Protein Phosphatases 1 and 2A Promote Raf-1 Activation by Regulating 14-3-3 Interactions

Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions

Montserrat Jaumot1 and John F Hancock*,1

1Laboratory of Experimental Oncology, Department of Pathology, University of Queensland Medical School, Herston Road, Queensland 4006, Australia

Raf-1 activation is a complex process which involves nucleus where it phosphorylates and activates tran- plasma membrane recruitment, phosphorylation, protein- scription factors which mediate diverse cellular re- protein and lipid-protein interactions. We now show that sponses, including proliferation, dierentiation, PP1 and PP2A serine-threonine phosphatases also have oncogenic transformation, cell cycle arrest or senes- a positive role in Ras dependent Raf-1 activation. cence (Marshall, 1995b; Woods et al., 1997; Zhu et al., General serine-threonine phosphatase inhibitors such 1998). sodium ¯uoride, or û-glycerophosphate and sodium In mammals, three closely related Raf isoforms (Raf- pyrophosphate, or speci®c PP1 and PP2A inhibitors 1 (or c-Raf), A-Raf and B-Raf) have been identi®ed including microcystin-LR, protein phosphatase 2A in- which exhibit regulatory similarities and dierences hibitor I1 or protein phosphatase inhibitor 2 all abrogate (Marais et al., 1997; Mason et al., 1999; Sithanandam H-Ras and K-Ras dependent Raf-1 activation in vitro.A et al., 1990). The Raf-1 molecule (human c-Raf; 648 critical Raf-1 target residue for PP1 and PP2A is S259. aa) can be divided into two functional domains, the Serine phosphatase inhibitors block the dephosphoryla- amino-terminal regulatory domain and the carboxy- tion of S259, which accompanies Raf-1 activation, and terminal kinase domain. The regulatory domain Ras dependent activation of mutant Raf259A is contains Conserved Region 1 (CR1, residues 62 ± 194) relatively resistant to serine phosphatase inhibitors. and CR2 (residues 254 ± 269) and the kinase domain is Sucrose gradient analysis demonstrates that serine represented by the CR3 (residues 330 ± 627). Although phosphatase inhibition increases the total amount of Raf-1 regulation is a complex process with many of the 14-3-3 and Raf-1 associated with the plasma membrane exact mechanisms involved still under intense investiga- and signi®cantly alters the distribution of 14-3-3 and tion, the sequence of certain critical events that lead to Raf-1 across dierent plasma membrane microdomains. Raf-1 activation is now clear. The initial event is Ras These observations suggest that dephosphorylation of dependent recruitment of inactive Raf-1 from the S259 is a critical early step in Ras dependent Raf-1 cytosol to the plasma membrane (Leevers et al., 1994; activation which facilitates 14-3-3 displacement. Inhibi- Stokoe et al., 1994; Traverse et al., 1993; Wartmann tion of PP1 and PP2A therefore causes plasma and Davis, 1994). The interaction between the Ras- membrane accumulation of Raf-1/14-3-3 complexes binding domain (RBD) of Raf-1 (residues 55 ± 131) which cannot be activated. Oncogene (2001) 20, with the switch 1 region of activated Ras-GTP is 3949 ± 3958. necessary for membrane recruitment. A second Ras binding domain, the Cysteine-Rich Domain (CRD, Keywords: Ras; Raf; 14-3-3; phophatases; plasma residues 130 ± 184) is also involved in Raf-1 activation, membrane although it is not required for membrane recruitment (Drugan et al., 1996; Luo et al., 1997; Mineo et al., 1997; Roy et al., 1997). Introduction Once Raf-1 has been localized at the plasma membrane, additional mechanisms complete full acti- The serine/threonine kinase Raf plays a critical role in vation. These include serine, threonine and tyrosine signal transduction as a downstream eector of Ras. phosphorylation, phospholipid binding, interactions Raf phosphorylates and activates MAP kinase kinase with 14-3-3, and possibly dimerization. Recently, it (MAPKK or MEK) which in turn activates mitogen- has been reported that phosphorylation of S338 and activated protein kinase (MAPK or ERK) (Marais and Y341 co-operate for Raf-1 activation (Mason et al., Marshall, 1996; Marshall, 1995a; Morrison and Cutler, 1999): the Ras-Raf-1 interaction and plasma membrane 1997). As a consequence, MAPK translocates to the localization are necessary for successful phosphorylation of these residues. The region which contains S338 and Y341 is located at the N-terminus of the catalytic CR3 domain and has been denoted the Negative- *Correspondence: JF Hancock Received 22 December 2000; revised 6 April 2001; accepted 9 April charge regulatory region or N-region (Mason et al., 2001 1999). The kinases Pak1 and Pak3 may be responsible Identification of PP2A and PP1 as Raf-1 regulators M Jaumot and JF Hancock 3950 for S338 phosphorylation (Chaudhary et al., 2000; the Raf-1 kinase domain in a conformation that is King et al., 1998; Sun et al., 2000) whereas several competent for activation by Ras. A concept which is kinases have been implicated in tyrosine phosphoryla- consistent with the data of Tzivion et al. (1998) and tion (Fabian et al., 1993; Marais et al., 1995; Xia et al., our own studies (Roy et al., 1998). There remains some 1996). Since Pak1 and Pak3 are activated by the debate as to whether 14-3-3 must remain bound to plasma membrane-localized small GTP-binding pro- activated Raf-1, again via S621 to maintain the active teins