Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas G12D , a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas G12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.Ras dimer | MAPK signaling | cancer | single molecule imaging | superresolution microscopy T he canonical rat sarcoma (Ras) GTPase family members H-, N-, and K-ras are frequently activated in human cancers (1-4) by recurrent point mutations at codons 12, 13, or 61. These mutations result in constitutive binding of Ras to GTP due to impaired GTP hydrolysis (5). Despite nearly identical G-domains, mammalian Ras isoforms serve nonredundant biological roles and exhibit different mutational spectra in human cancers (1,4,6). These functional differences are in part attributed to distinctions in the membranetethering motif at the C-terminal of Ras known as the hyper-variable region [HVR, which includes the "CAAX" (C, cysteine; A, aliphatic; X, any amino acid) motif] (6, 7). Although mechanisms regulating Ras-GTP levels in cells have been examined extensively, details of how Ras organizes and operates on the cell membrane have been elusive. Efforts on targeting mutant Ras to therapeutic benefits in human cancers by inhibiting membrane localization or GTP binding have not been successful, leaving mutant Ras an intractable drug target (8). Hence, identification of new mechanisms that regulate Ras oncogenesis is crucial to combating mutant Ras-driven cancers.Recent studies using immuno electron microscopy (immuno-EM) have implicated a previously unappreciated spatial mechanism in regulating the biological functions of Ras. In particular, Ras proteins were found to form 5-to 8-membered nanoclusters that serve as signaling scaffolds for recruiting and activating downstream effectors such as Raf and PI3K on the cell membr...

The functions ascribed to PTEN have become more diverse since its discovery as a putative phosphatase mutated in many human tumors. Although it can dephosphorylate lipids and proteins, it also has functions independent of phosphatase activity in normal and pathological states. In addition, control of PTEN function is very complex. It is positively and negatively regulated at the transcriptional level, as well as post-translationally by phosphorylation, ubiquitylation, oxidation and acetylation. Although most of its tumor suppressor activity is likely to be caused by lipid dephosphorylation at the plasma membrane, PTEN also resides in the cytoplasm and nucleus, and its subcellular distribution is under strict control. Deregulation of PTEN function is implicated in other human diseases in addition to cancer, including diabetes and autism. Journal of Cell Science 4072 and protein levels (but decreases phosphorylation of PTEN at S370 and S385, and its stability), and the ability of SPRY2 to restrain proliferation is abolished in PTEN-negative cells (Edwin et al., 2006).Resistin, a cytokine involved in inflammation and insulin resistance, increases PTEN expression in human aortic vascular endothelial cells by activating p38 MAPK and activating transcription factor 2 (ATF2), which leads to decreased activation of PKB and of its target, endothelial nitric oxide synthase (eNOS, also known as NOS3). This provides a potential mechanism for resistin-mediated impairment of insulin signaling and of its role in cardiovascular diseases (Shen et al., 2006). Furthermore, phytoestrogens such as genistein (in soy), resveratrol (in red wine) and quercetin (in fruit and vegetables) also lead to increased PTEN expression, with a concomitant decrease in phospho-PKB levels and increase in levels of the CDK inhibitor p27 (Waite et al., 2005). Epidemiological data suggest that phytoestrogen consumption can protect against cancer (Park and Surh, 2004;Verheus et al., 2007). Upregulation of PTEN expression might therefore be one basis for these beneficial effects. Indeed, mammary glands of rats fed with soy protein or a genistein-supplemented diet display higher PTEN levels and a higher apoptotic index (Dave et al., 2005). The same is true for MCF-7 cells cultured in serum from such rats (Dave et al., 2005). Similarly, indole-3-carbinol, a phytochemical derived from cruciferous vegetables such as broccoli, upregulates PTEN expression in the cervical epithelium in a mouse model for cervical cancer (Qi et al., 2005). Negative regulation of PTEN transcriptionMost of the data so far have demonstrated positive regulation of PTEN transcription. Negative regulation of PTEN expression has also been shown, however (Fig. 1). Mitogenactivated protein kinase kinase-4 (MKK4) inhibits PTEN transcription by activating NFB, which binds to a site in the PTEN promoter (Xia et al., 2007). Transforming growth factor (TGF)␤ also decreases PTEN transcription in pancreatic cancer cells (Chow et al., 2007) , 2006). Phytoestrogens, such as genistein from soybea...

The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF-CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.cancer signaling | single molecule imaging | superresolution optical microscopy T he RAF serine/threonine protein kinase is a component of the three-tiered MAPK signaling pathway that regulates cell growth and many other essential biological processes (1, 2). In normal cells, extracellular mitogenic stimuli are transmitted to the nucleus via the receptor-RAS-RAF-MAPK cascade (2). Abnormal activation of this pathway is a central event in many human cancers and results from activating mutations in BRAF itself or in upstream factors (such as the RAS genes) (3, 4). As the RAS proteins so far are intractable pharmacologic targets (5), attention has shifted to development of small-molecule RAF inhibitors (RAFis) as antitumor therapeutic agents (6). To date, RAFi clinical efficacy has been demonstrated only for BRAF V600E melanoma (6-8). In tumors with WT BRAF or mutant RAS, most RAFis paradoxically promote growth, at times malignant in nature (6). Moreover, BRAF mutant melanomas that are initially sensitive to RAFi rapidly become resistant by using a variety of compensatory mechanisms including RAF isoform switching and activation of other pathways including RTKs, RAS, or PI3K (9). In addition, some RAFis (e.g., vemurafenib) accelerate the occurrence of secondary squamous cell carcinomas (10).Several lines of investigation suggest that multimer formation plays an important role in RAF activation and tumor responses to RAFi (11-16). RAF dimerization-mediated signaling was first suggested by the observation that ar...