The Role of PAK-1 in Activation of MAP Kinase Cascade and Oncogenic Transformation by Akt

Oncogene (2009) 28, 2365–2369 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE The role of PAK-1 in activation of MAP kinase cascade and oncogenic transformation by Akt

PR Somanath1,3, J Vijai2,3, JV Kichina2, T Byzova1 and ES Kandel2

1Department of Molecular Cardiology, The Cleveland Clinic, Lerner Research Institute, Cleveland, OH, USA and 2Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY, USA

The activity of protein kinase B, also known as Akt, is quently, cRaf phosphorylates and activates dual speci- commonly elevated in human malignancies and plays a ficity MEKs (‘MAP/ERK Kinases’, also known as crucial role in oncogenic transformation. The relationship ‘MAP kinase kinases’), which, in turn, phosphorylate between Akt and the mitogen-activated protein kinase and activate MAP kinases such as extracellular signal- cascade, which is also frequently associated with oncogen- regulated kinases (ERKs). Activated ERKs control the esis, remains controversial. We report here examples of function of various transcription factors, such as the cooperation between Akt and cRaf in oncogenic transfor- ones belonging to the Ets family, and additional kinases mation, which was accompanied by elevated activity of (for example, ribosomal protein S6 kinases). Several extracellular signal-regulated mitogen-activated protein other factors have been implicated in the control of kinases. The effect of Akt on extracellular signal- this pathway. For example, the function of cRaf may regulated kinases depended on the status of p21-activated be influenced by the status of 14-3-3 proteins and by p21- kinase (PAK). Importantly, disruption of the function of activated kinases PAK-1 and PAK-3 (King et al., 1998; PAK not only uncoupled the activation of Akt fromthat Chaudhary et al., 2000). In various models, continuous of extracellular signal-regulated kinases, but also greatly engagement of this signaling pathway contributes to the reduced the capacity of Akt to act as a transforming survival and proliferation of cancer cells. oncogene. For the malignancies with hyperactive Akt, our Similarly, a multitude of effectors are capable of observations support the role for PAK-1 as a potential elevating the phosphoinositol-3-kinase activity in a cell, target for therapeutic intervention. leading to accumulation of phospholipids that act to Oncogene (2009) 28, 2365–2369; doi:10.1038/onc.2009.114; recruit members of the Akt family to the plasma published online 4 May 2009 membrane. The membrane-bound Akt undergoes full activation after phosphorylation by phosphotidyl inositol- Keywords: extracellular signal-regulated MAP kinases; dependent kinases (PDKs)and is capable of phosphor- protein kinase B; neoplastic cell transformation; ylating a plethora of cellular proteins. The precise p21-activated kinases number, identity and biological relevance of such targets are a matter of intense research and debate. It is clear that transcription, translation, carbohydrate and lipid meta- bolism, cell adhesion, motility and death are all influenced The classical mitogen-activated protein (MAP)kinase by the status of this pathway. Constitutive activation cascade and the PI-3-K-Akt pathway are two signaling of Akt in cancer is achieved through amplification or mechanisms that are commonly found activated in mutation of the corresponding genes, overexpression of human malignancies, are actively pursued as therapeutic the catalytic subunit of PI-3-K, mutation or overexpres- targets, and are the subjects of an immense body of sion of various growth factor receptors, and the loss of research literature reviewed elsewhere (for example, negative regulator PTEN. As an oncogene, hyperactive Kandel and Hay, 1999; McCubrey et al., 2006; Roberts