Cdc42 and Rac, there is potential for crosstalk conformation, although diering experimental proto- between Ras and Rho GTPases at the level of Raf-1 cols may underlay some of the apparently con¯icting activation. A-Raf appears to be regulated in a similar data (McPherson et al., 1999; Thorson et al., 1998; manner to Raf-1, but the N-region of B-Raf has an Tzivion et al., 1998; Yip-Schneider et al., 2000). Either aspartate in place of the equivalent tyrosine residue way it is an attractive hypothesis that re-phosphoryla- (Y341) in Raf-1, and phosphorylation of S445 tion of S259 and S621 at the cell membrane, associated (equivalent to S338 in Raf-1) is constitutive (Mason with the switching o of kinase activity, permits 14-3-3 et al., 1999). Additional kinases have been reported to to rebind to now inactive Raf-1 and sequester it to the phosphorylate and participate in Raf-1 regulation: cytosol. protein kinase C increases Raf-1 activity by phosphor- Despite the increasing database on Raf-1 phosphor- ylating S499 and S497 (Carroll and May, 1994; Kolch ylation sites and the identi®cation of some of the et al., 1993; Schonwasser et al., 1998). In contrast, kinases involved, the de-phosphorylation mechanisms protein kinase A inhibits Raf-1 activation through the remain largely obscure. The aim of this study was to phosphorylation of S43 (Sidovar et al., 2000). T269 is investigate whether serine-threonine phosphatases the target of the ceramide activated protein kinase could potentiate Raf-1 kinase activation. We report (Yao et al., 1995). the use of an in vitro Raf-1 activation assay to identify Phosphorylated serines, S259 and S621, and a non- serine-threonine phosphatases involved in Raf-1 activa- phosphorylated domain within the CRD have been tion. We show that PP1 and PP2A contribute to Raf-1 identi®ed as 14-3-3 binding sites (Clark et al., 1997; activation by dephosphorylating S259. Inhibiting these Michaud et al., 1995; Muslin et al., 1996). The role of phosphatases blocks Raf-1 activation and results in a 14-3-3 in Raf-1 regulation is controversial and several redistribution of Raf-1 and 14-3-3 into dierent plasma models have been proposed. In a previous study we membrane microdomains, coupled with defective demonstrated that 14-3-3 must be complexed with Raf- recycling of Raf-1 from the plasma membrane back 1 for ecient membrane recruitment and activation by to the cytosol. Ras, but that 14-3-3 is completely displaced from active Raf-1 at the plasma membrane (Roy et al., 1998), a model consistent with recent observations in mast cells Results (Cissel and Beaven, 2000). More recently we proposed that the interaction between the Raf-1 CRD and the In vitro activation of Raf-1 is inhibited by general membrane lipid phosphatidylserine (PS) destabilizes the serine-threonine phosphatase inhibitors Raf-1/14-3-3 complex and may facilitate displacement of 14-3-3 at the plasma membrane (McPherson et al., In order to study the putative role of serine-threonine 1999). Raf-1 complexed with 14-3-3 is protected from phosphatases in the regulation of Raf-1, we ®rst tested dephosphorylation by serine and tyrosine phosphatases how the presence or absence of general serine-threonine (Dent et al., 1995), therefore as a consequence of 14-3- phosphatase inhibitors aected Raf-1 activation. We 3 displacement at the plasma membrane S259 and S621 used an assay which reproduces in vitro the Ras- may become accessible to serine phosphatases. This is dependent membrane recruitment and activation of important because phosphorylation of both these Raf-1 that are evident in vivo (Roy et al., 1998). serines negatively regulates Raf-1 activity. For exam- Membranes from COS cells expressing activated H- ple, mutation of S259 to alanine generates a constitu- RasG12V, or K-RasG12V and cytosol from COS cells tively active kinase (Clark et al., 1997; Michaud et al., expressing epitope tagged FLAGRaf-1 were prepared 1995; Rommel et al., 1996) and phosphorylation of in the presence or absence of sodium ¯uoride, a general S259 by Akt downregulates Raf-1 activity (Rommel et serine-threonine phosphatase inhibitor. The membranes al., 1999; Zimmermann and Moelling, 1999). S621 is a were incubated with the cytosol at 308C for 20 min and Raf-1 autophosphorylation site (Michaud et al., 1995; were re-isolated by centrifugation. Ras expression and Morrison et al., 1993; Thorson et al., 1998), although Raf-1 recruitment to the membrane fractions were trans-phosphorylation of this site by protein kinase A measured by quantitative Western blotting and the Raf downregulates Raf-1 kinase activity (Hafner et al., activity associated with the membranes was measured 1994; Mischak et al., 1996). Mutational analysis of in a coupled MEK-ERK assay. Interestingly, the S621 is problematic because mutation to any other presence of sodium ¯uoride during the in vitro residue causes a complete loss of kinase activity incubation signi®cantly inhibited Raf-1 activation by (Hafner et al., 1994; Morrison et al., 1993), despite both H-RasG12V and K-RasG12V-containing mem- S621 being outside the Raf-1 kinase domain. Thorson branes (Figure 1a,b). The eect on K-Ras mediated et al. (1998) have argued that the interaction of 14-3-3 Raf-1 activation was consistently less than that on H- with phosphorylated serine 621 is essential to maintain ras mediated Raf-1 activation.

