Coordinating ERK signaling via the molecular scaffold Kinase Suppressor of Ras

Many cancers, including those of the colon, lung, and pancreas, depend upon the signaling pathways induced by mutated and constitutively active Ras. The molecular scaffolds Kinase Suppressor of Ras 1 and 2 (KSR1 and KSR2) play potent roles in promoting Ras-mediated signaling through the Raf/MEK/ERK kinase cascade. Here we summarize the canonical role of KSR in cells, including its central role as a scaffold protein for the Raf/MEK/ERK kinase cascade, its regulation of various cellular pathways mediated through different binding partners, and the phenotypic consequences of KSR1 or KSR2 genetic inactivation. Mammalian KSR proteins have a demonstrated role in cellular and organismal energy balance with implications for cancer and obesity. Targeting KSR1 in cancer using small molecule inhibitors has potential for therapy with reduced toxicity to the patient. RNAi and small molecule screens using KSR1 as a reference standard have the potential to expose and target vulnerabilities in cancer. Interestingly, although KSR1 and KSR2 are similar in structure, KSR2 has a distinct physiological role in regulating energy balance. Although KSR proteins have been studied for two decades, additional analysis is required to elucidate both the regulation of these molecular scaffolds and their potent effect on the spatial and temporal control of ERK activation in health and disease.


Introduction
The three Ras genes (K-Ras, H-Ras, and N-Ras) regulate diverse cellular functions through multiple pathways and are also commonly mutated in human cancer to yield constitutively active small GTPases. Activating Ras mutations are found in approximately 25% of human tumors, though these three small GTPases are not mutated at equivalent frequencies in cancer. A total of 85% of Rasdriven cancers have activating mutations in K-Ras, while N-Ras and H-Ras are mutated in 12% and 3%, respectively, of these cancers (http://cancer.sanger.ac.uk/cosmic). Ras mutations are most common in pancreatic ductal adenocarcinomas (95%), colorectal adenocarcinomas (52%), and lung adenocarcinomas (31%). Intensive analysis revealed that multiple effectors with Ras-binding domains (or Ras association domains) were capable of interacting with the Ras effector loop and mediating its biological effects 1 . Observations that activating Ras and Raf mutations are typically mutually exclusive [2][3][4] , and that only components of the Raf/MEK/ERK pathway rescue growth in "Rasless" mouse embryo fibroblasts (MEFs) 5 , suggest that the interaction of Ras with Raf, and the activation of MEK1/2 and ERK1/2, may be most critical to Ras-driven cancers.
Kinase Suppressor of Ras 1 (KSR1) interacts with Raf, MEK, and ERK 6-12 , mediates ERK activation and signaling in a dosedependent fashion (discussed in greater detail below), and is essential for the transformation of MEFs by oncogenic Ras 8,13 . These discoveries revealed a critical role played by this molecular scaffold in transformation and tumorigenesis. However, KSR1 -/mice are fertile and show inconsequential developmental alterations 12,14 . These observations suggest that KSR1 may play a prominent role in cancers that are dependent upon Ras and ERK signaling and that it might be exploited therapeutically with minimal toxicity to the patient. Here we review the biochemistry and biology of KSR1 and its paralog, KSR2, and discuss their potential as therapeutic targets.

