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

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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 1 0.8 pBH/pBP pBH/pBPcRaf 0.6 0.4 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,
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