Oncogene Identification of PP2A and PP1 as Raf-1 regulators M Jaumot and JF Hancock 3951

Figure 1 Sodium ¯uoride inhibits the in vitro activation of Raf-1 in cell membranes containing H-RasG12V or K-RasG12V. (a,b) Membrane fractions from COS cells transfected with H-RasG12V, K-RasG12V or with empty EXV plasmid (V) were mixed on ice with cytosol prepared from COS cells expressing FLAG Raf-1. The samples were incubated at 308C for 20 min and membranes were reisolated by centrifugation. These steps were carried out in presence or absence of 25 mM sodium ¯uoride (NaF) (general serine-threonine phosphatase inhibitor). The Raf activity associated with the membranes was then measured in a coupled MEK- ERK assay as described in Materials and methods. Raf activities have been expressed relative to the Raf activity measured in the incubation without sodium ¯uoride (=1). (c) Western blots showing the amount of Raf-1 and Ras bound to the membranes after these incubations. (d) To study endogenous Raf-1 activation, membrane fractions from COS cells transfected with H-RasG12V were incubated with buer A in the presence or absence of 25 mM sodium ¯uoride (NaF) instead of Raf-1 cytosol at 308C for 20 min. The Raf activity and the amount of Raf-1 and Ras associated with the membranes were measured as described above. Raf kinase activities have been expressed relative to the Raf activity measured in the incubation without sodium ¯uoride (=1). These data are representative of experiments that were replicated six times with similar results

We next investigated whether sodium ¯uoride would (1.5+0.82-fold, mean of six dierent experiments) aect the activation of endogenous Raf-1 pre-recruited (Figure 1d). Thus incubation with sodium ¯uoride in onto the membranes by activated Ras prior to cell the conditions of this assay signi®cantly decreases the harvesting and fractionation. To this end H-RasG12V speci®c activity of membrane associated Raf-1 and containing membranes were incubated in vitro with increases the amount of Raf-1 recruited onto the cell buer instead of Raf-1 cytosol at 308C for 20 min, re- membranes. isolated by centrifugation and assayed. Figure 1d To discern whether Raf-1 activation could be shows that Raf activity is readily detected when the inhibited by other general serine-threonine phosphatase incubation is carried out in the absence of sodium inhibitors, we repeated the assays described in Figure 1 ¯uoride, but Raf activity is almost completely abol- in the presence or absence of b-glycerophosphate and ished in the presence of sodium ¯uoride. To better sodium pyrophosphate. As shown in Figure 2a, Raf understand the inhibitory mechanism, we examined if speci®c activity was again signi®cantly reduced under sodium ¯uoride aected Raf-1 recruitment onto plasma these experimental conditions. The degree of inhibition membranes. Western blotting analysis showed that was greater in the presence of sodium ¯uoride than in there was a small but consistent increase in the amount the presence of b-glycerophosphate and sodium of endogenous Raf-1 remaining associated with the pyrophosphate, but was still highly signi®cant. To- membranes in the presence of sodium ¯uoride gether these results demonstrate that Raf activation

Oncogene Identification of PP2A and PP1 as Raf-1 regulators M Jaumot and JF Hancock 3952 eect of microcystin-LR re¯ected inhibition of PP2A or PP1 or both, we tested two selective inhibitors of these phosphatases. Protein phosphatase 2A inhibitor I1 which speci®cally inhibits PP2A, and protein phosphatase inhibitor 2 which speci®cally inhibits PP1, both clearly decreased Raf-1 activation, although the inhibition was not as complete as with sodium ¯uoride (Figure 2c). From these experiments we conclude that both PP2A and PP1 might cooperate in the activation of plasma membrane recruited Raf-1.