Akt may enhance resistance to growth-arresting and and Der, 2007; Yuan and Cantley, 2008). Both pro-apoptotic impacts (Kennedy et al., 1999; Mirza et al., mechanisms are engaged after activation of various 2000), and facilitate acquisition of additional mutations in growth factor receptors and, at least in some cells, both some conditions of genotoxic stress (Kandel et al., 2002). could be turned on by activated Ras. The interplay between the two signaling cascades The MAP kinase cascade typically ensues from remains controversial. As both pathways could be simulta- recruitment and activation at the plasma membrane of neously engaged and, apparently, contribute to the same a ‘MAP kinase kinase kinase’, such as cRaf. Conse- features of cancer cells, it would appear that a positive cooperation between the two might exist. Unexpectedly, an Correspondence: Dr ES Kandel, Department of Cell Stress Biology, early report claimed that Akt directly phosphorylates and Roswell Park Cancer Institute, BLSC L3-318, Elm and Carlton inactivates cRaf (Zimmermann and Moelling, 1999). Based Streets, Buffalo, NY 14263, USA. on the stated direct nature of this interaction, one might E-mail: [email protected] 3These authors contributed equally to this work. thinkthatanincreaseinAktactivitywouldbealways Received 26 January 2009; revised 23 March 2009; accepted 15 April inhibitory to Raf, yet the same group has reported that 2009; published online 4 May 2009 the effect of Akt activation on Raf varies dramatically Oncogenic Akt requires the function of PAK-1 PR Somanath et al 2366 depending on the conditions of treatment (Moelling et al., Thus, the specifics of ERK activation may differ 2002). Others have suggested that Akt interferes with ERK depending on whether this is achieved by activation of activation, but the point of interference is downstream of Ras or through cooperation of mAkt and cRaf. Ras, Raf and MEK (Galetic et al., 2003). In contrast, Importantly, in these experiments we assay the steady- several reports described cooperation between the two state condition of the signaling pathways in genetically pathways in acquiring growth factor independence and in engineered cells, which resemble cancerous cells harbor- cell-cycle progression (McCubrey et al., 2001; Sheng et al., ing activated oncogenes, but may be distinct from the 2001; Mirza et al., 2004). cells transiently treated with growth factors. We examined the status of cRaf protein in mouse We hypothesized that an apparently similar effect on the embryonic fibroblasts that did or did not express a exogenous and endogenous proteins, which are being constitutive form of mouse Akt1 (mAkt)(Figure 1a). expressed from different promoters in the context of We observed that the levels of endogenous cRaf different transcripts, may point to a post-transcriptional increased upon mAkt expression. This was seen even effect on cRaf abundance. We also hypothesized that such a in the cells in which cRaf expression was greatly elevated change may reflect participation of cRaf in a different set of through the introduction of human cRaf. Importantly, interactions, which may become visible as intercellular co-expression of mAkt and cRaf resulted in a noticeable redistribution of the protein. To test this hypothesis, we increase in the activity of MAP kinase cascade, as is investigated the localization of cRaf as a function of Akt evidenced by the increase in phosphorylation of ERK activity. We observed distinct high-intensity staining at the kinases. Interestingly, ERK activation was readily periphery of the cells that expressed mAkt. Some accumula- achieved by expression of an activated Harvey Ras tion of cRaf at this location was occasionally seen in the protein, but without an increase in the level of cRaf. cells that were fed with serum-containing medium, but not