The role of KSR proteins in the Raf/MEK/ERK cascade
A single ksr gene was identified as necessary for the rougheye phenotype of activated Ras in Drosophila 15 . Two ksr genes (ksr1 and ksr2) are expressed in Caenorhabditis elegans 16,17 and mammals 8,15,17-20 . KSR1 and KSR2 proteins facilitate Raf phosphorylation of MEK, leading to increased ERK activation in response to Ras activation or calcium influx 8,9,12,15,[21][22][23][24][25] . KSR proteins have properties expected of molecular scaffolds 6-12 . As expected of true scaffolds, increasing KSR1 allows for increased ERK activation until KSR1 reaches an optimal level. Surprisingly, in MEFs the level of KSR1 that maximizes ERK activation and signaling is approximately 12 times the endogenous level of expression. Further increasing KSR1 causes a decrease in ERK activation because the cellular concentration of KSR1 exceeds the amount of scaffold that can coordinate signaling with Raf, MEK,and ERK 8,26 . This suggests that overexpression of KSR1 sequesters individual components of the MAPK cascade such that they are unable to interact, which reduces MAPK signaling. However, overexpressing additional individual components of the MAPK pathway can suppress the inhibitory effects of scaffold excess 9 . This observation likely explains why early studies in which ectopic KSR1 was overexpressed suggested that KSR1 inhibited Ras-driven transformation [27][28][29][30] . Importantly, the level of KSR1 expression that optimizes ERK activation is the same level that maximizes the transforming activity of oncogenic Ras and the proliferative effects of growth factors 8 .
Phosphorylation sites and determinants of protein-protein interaction have been mapped extensively on KSR1 and KSR2 and have been shown to regulate KSR1 in part through subcellular localization 31-37 . Analysis of these phosphorylation sites and interactions with effectors and modifiers suggest dynamic regulation of KSR1 and its scaffold function. Interaction with the E3 ligase IMP promotes the redistribution of KSR1 to Tritonresistant punctate structures that sequester KSR1 and impair ERK activation 38,39 . Phosphorylation of KSR1 on Ser297 and Ser392 (Ser310 and Ser469 in KSR2) by the kinase C-TAK1 (MARK3) creates a 14-3-3 binding site that anchors KSR1 within this subcellular compartment ( Figure 1a) 13,18,25,28,38,[40][41][42] . Ras activation catalyzes IMP autopolyubiquitylation and proteasomal destruction ( Figure 1b) 39 . Stimuli that promote IMP degradation also promote the dephosphorylation of KSR1 at Ser392 by PP2A, which eliminates 14-3-3 binding ( Figure 1b) 35,41,43 . Calcineurin dephosphorylates 14-3-3 binding sites on KSR2 25 . These events promote the redistribution of KSR1 to the plasma membrane, facilitating the activation of MEK by Raf ( Figure 1c). MEK is bound to KSR1 in the absence of Ras activation 44 . Though identified as a loss-offunction mutation on KSR1 in C. elegans 15,16,42 , the mutation of KSR1 at Cys809 to tyrosine (C809Y) enhances the activation of ERK in mammalian cells 13 . These observations suggest that KSR proteins may sequester MEK in an inactivated state and present MEK for phosphorylation by Raf 21,45 . In this model, MEK does not need to be in complex with KSR1 to phosphorylate and activate ERK. However, KSR1 encodes a DEF domain 46 , which is essential to KSR1-mediated ERK interaction and critical for competent signal transduction 13,34 . KSR1 (but not KSR2) also encodes a caveolin-binding domain 47 , which is required for binding to caveolin-1, localization to the plasma membrane, and ERK activation ( Figure 1c and 1d) 47,48 . Another level of KSR1 regulation exists in its degradation. Recently, it has been shown that KSR1 is polyubiquitinated by praja2, which promotes KSR1 degradation, causing a decrease in ERK signaling 49 . Reconciling these observations implies that KSR1 coordinates a dynamic mechanism that provides spatial and temporal control of signaling through the Raf/MEK/ ERK kinase cascade.

Regulation of MEK and ERK by KSR and Raf heterodimers
The dimerization of Raf proteins is an essential step in wild-type Raf activation. KSR and Raf proteins share homology within the region required for Raf dimerization, and KSR has been shown to form heterodimers with Raf, particularly B-Raf 24,36,50 . Based on modeling, KSR dimerization with Raf induces a conformational change in KSR that induces the exposure of the MEK activation loop and facilitates its phosphorylation 24 . However, the dimerization of KSR and Raf orients the Raf protein such that the catalytic site of Raf is not in close proximity to its phosphorylation target site on MEK. Therefore, MEK phosphorylation must be completed by another Raf protein 24,36 . KSR2 also forms homodimers through a side-to-side interface that is specifically dependent upon Arg718  Apart from minor deficits, ksr1 -/knockout mice are fertile and developmentally normal. Ksr1 -/mice have hair follicle defects similar to the phenotype of egfr -/mice, supporting the suggestion that these proteins function within the same pathway 12,14,53 . As a result of reduced ERK signaling, ksr1 -/mice have a marginally impaired immunological response 6,12,54,55 . The most profound and translationally significant phenotype of ksr1 -/mice is resistance to Ras-dependent tumor formation. Skin tumor induction by v-Ha-Ras is lost in ksr1 -/mice 14 , and mammary tumor burden is markedly reduced by KSR1 disruption in mice expressing transgenic polyomavirus Middle T-Antigen 12 . These observations demonstrate that KSR modulates Ras signaling in vivo, but it is largely dispensable for normal cell survival. The requirement for KSR1 in Ras-driven tumor formation, but not normal development, reveals KSR1 as a potential target for therapeutic intervention.
In contrast to the mild phenotype of ksr1 -/mice, ksr2 -/mice have reduced fertility and become spontaneously obese [56][57][58][59] . Pathways regulating adaptive thermogenesis, metabolic rate, and leptinsensitive food consumption are implicated in KSR2-dependent energy balance [56][57][58][59][60] . Consistent with observations in the knockout mice, humans with ksr2 mutations show severe early onset obesity 60 . Ksr2 variants in humans that impair Ras signaling or inhibit KSR2 interaction with AMPK also disrupt glucose metabolism and fatty acid oxidation 60 . Interestingly, KSR2 is almost exclusively expressed in the brain and pituitary 19,58 . Brainspecific disruption of KSR2 is sufficient to cause obesity and glucose intolerance in mice, though it does not perfectly recapitulate the phenotype of ksr2 -/mice 19 . These observations show that KSR2 function in the brain plays a potent role in the regulation of energy balance.