Phosphatase inhibitors alter 14-3-3 membrane distribution Previous studies have demonstrated that phosphorylated serines 259 and 621 comprise the core of two 14- 3-3 binding sites in the Raf-1 molecule (Muslin et al., 1996). We previously showed that 14-3-3 is largely displaced from Raf-1 at the plasma membrane when Raf-1 is activated (Roy et al., 1998). Given these data we next investigated whether Ser259 and/or Ser621 were the target residues of the regulating serine phosphatase and if so whether the plasma membrane distribution of Raf-1 and 14-3-3 could be altered by serine phosphatase inhibitors. In order to test this hypothesis we obtained postnuclear-supernatants (PNS) from cells expressing H- RasG12V and from cells transfected with the empty Figure 2 b-Glycerophosphate and Na Pyrophosphate, Micro- vector as a control. The cells were harvested and then cystin LR, and speci®c PP1 or PP2A inhibitors, but not incubated at 308C for 5 min in presence or absence of Cypermethrin inhibit the in vitro activation of endogenous Raf- sodium ¯uoride. The PNS were ¯oated through a 28 ± 1 in cell membranes expressing H-RasG12V. Membrane fractions 45% w/v continuous sucrose gradient, which resolves from COS cells transfected with H-RasG12V were incubated with buer A at 308C for 20 min. The Raf activity associated with the light membranes in the top fractions from denser membranes after the incubation was measured as described under membranes in the bottom fractions (Figure 3c). As well Materials and methods. Raf activities have been expressed relative as the homogenization buer, the stocks of concen- to the Raf activity measured in the incubation without inhibitors trated sucrose used to create the gradients were also (=1). (a) In vitro incubations were carried out in the presence or prepared in presence or absence of sodium ¯uoride. absence of 25 mM sodium ¯uoride or 25 mM b-glycerophosphate and 2 mM sodium pyrophosphate (general serine-threonine Ten fractions were collected from the top to the phosphatase inhibitors). (b) In vitro incubations were carried out bottom of each gradient and membranes were isolated in presence or absence of 25 mM sodium ¯uoride (NaF), 4 nM from each fraction by centrifugation as described in cypermethrin a PP2B inhibitor, or 10 mM microcystin-LR a dual Materials and methods. The membrane fractions were PP1 and PP2A inhibitor. (c) In vitro incubations were carried out in presence or absence of 25 mM sodium ¯uoride, 1.5 mM protein analysed for Raf kinase activity using a MEK/ERK phosphatase 2A inhibitor I1 (PP2Ainh), or 100 nM protein coupled assay and immunoblotted for Raf-1 and 14-3- phosphatase Inhibitor 2 (PP1inh). These data are representative 3. of experiments that were replicated three times with similar results Figure 3 shows that both the amount and distribution of Raf-1 associated with the gradient fractions diered between the two incubation conditions. The can be inhibited by general serine-threonine phospha- total amount of Raf-1 associated with membranes tase inhibitors. One interpretation of these data is that incubated with sodium ¯uoride was twofold greater serine-threonine phosphatases have a positive role in than that associated with control membranes incubated Raf-1 activation. without sodium ¯uoride (Figure 3b) consistent with the results obtained in the in vitro Raf-1 activation assays (Figure 1d). In addition, Raf-1 was resolved into two PP2A and PP1 are implicated in the in vitro activation of peaks along the gradient: a smaller peak in the light Raf-1 membranes (#3 ± 4) and a larger peak in the denser We next performed the in vitro Raf-1 activation assays membranes (#7 ± 10). Incubation with sodium ¯uoride in presence or absence of more speci®c PP1, PP2A or resulted in a single large peak concentrated over PP2B inhibitors. Cypermethrin, a potent inhibitor of fraction #6. Consistent with the previous results the PP2B had no eect on Raf-1 activation (Figure 2b), total amount of Raf activity associated with control whereas microcystin-LR, which inhibits PP1 and membranes was 6.5-fold greater than that associated PP2A, reduced Raf kinase activity to a similar extent with membranes incubated with sodium ¯uoride as sodium ¯uoride (Figure 2b). To clarify whether the (Figure 3a). The speci®c activity of the control Raf-1

Oncogene Identification of PP2A and PP1 as Raf-1 regulators M Jaumot and JF Hancock 3953