1st pBH pBH pBH pBH pBH construct mAkt Ras mAkt 2nd pBP pBP pBP pBP pBP construct cRaf cRaf

p-ERK

ERK

cRaf

mAkt vector

1.2

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0.8 pBH/pBP pBH/pBPcRaf 0.6

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0.2 relative frequency of positive cells serum + + + + - - - LY - + - + - - + mAkt - - + + - + +

pBHmAkt/pBP pBHmAkt/pBPcRaf pBHRas/pBP

Figure 1 The evidence of interplay between Akt and cRaf in mouse fibroblasts. (a)Akt and cRaf cooperatively activate ERKs)in MEF-WT cells. The lysates of cells transduced with the indicated plasmids were probed by western blotting using antibodies against phospho-ERK, total ERK and total cRaf protein. pBH, pBP-retroviral vectors pBabeHygro and pBabePuro (Morgenstern and Land, 1990). mAkt-activated (myristoylated) mouse Akt-1. cRaf—human cRaf. Ras—v-Ha-Ras. (b–d)The status of Akt affects the intracellular distribution of cRaf. NIH3T3 cells were transfected with pBabePuro or pBabePuro-mAkt, plated on coverslips 18 h later, incubated for additional 12 h, subjected for serum starvation or treatment with LY294002 for 4 h, fixed and stained for cRaf. Examples of the cells that do (b)or do not ( c)show the peripheral staining (indicated by arrowheads)are shown. The incidence of cells with peripheral staining is shown relative to that in serum-treated vector-transfected cultures. The averages and standard deviations of triplicates are shown (d).(e, f)Cooperative transformation of MEF-WT by co-expression of activated Akt and cRaf. Typical appearances of confluent cultures of mouse embryonic fibroblasts transduced with the indicated constructs are shown. The cultures were photographed 1 week after reaching confluence.

Oncogene Oncogenic Akt requires the function of PAK-1 PR Somanath et al 2367 in the presence of LY294002 (‘LY’), a compound that was readily achieved upon their co-expression inhibits PI3 kinase and endogenous Akt. Therefore, Akt (Figures 1b–f). The metabolic shutdown, which is activity in these cells facilitates targeting of a subset of cRaf characteristic of contact-inhibited cells (Bereiter-Hahn molecules to a specific compartment at the cell surface. This et al., 1998), was also lost when mAkt and cRaf were co- observation may be significant, because peripheral localiza- expressed; and the doubly infected cells rapidly acidified tion of cRaf has been associated with elevated oncogenic growth medium. Similar results were obtained with rat activity of this kinase before (Leevers et al., 1994; Stokoe intestinal epithelial cells RIE-1 (data not shown). We et al., 1994)and resembles the Akt-dependent localization concluded that the observed biochemical cooperation of Rac-1 (Somanath and Byzova, 2009). between Akt and cRaf correlates with multiple biologi- The cooperation between overexpressed cRaf and cal features of oncogenic transformation. mAkt extended beyond activation of ERKs. Although Unlike the earlier report that observed cooperation none of the protein was able to relieve our mouse between the activated forms of the two kinases (Sheng embryonic fibroblast culture of contact inhibition, this et al., 2001), cRaf used in our system was a wild-type

control L Y controlCA RacdnRac controlCA PAKdnPAK p-Akt p-ERK p-ERK

p-PAK ERK ERK p-ERK Rac1 ERK PAK1

p-cRaf (S338) 6 5 cRaf 4 3 2 activity 1 0 Relative luciferase

mAkt vector dnAkt dnRac CA Rac dnPAK ector+LY CA PAK

6 5 5 4 4 3 3

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activity 2 1 1 Relative luciferase

0 Relative luciferase K K K 0 K K vector mAkt dnAkt

vectordnPAKwtAkt1 mAkt1 ddAkt mAkt + dnPAK dnAkt + CA PAmAkt + CAdnAkt PA + dnPA wtAkt + dnPAmAkt + dnPAKddAkt + dnPA Figure 2 PAK-1-dependent modulation of MAP kinase cascade by Akt. (a)PAK-1 and ERK activity depends on the PI3-K pathway. The status of Akt, PAK-1 and ERKs is tested using the respective phospho-specific antibodies in the lysates of untreated and LY294002-treated HEK293 cells. (b–c)ERK activity depends on the status of PAK-1 and Rac-1. The lysates from HEK293 cells, untransfected or transiently transfected with expression constructs for the constitutively active (‘CA’)and dominion-negative (‘dn’) forms of Rac-1 and PAK-1, were probed with antibodies to the indicated proteins. (d). Activities of downstream effectors of the MAP kinase cascade depend on the status of PI3-K, Akt, Rac-1 and PAK-1. The effect of the indicated expression constructs or the respective empty vector (with or without treatment with LY294002)was compared using co-transfected Ets reporter. The activity of an Ets-driven firefly luciferase construct was normalized for that of co-transfected Renilla luciferase driven by a constitutive CMV promoter, and is shown relative to the value in untreated vector control. Means and standard deviations from quadruplicates are shown. (e, f)The effect of Akt on Ets activity is modulated by PAK1. Luciferase reporter assays with the indicated constructs were as in (a).