Structural properties of KSR proteins
KSR1 and KSR2 proteins are highly conserved in invertebrates and mammals 9,15 . KSR proteins are structurally related to Raf proteins in five conserved areas, CA1-CA5 15 . CA1 is located on the N-terminus end. It contains 40 amino acids that contribute to B-Raf binding by KSR1 and encode coiled-coil and sterile-α-motif (SAM) structures that promote KSR1 membrane association 13,34,44,61 . CA2 is a proline-rich region without known function. A region in KSR2 between CA2 and CA3 is required for KSR interaction with AMPK, and mutations in this region inhibit this interaction 19,31,58 . CA3 is a cysteine-rich region containing an atypical C1 motif homologous to the cysteine-rich CR1 region in Raf that also contributes to KSR1 membrane localization 21,62 . CA3 mediates the membrane localization of KSR by recruiting phospholipids but does not react to phorbol esters or ceramide or interact directly with Ras 63 . CA4 is a serine/threonine-rich region that mediates interactions with ERK through an FXFP motif 32 . This interaction is not constitutive but requires Ras activation 21,33,42,61,63 . The CA5 domain in KSR proteins encodes a kinase (or pseudokinase) domain highly homologous to Raf family CR3 kinase domains 15,17 . Mammalian KSR proteins contain multiple alterations in amino acids typically required for catalytic activity including arginine in place of the lysine that coordinates the gamma phosphate of ATP 15,61 . Substantial effort has been exerted to clarify if KSR can or does phosphorylate cellular substrates and, if so, whether or not this activity contributes to the downstream effects of KSR 21,24,37,64 . KSR1 substrates and the biological relevance of any residual phosphotransferase activity have yet to be validated.
The CA5 region contributes to KSR interaction with MEK in both quiescent and growth factor-activated cells 31,44,65 . Amino acid substitutions within the CA5 region that diminish interaction with MEK also reduce ERK signaling [15][16][17]44,65 . However, these alterations are within or near the ATP-binding domain and may disrupt ATP binding, potentially affecting interaction with MEK secondarily. The CA5 domain also interacts with Raf, but the mechanism is incompletely understood 34 . Thus, there may be unidentified dynamic interactions between the CA1 and CA5 domains of KSR proteins and B-Raf that regulate Raf kinase activation, MEK phosphorylation, and signal transduction through the kinase cascade.  69,70 . These observations suggest the existence of multiple effectors that may be used to support the survival in tumor cells in a manner distinct from their role in normal tissue. Careful validation of these potential targets may yield additional therapeutic strategies.

Conclusions
KSR proteins are established as scaffolds for the Raf/MEK/ ERK kinase cascade, though a detailed understanding of KSRdependent temporal and spatial control of ERK signaling is still lacking. The physiology regulated by KSR1 and KSR2 is also incompletely understood, though disruption of KSR2 in mice and its mutation in humans show that it plays an important role in mammalian energy balance. Given the minimal phenotype of ksr1 -/mice, the normal function of KSR1 is unclear.
However, KSR1 is demonstrably physiologically important as ksr1 -/-ksr2 -/mice do not survive beyond 21 days of age (Costanzo-Garvey and Lewis, unpublished results), while the single gene knockouts survive into adulthood. KSR1 and KSR2 are primarily expressed in brain, though KSR1 is expressed at lower levels throughout the body. Thus, the most profound function of KSR proteins is likely found in the central nervous system. The obesity phenotype caused by brain-specific knockout of KSR2 demonstrates its potent role in cell non-autonomous regulation of energy balance. However, the role of KSR2 as a scaffold and its regulation of ERK signaling in support of brain-regulated development and maintenance of metabolism are unknown. Relevant in vitro cell models that express both KSR1 and KSR2 will be crucial to further our understanding of the cell biology controlled by these molecular scaffolds.