Figure 3 Serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with cell membranes and alters the 14-3-3 and Raf-1 membrane distribution across a sucrose density gradient. Postnuclear supernatants obtained from COS cells transfected with H-RasG12V and with empty vector as a control were incubated at 308C for 5 min in the presence or absence of 25 mM sodium ¯uoride and then fractionated over sucrose gradients. Ten fractions were collected and the membranes in each fraction were reisolated by centrifugation and resuspended as described. Fractionated membranes were then assayed for Raf kinase activity in a coupled MEK-ERK assay (a) and immunoblotted for 14-3-3 and Raf-1 (b). (c) The graph shows the percentage of sucrose (w/v) of each fraction of the gradient is even higher given the reduced amount of Raf-1 gradients were observed when the membranes were associated with these membranes. In good correlation obtained from cells transfected with empty vector with the Raf-1 immunoblots there were two peaks of (Figure 3b). Thus the accumulation of 14-3-3 in cell Raf activity in the control membranes, but essentially membranes in the absence of sodium ¯uoride is no activity was associated with the major Raf-1 peak dependent on Ras expression and hence Raf-1 detected on the immunoblots from membranes incu- recruitment. bated with sodium ¯uoride: the small amount of Raf Together these data strongly suggest that inhibition kinase activity that was detected was con®ned to of serine phosphatase activity prevents de-phosphor- fraction 10 (Figure 3a). ylation of phospho-Ser 259 and/or phospho-Ser621, Western blotting analysis also revealed dramatic this prevents the release of 14-3-3 from membrane- dierences in the total amount and distribution of recruited Raf-1 which in turn impairs full Raf-1 14-3-3 across the gradients (Figure 3b). 7.7-fold more activation. 14-3-3 was associated with membranes incubated with sodium ¯uoride than with the control membranes. The The phosphorylation of Ser 259 is affected by phosphatase small amount of 14-3-3 associated with these control inhibition whereas Ser621 phosphorylation is not membranes was con®ned to the bottom fractions of the gradient (#8 ± 10). In contrast, incubation in sodium With the aim of clarifying whether S259 or S621 are ¯uoride resulted in a substantial re-distribution of 14-3- the targets of the activating phosphatase we ®rst 3 into two pools, one associated with low density evaluated the phosphorylation state of these residues membranes (#3 and 4) and a large pool associated with in gradient fractions using phospho-speci®c antisera denser membranes (#6 ± 9). Thus incubation in sodium generated and characterized by Thorson et al. (1998). ¯uoride results in a substantial increase in the amount There was no detectable change in the amount of of 14-3-3 associated with the Raf-1-containing fractions phosphorylated Ser621 in the presence or absence of that lack Raf kinase activity. Importantly no dier- sodium ¯uoride. On the contrary, Ser259 phosphoryla- ences in the amount or distribution of 14-3-3 along the tion was only detectable when cell lysates had been

Oncogene Identification of PP2A and PP1 as Raf-1 regulators M Jaumot and JF Hancock 3954 incubated in sodium ¯uoride (Figure 4). Moreover the peak of 259 phosphorylated Raf-1 coincided exactly with the peak of inactive Raf centred on fraction #6 (Figure 3a,b). To extend and con®rm these observations we investigated whether the kinase activity of Raf-1 with a S259A mutation was still sensitive to phosphatase regulation. H-RasG12V was co-transfected with wild type Raf-1 or RafS259A. Cell membranes were isolated and the Raf kinase activity was measured after an in vitro incubation in the presence or absence of Protein Phosphatase inhibitor 2 (speci®c PP1 inhibitor). While wild type Raf-1 and mutant Raf259A were both aected by the PP1 inhibitor, Figure 5 shows that RafS259A