Oncogene Oncogenic Akt requires the function of PAK-1 PR Somanath et al 2368 protein and on its own was unable to significantly affect mAkt in this cell line, alone or with dominant-negative the activity of ERKs. It appears that at least two Akt- PAK-1. Activated Akt elevated the levels of active ERKs, dependent signals, one of them requiring Raf activation, and this effect was abolished by co-expression of dominant- are necessary for transformation in these cells. Indeed, negative PAK-1 without a significant loss of phosphoryla- the activated form of MEK kinase, which mimics the tion on either of the PDK sites (Figure 3a). When grown effect of activation by Raf, relieved contact inhibition, past confluence, original Rat-1 form a flat monolayer, but only in cooperation with activated Akt (data not while their transformed derivatives continue proliferation, shown). This is similar to the observation that activated forming multilayer foci that could be easily detected on MEK and Akt cooperatively relieve hematopoietic cells methylene blue staining. The loss of contact inhibition, a of cytokine dependence (Shelton et al., 2004). classic feature of oncogenic transformation in fibroblasts The biological consequences of activation of ERKs (Weinberg, 2007), was readily induced by mAkt, but was ensue, in part, from activation of the Ets family of dramatically reduced in the presence of dominant-negative transcription factors which, at least in some case, is PAK-1 (Figure 3b). Similar results were obtained in required for the transformed phenotype (Galang et al., experiments that used different sets of expression constructs 1996). We examined the regulation of this pathway for mAkt and dnPAK (data not shown), and the ability of using transient transfection of an Ets-dependent repor- mAkt to facilitate tumor growth was significantly dimin- ter construct in HEK293T cells. It has been suggested that PI3K may affect cRaf through activation of PAK-1, which phosphorylates cRaf on serine 338 pBP pBPmAkt 100

