in activating ERK (Figure 6A) and with the ability of Shoc2 to regulate the specific activity of the Ras bound Raf, even under resting conditions (Figure 6C and Figure S2). In C. elegans, Shoc2/SUR8, unlike Raf/LIN45 or ERK/ SUR1, is not essential for organ development, but loss of Shoc2 function strongly reduces the penetrance of the active Ras phenotypes (Moghal and Sternberg, 2003). In humans, the MAPK pathway plays a critical role in the development of many malignancies and is an important target for drug discovery (Sebolt-Leopold and Herrera, 2004). Ras activation of ERK in human tumors relies on Shoc2 function to cooperate in the activation of Raf. Similarly, Shoc2 is critically required for ERK activation by receptor tyrosine kinases. Consequently, the Shoc2-PP1c holoenzyme may provide an attractive target for drug development for inhibition of the MAPK pathway. It has proved difficult to design inhibitors against the exposed catalytic pocket of free PP1c. However, the crystal structure of the MYPT1-PP1c complex illustrates how regulatory subunits can modulate phosphatase specificity by reshaping the catalytic site of the holoenzyme. Thus, it is possible that phosphatase holoenzymes, with their structurally distinct catalytic pockets, in addition to being more specific targets, may prove more amenable to drug inhibition. In summary, we have identified a Shoc2-PP1c holoenzyme as a highly specific effector of M-Ras involved in Raf activation. M-Ras can target a Shoc2-PP1c complex to stimulate the activity of Raf kinases bound to other molecules of active M-Ras and/or Ras (and other RFGs). Therefore, several independent, direct effector inputs from different RFGs can simultaneously contribute to Raf/MAPK pathway activation by coordinating the assembly of specialized signaling complexes and regulating the specific activity of Raf kinases in those complexes. Furthermore, Shoc2 function is essential for MAPK pathway activation by GFs and mutant Ras. We propose that Shoc2 may provide an attractive new target of therapeutic intervention in the treatment of the many human malignancies with up-regulated MAPK activity. Experimental Procedures Constructs, antibodies, TAP purifications and mass spectrometry analysis are described in detail in the Supplemental Data. In Vitro Interaction Assays His-M-Ras, His-PP1cA, and Gst-PP1cA were expressed and purified from bacteria. Flag-Shoc2 and Gst-Shoc2 were purified from 15 3 15 cm 2 dishes of transiently transfected 293T cells. For detailed in vitro interaction protocol, see the Supplemental Data. Transient Transfections and Interaction Assays Cells were seeded the day before in 6-well dishes and transfected with 2 mg total plasmid DNA and 5 ml of Lipofectamine 2000 (Life Techonologies) according to manufacturer s instructions. If appropriate, cells were serum-starved (0.5% FBS) the next day and lysed 2 days after transfection in 350 ml of TNM lysis buffer. Gst-tagged proteins were pulled down from cleared lysates with glutathione Sepharose beads (Amersham), washed three times with 1% TX100- TNM, drained, and resuspended in sample buffer as described in detail in (Rodriguez-Viciana and McCormick, 2006). Bound proteins were detected by WB with ECL (Amersham) or Visualizer (Upstate).