activity was relatively resistant (35% inhibited) compared to wild type Raf (75% inhibited). The same experimental approach to investigate Ser621 as a putative target of phosphatase is not possible because a S621A substitution results in an inactive kinase (Morrison et al., 1993). Figure 5 The kinase activity of the S259A Raf-1 mutant is less aected by PP1 inhibition than wild type Raf-1. Membranes from Discussion COS cells co-transfected with H-RasG12V and either FLAG Raf- 1 (wt) or mutant Raf259A, or transfected with empty EXV To directly address the role of serine-threonine plasmid (V), were isolated and incubated with buer A at 308C for 20 min in the presence or absence of 100 nM of protein phosphatases in Raf-1 activation we have used a well phosphatase inhibitor 2. The membranes were reisolated by characterized reconstituted in vitro model system in centrifugation and measured for Raf kinase activity in a coupled which Raf-1 is recruited to Ras-containing membranes MER-ERK assay. (a) Phosphorylation of 16 mg of MBP from the and undergoes activation. We show that in vitro Ras- Raf kinase assay. (b) Graph representative of relative Raf activity under the dierent conditions. The kinase activities of wt Raf-1 dependent activation of membrane recruited Raf-1 is and 259ARaf-1 were calculated by subtracting the activity present inhibited by a wide range of non-speci®c serine- in the control (H-RasG12V+empty vector) from the activity threonine phosphatase inhibitors. Moreover through present in the Ras and Raf-1 co-transfected samples the use of highly speci®c chemical and peptide phosphatase inhibitors we provide evidence that PP1 and PP2A are both implicated in Raf-1 activation. phosphatase inhibitors. These data are consistent with However, since the eects of sodium ¯uoride, b- earlier studies showing that alanine or aspartate glycerophosphate and sodium pyrophosphate were substitutions of S259 partially activate Raf-1 and more pronounced than any combination of the more conversely that phosphorylation of S259 by Akt speci®c PP1 and PP2A inhibitors, we can not exclude downregulates Raf activity (Rommel et al., 1999; the participation of other serine phosphatases in Raf-1 Zimmermann and Moelling, 1999). In addition during activation. Two sets of experimental data indicate that the preparation of this paper, Abraham et al. (2000) S259 is one target of these phosphatases: phosphoryla- showed that PP2A can be co-immunoprecipitated with tion of S259 monitored with phosphospeci®c antisera Raf-1 and is involved in Raf-1 activation in vivo correlates inversely with Raf-1 activation and secondly, through the de-phosphorylation of Ser259, observa- in vitro activation of S259A mutant Raf-1 is substan- tions which clearly support and complement our tially resistant to speci®c PP1 and non-speci®c observations in the in vitro activation system. The biological relevance of these biochemical results is underscored by invertebrate genetic data which show that PP2A positively regulates Ras-mediated signaling during C. Elegans vulvar induction (Sieburth et al., 1999) and PP2A promotes Ras mediated photoreceptor development in Drosophila (Wassarman et al., 1996). Our data imply that not all serine residues on membrane recruited Raf-1 are equally sensitive to phosphatase action. We show directly that de-phosphorylation of S259 is inhibited by general serine phosphatase, or speci®c PP1 or PP2A inhibitors, but Figure 4 Phosphorylation of S259 is aected by serine phos- phosphorylation of S621 is relatively unaected, at phatase inhibition whereas S621 phosphorylation is not. The least in the conditions of our assay. In addition since membrane fractions from the sucrose gradients presented in Figure 3 were immunoblotted using antibodies against phos- the assay measures Ras-dependent Raf activation, phorylated S259 or phosphorylated S621 which correlates directly with S338 phosphorylation