(Chaudhary et al., 2000; Sun et al., 2000). We observed pBH pBH pBH pBH 80 that phosphorylation of both PAK-1 and ERKs was dnPAK dnPAK 60 inhibited by LY (Figure 2a). PAK-1 itself is known to be pAKT (S473) 40 regulated by Rac-1. Predictably, phosphorylation of pAKT (T308) ERKs was sensitive to dominant-negative forms of 20 AKT Rac-1 (RacS17N)and PAK-1 (PAK K299R),and was relative number of foci 0 P-ERK pBPmAkt pBPmAkt induced by the constitutively active forms of these + + proteins (Rac-1 Q61 L and PAK T493E, respectively) pBH pBHdnPAK (Figures 2b and c), whereas the status of serine 338 on cRaf was PAK-1-dependent (Figure 2c). Ets activity 1.2 100 1 also responded profoundly to the manipulations of 80 PAK-1 and Rac-1 (Figure 2d). Inhibition of endogenous 0.8 60 Akt by LY or by expression of the dominant-negative 0.6 40 form of this kinase (Akt1 K179D)reduced Ets activity, 0.4 whereas mAkt activated it (Figure 2d). The effect of 0.2 20 relative tumor volume dominant-negative Akt was partially alleviated by relative number of foci 0 0 simultaneous expression of the constitutively active pBPmAkt pBPmAkt pBHmAkt pBHmAkt form of PAK-1. On the other hand, dominant-negative + + + + Scrambled shRNA-PAK PAK-1 abolished induction of Ets activity by mAkt pBH pBHdnPAK (Figure 2e). Importantly, dominant-negative PAK-1 was Figure 3 PAK-1 is required for oncogenic transformation by Akt. able to ablate the Ets-stimulating activity even in the (a)Dominant-negative PAK-1 prevents activation of ERKs by constitutive Akt. Rat-1a cells were transduced with a combination case of ddAkt (Figure 2f). This Akt variant has the of pBabePuromAkt (‘pBPmAkt’)or pBabePuro (‘pBP’)with either PDK-sensitive serine and threonine of wild-type protein pBabeHygro (‘pBH’)or pBabeHygro-dnPAK (‘pBHdnPAK’).The substituted for aspartates to mimic constitutive phos- indicated proteins were assayed by immunoblotting. (b)Dominant- phorylation by PDKs. It lacks the constitutive mem- negative PAK-1 restores contact inhibition in cells transformed by activated Akt. One million cells from each culture were seeded per brane localization signal of mAkt and retains the native 10 cm plate and allowed to reach confluence. Two weeks later the PH domain. Although we cannot rule out multiple cells were fixed and stained with methylene blue. The foci of mutual interactions between PAK-1 and the PI3K-Akt multilayer culture, which appear as darker areas on the images of pathway, our data suggest that there is at least one the plates, were automatically counted using Image Pro software. PAK-1-dependent step in the process of ERK activation Results of triplicate experiments are shown. No foci were detected in the absence of mAkt. (c)Tumorigenic growth of cells by Akt. Overall, our observations are compatible with transformed by activated Akt is inhibited by dominant-negative signaling from activated Akt to the Rac-1/PAK-1 PAK-1. Five aliquots of 2.5 Â 105 cells each from the indicated complex to the MAP kinase cascade and on to the cultures were injected subcutaneously into nude mice. Five weeks respective target transcription factors. later, the tumor volumes were estimated using the prolate spheroid model (Euhus et al., 1986). For each group, the data are shown as PAK-1 is essential for Ras-induced upregulation of an average and standard deviation, using the average volume of cyclin D1 (Nheu et al., 2004)and is an important ‘pBPmAkt þ pBH’ tumors as a normalization factor. (d)Suppres- intermediary in Ras-mediated oncogenic transformation sion of PAK-1 through RNA interference restores contact of Rat-1 fibroblasts (Tang et al., 1997). These cells are also inhibition in cells transformed by activated Akt. Confluent cultures prone to transformation by expression of activated Akt of Rat-1 cells transduced with pBabeHygro-mAkt and either pSM2c-PAK-1-shRNAmir (‘shRNA-PAK’; Open Biosystems, (Mirza et al., 2000). Our observations predict that Huntsville, AL, USA; Cat # RMM1766-96744645)or the respective transformation of these cells by Akt also depends on the non-silencing control (‘scrambled’; Open Biosystems Cat # function of PAK-1. To test this prediction, we expressed RHS1707)were maintained for 20 days and analysed as in ( b).

Oncogene Oncogenic Akt requires the function of PAK-1 PR Somanath et al 2369 ished by expression of dnPAK (Figure 3c). Transforming oncogene. Considering the prevalence of Akt activation ability of mAkt was also reduced by co-expression of in human malignancies, out findings predict that an shRNA against PAK-1. Although the use of RNA PAK-1 is a strong candidate target for therapeutic interference bares some risk of artifacts (Gartel and intervention. Kandel, 2006), the concordance between the dnPAK and shRNA experiments proves that Akt-mediated transforma- tion of these cells is PAK-1 dependent. Conflict of interest Our observations confirm that at least in some cells constitutive activation of Akt can cooperate with the The authors declare no conflict of interest. MAP kinase cascade in oncogenic transformation, thus explaining the co-activation of both pathways in human cancers. Furthermore, we have observed that Acknowledgements in our models the hyperactivation of elements of the We thank Dr Nikolay Neznanov for the Ets-reporter MAP kinase cascade is necessary, albeit not sufficient, construct, Dr Channing Der for RIE-1 cells, Dr George Stark for full oncogenic activity of Akt. Importantly, we have for the MEF cells and Dr Nissim Hay for various Akt- observed that disrupting the function of PAK-1 expressing constructs. This work was supported by the uncoupled activation of Akt from that of ERKs, Howard Temin Award to ESK, NIH Grant HL071625 to and prevented Akt from acting as a transforming TB and American Heart Association grant 0830326N to PRS.

References

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