Oncogene Identification of PP2A and PP1 as Raf-1 regulators M Jaumot and JF Hancock 3955 (Mason et al., 1999), de-phosphorylation of S338 previous study (Roy et al., 1998) we stimulated cells cannot be occurring in the phosphatase inhibitor-free with EGF and examined the time course of endogen- incubations. ous Raf-1, 14-3-3 and Raf activity associated with cell One interpretation of these results is that phospha- membranes at dierent time points. The maximum tases do not have equal access to dierent serine recruitment of 14-3-3 to the plasma membrane residues: this may re¯ect the presence of plasma correlated most closely with Raf-1 dissociation. These membrane proteins or Raf-1-associated proteins that and other data indicated that the re-association of 14- impede their access. In respect of S259 and S621, 14-3- 3-3 with Raf-1 following deactivation could have a role 3 proteins could serve this role; if so, this raises the in removing Raf-1 from the plasma membrane. Taking critical question of how 14-3-3 is displaced from the studies together, we would contend that a phospho-S259 to allow the residue to be de-phos- secondary eect of serine phosphatase inhibition is phorylated. Several studies have shown that the impairment of normal Raf-1 turnover from the plasma RafCRD is essential for Raf-1 activation (Clark et membrane back to the cytosol. In this context the al., 1997; Luo et al., 1997; Roy et al., 1997). This dierent distributions of Raf-1 across the gradient domain has overlapping binding sites for phosphati- fractions in the presence or absence of phosphatase dylserine (PS), Ras and 14-3-3 (Clark et al., 1997; inhibitors may re¯ect association of Raf-1 with Daub et al., 1998; Ghosh et al., 1996; Improta-Brears dierent plasma membrane microdomains and/or et al., 1999; Mott et al., 1996; Okada et al., 1999; dierent cellular membranes. Thus, if S259 depho- Williams et al., 2000). It has been shown that the sphorylation is an early step in Raf-1 activation, then interaction of the CRD with 14-3-3 stabilizes the Raf- PP1/PP2A inhibitors, which block 14-3-3 removal, trap 1/14-3-3 complex and that the incubation of Raf-1 with Raf-1 in the plasma membrane microdomain to which PS in vitro completely displaces 14-3-3 with a it is initially recruited. If 14-3-3 is displaced then Raf-1 concomitant increase in Raf kinase activity (McPher- tracs to other plasma membrane microdomains son et al., 1999). Taking these results together it is where additional activation events proceed: marked reasonable to postulate that interactions of Ras and/or by the two peaks of Raf activity devoid of 14-3-3 seen the plasma membrane enriched phospholipid, PS with in the absence of phosphatase inhibitors. Conversely, the RafCRD displaces 14-3-3 from membrane recruited the co-localization of Raf-1 of low activity with 14-3-3 Raf-1 allowing access of PP1/PP2A to S259. Once S259 in denser fractions of the gradient, which also contain has been dephosphorylated then interactions of Ras the endocytic markers Rab5 and a-adaptin 2 (data not and/or PS with the CRD may no longer be required to shown), may indicate a pool of endocytosed Raf-1 that progress Raf-1 activation. The actual removal of is in the process of deactivation and enroute to the phosphate from S259 to destroy the 14-3-3 binding cytosol (Ceresa and Schmid, 2000; Di Guglielmo et al., site may be especially important if the Ras- and PS- 1994; Pol et al., 1998, 2000; Rizzo et al., 2000). RafCRD interactions are relatively transient at the While this interpretation remains speculative in the plasma membrane. absence of more detailed micro-localization of active Strong support for these concepts comes from the and inactive Raf-1 at the plasma membrane, it is sucrose gradient analysis of interactions between Raf-1 compatible with current views on the micro-organiza- and 14-3-3 at the plasma membrane under conditions tion of the plasma membrane. Cholesterol rich where dephosphorylation of S259 is inhibited. The microdomains or lipid rafts have been proposed to sucrose gradient experiments are especially informative function as molecular platforms for signal transduction because the separation of cell membranes into dierent where speci®c proteins are recruited into the appro- subfractions permits detection of subtle variations that priate environment for successful signaling interactions are not detectable in whole membrane preparations. (Simons and Ikonen, 1997). Given that many signaling When cell lysates were incubated in the presence of proteins including Ras, tyrosine and serine kinases and phosphatase inhibitors prior to fractionation the total phosphatases are dierentially localized between raft amount of 14-3-3 and Raf-1 associated with membrane and non-raft microdomains (Cary and Cooper, 2000; fractions substantially increased. Most compelling was Couet et al., 1997; Kurzchalia and Parton, 1999; Liu et the dierential distribution of Raf activity across the al., 1996, 1997; Okamoto et al., 1998; Plyte et al., 2000; gradient fractions. There was no activity associated Roy et al., 1999), it is not unreasonable to speculate with any of the Raf-1 recruited to the membrane that Raf-1 initially recruited by H-Ras to cholesterol fractions in the presence of phosphatase inhibitors, rich lipid rafts may need to access other non-raft which all contained high amounts of 14-3-3: the only domains for ecient activation. If so, the results fraction with any Raf activity was that which presented here showing that serine-threonine phospha- contained the least amount of 14-3-3. The simplest tase inhibition has a less dramatic eect on K-Ras than interpretation of these results is that dephosphorylation H-Ras-dependent Raf-1 activation (Figure 1a, b) could of S259 is critical for displacement of 14-3-3 at the re¯ect dierences in microlocalization of these Ras plasma membrane and in turn for subsequent ecient isoforms (Prior et al., 2001; Roy et al., 1999) Raf-1 activation. From the data presented in this study and our What is the signi®cance of the increased amount of previous results we propose the following sequence for Raf-1 recruited to the plasma membrane in the Raf-1 activation (Figure 6). A Raf-1/14-3-3 complex is presence of serine phosphatase inhibitors? In a recruited by Ras to a speci®c microdomain of the

Oncogene Identification of PP2A and PP1 as Raf-1 regulators M Jaumot and JF Hancock 3956

Figure 6 Model of the H-Ras-dependent Raf-1 activation. Inactive Raf-1 is complexed with 14-3-3 in the cytosol. GTP-Ras recruits 14-3-3-Raf-1 into a de®ned microdomain of the plasma membrane, at this point P-S259 is protected from dephosphorylation. The RafCRD interacts with phosphatidylserine (PS) and/or Ras which transiently displaces 14-3-3 and allows PP1/PP2A to dephosphorylate P-S259. 14-3-3 is then released from the N-terminal domain of Raf-1. These events are accompanied by the displacement of Raf-1 to a dierent membrane microdomain where it becomes accessible to kinases responsible for Y341 and S338 phosphorylation and Raf-1 is fully activated. 14-3-3 rebinding to Raf-1 sequesters it back to the cytosol once it has been inactivated. Based on the observations of Roy et al. (1999) and Priov et al. (2001) microdomain A could be cholesterol-rich lipid rafts and microdomain B, non-raft plasma membrane

plasma membrane where an initial Ras and/or PS- glycerophosphate and 2 mM sodium pyrophosphate, or CRD interaction transiently displaces 14-3-3 allowing 4nM cypermethrin, or 10 mM microcystin-LR, or 1.5 mM S259 dephosphorylation and the loss of 14-3-3 from protein phosphatase 2A inhibitor I1, or 100 nM protein the N-terminal regulatory domain of Raf-1. This is phosphatase inhibitor 2. The protein content was measured accompanied by a displacement of the Ras/Raf-1 by the Bradford reaction. complex to other membrane microdomains where tyrosine and serine phosphorylation and other events In vitro activation assay fully activate Raf-1. At some point 14-3-3 is also One hundred and twenty-®ve mg of fresh P100 from EXV- released from the Raf-1 C-terminus. During deactiva- Ha-RasG12V or EXV-Ki-RasG12V transfected cells was tion, perhaps on the endosome, 14-3-3 rebinds to mixed on ice with 150 mg of fresh S100 from EXV- inactive Raf-1 and sequesters it back to the cytosol. FLAGRaf-1 transfected cells. To measure endogenous Raf- Notwithstanding the details of this model the data 1 activation, 125 mg of P100 was mixed with Buer A. KCl presented here clearly demonstrate a critical role for (®nal concentration 100 mM) was added and the volumes serine-threonine phosphatases in Raf-1 activation. were adjusted to 95 ml with Buer A. The mixture was incubated at 308C on a shaking heating block for 20 min. The samples were spun immediately at 100 000 g at 48C for 15 min. All the steps which involve Buer A were carried out in presence or absence of the serine-threonine phosphatase Materials and methods inhibitors listed above. The supernatant was discarded and the pellet was rinsed and then resuspended by sonication in Cell transfection and fractionation 55 ml of Buer A. Twenty ml of the resuspended membranes COS-1 cells were electroporated as previously described were assayed for Raf activity and 20 ml were analysed by (Huang et al., 1993) using 10 ± 20 mg of the following quantitative Western blotting for Ras and Raf-1. expression plasmids: EXV-FLAG-Raf-1 or EXV-Myc- Raf259A, and/or EXV-Ha-RasG12V, EXV-Ki-RasG12V or Determination of Raf activity EXV empty vector as control. After 48 h, cells were incubated for 18 h in serum free media and then washed Raf activity was measured in a coupled two-step MEK/ERK with cold phosphate buered saline (PBS) and scraped on ice assay with MBP phosphorylation as read out, exactly as into 0.2 ml of Buer A (10 mM Tris-Cl, pH 7.5, 5 mM described previously (Roy et al., 1997). MgCl2,1mM EGTA, 1 mM DTT, 100 mM NaVO4,10mg/ ml leupeptin). After homogenization through a 23 gauge Gel electrophoresis and Western blotting needle the nuclei were removed by low-speed centrifugation and the postnuclear supernatants were spun at 100 000 g at Proteins were separated in sodium dodecyl sulfate-polyacry- 48C for 30 min. The soluble fraction (S100), which contains lamide gels (SDS ± PAGE) and transferred to polyvinylidene cytosolic proteins, was collected, and the sedimented fraction di¯uoride membranes using semidry transfer. Western (P100), which contains cellular membranes, was rinsed, and blotting was carried out with the following antibodies: anti- resuspended by sonication in 80 ml of Buer A in presence or Raf-1 (c-Raf-1 Transduction Laboratories, #R19120), anti- absence of the following serine-threonine phosphatase 14-3-3 (b/pan 14-3-3; Santa Cruz Biotechnology, #sc-629), inhibitors: 25 mM sodium ¯uoride (NaF), or 25 mM b- anti-Ras (Y13-259) or anti-Raf-1-Phospho-Serine 259 and

Oncogene Identification of PP2A and PP1 as Raf-1 regulators M Jaumot and JF Hancock 3957 anti- Raf-1-Phospho-Serine 621 (a kind gift from Andrey equal volume of 85% w/v sucrose (2.5 M) in HB. 0.6 ml of Shaw, Washington University, St Louis, USA). Immunoblots 42.5% sucrose/PNS was overlaid sequentially with 0.9 ml were developed using enhanced chemiluminescence (ECL) 35% sucrose and 0.6 ml of 25% sucrose and spun in a (SuperSignal; Pierce) and quanti®ed by phosphorimaging Beckman SW-55 rotor for 16 h at 35 000 r.p.m. Ten 0.2 ml with a CH-screen (BioRad). fractions were collected from the top and diluted into 1 ml of buer A. The diluted fractions were spun at 100 000 g at 48C for 30 min. Membrane pellets were resuspended by sonication Sucrose density gradient centrifugation into 40 ml Buer A, 10 ml were used for Western blotting COS cells transfected with EXV-Ha-RasG12V or EXV as a analysis and 10 ml for determination of Raf kinase activity. control were washed and resuspended in 1 ml PBS. The cells were spun at 1000 r.p.m. at 48C for 5 min and gently resuspended in 3 ml of homogenization buer (HB=250 mM Acknowledgments sucrose, 10 mM HEPES, pH 7.4, 100 mM NaVO4,10mg/ml leupeptin). After pelleting at 2500 r.p.m at 48C for 10 min the We thank Andrey Shaw for supplying the phosphospeci®c cells were gently resuspended by pippeting in 0.5 ml of HB. Raf antisera and the Raf259A expression plasmid and After homogenization through a 23 gauge needle the broken Brian Gabrielli, Albert Pol and other members of the cells were spun at 2500 r.p.m. at 48C for 15 min and the Oncology Laboratory for helpful comments on the manu- postnuclear supernatant (PNS) was collected and then script. This work was supported by a grant from the incubated at 308C for 5 min in the presence or absence of National Health and Medical Research Council of Aus- 25 mM sodium ¯uoride. The protein content was measured tralia. JF Hancock is also supported by the Royal by the Bradford reaction. 0.3 ml of PNS was mixed with an Children's Hospital Foundation, Queensland, Australia.

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Oncogene