A KSR/CNK complex mediated by HYP, a novel SAM domain-containing protein, regulates RAS-dependent RAF activation in <i>Drosophila</i>

Douziech, Mélanie; Sahmi, Malha; Laberge, Gino; Therrien, Marc

doi:10.1101/gad.1390406

Cited by 53 publications

(53 citation statements)

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“…Activation of RAF kinase results in phosphorylation of its substrate MEK that can be detected by immunoblotting with an anti-phospho-MEK antibody. Using RNAi, we depleted endogenous dHYP and found that this abolishes MEK phosphorylation as reported (6) (Fig. 4E; SI Fig.…”

Section: Cnk/hyp Dimerization Is Essential For Raf Signaling In Vivomentioning

confidence: 99%

CNK and HYP form a discrete dimer by their SAM domains to mediate RAF kinase signaling

Rajakulendran

Sahmi

Kurinov

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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RAF kinase functions in the mitogen-activated protein kinase (MAPK) pathway to transmit growth signals to the downstream kinases MEK and ERK. Activation of RAF catalytic activity is facilitated by a regulatory complex comprising the proteins CNK (Connector enhancer of KSR), HYP (Hyphen), and KSR (Kinase Suppressor of Ras). The sterile ␣-motif (SAM) domain found in both CNK and HYP plays an essential role in complex formation. Here, we have determined the x-ray crystal structure of the SAM domain of CNK in complex with the SAM domain of HYP. The structure reveals a single-junction SAM domain dimer of 1:1 stoichiometry in which the binding mode is a variation of polymeric SAM domain interactions. Through in vitro and in vivo mutational analyses, we show that the specific mode of dimerization revealed by the crystal structure is essential for RAF signaling and facilitates the recruitment of KSR to form the CNK/HYP/KSR regulatory complex. We present two docking-site models to account for how SAM domain dimerization might influence the formation of a higher-order CNK/HYP/KSR complex. RAF kinases are downstream effectors of the RAS family of small GTPases (reviewed in ref. 4). Although the events leading to RAS activation are now well understood, the precise mechanism by which activated RAS in turn activates RAF to transduce signals to MEK and ERK remains unclear (5). Studies in Drosophila S2 cells revealed that activation of RAF kinase at sites of RAS-mediated signaling is facilitated by a regulatory complex comprising the proteins CNK (Connector enhancer of KSR), HYP (Hyphen, also known as Aveugle or AVE), and KSR (Kinase Suppressor of RAS) (6, 7). The sterile ␣-motif (SAM) domain, present in both CNK and HYP, is essential for the ability of CNK/HYP/KSR to associate and for signals to transduce through the RAF-MEK-ERK cascade (6); see Fig. 1A for schematic of domain architecture.The structural characterization of SAM domains has revealed the basis by which some SAM domains engage in polymeric protein-protein interactions (8-14), and the basis by which certain SAM domains bind RNA (15-17). A characteristic constellation of basic residues in the sequence of some SAM domains is diagnostic for binding RNA hairpins in a loop sequence-dependent manner (18). In contrast, SAM domains that mediate polymeric proteinprotein interactions cannot be readily recognized from their primary sequence alone. Polymer formation by SAM domains in general arises from the interaction of two distinct surfaces on the SAM domain termed the midloop (ML) and end-helix (EH) surfaces (8). Repeating ML/EH interactions of adjacent SAM domains lead to polymer extension. SAM domain-mediated polymerization has been shown to underlie many aspects of biological function. For example, SAM domain-mediated polymerization is essential for long-range transcriptional repression by the polycomb group proteins (9). In the TEL transcriptional repressor, which is a common target of chromosomal translocations in several hematalogical malignancies, the N-terminal SA...

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Section: Cnk/hyp Dimerization Is Essential For Raf Signaling In Vivomentioning

confidence: 99%

CNK and HYP form a discrete dimer by their SAM domains to mediate RAF kinase signaling

Rajakulendran

Sahmi

Kurinov

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Good examples for this conclusion are Prohibitin, which is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration, 54 Shoc2/Sur8, which functions in complex with the catalytic subunit of protein phosphatase 1 as M-Ras effector modulating Raf activity 55 and Hyphen, which in Drosophila is part of a novel Raf controlling complex consisting of the scaffold KSR and the Raf binding protein CNK. 56 In these multiprotein complexes Raf proteins may act as true kinases, but kinase-independent, scaffolding-like functions of Raf kinases have also been described. Work from several groups established that homo-and heterodimerization of Raf kinases clearly exist, [57][58][59] and that heterodimerization can be Ras induced.…”

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Raf kinases: Oncogenesis and drug discovery

Schreck

Rapp

2006

Intl Journal of Cancer

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Raf kinase signaling has been thoroughly investigated over the last 20 years. A-Raf, B-Raf and C-Raf, the 3 mammalian members of the Raf family, are involved in a variety of cellular processes such as growth, proliferation, survival, differentiation and transformation. The detection of B-RAF mutations in a wide variety of human cancers, the description of wildtype and mutant B-RAF as tumor antigens in melanoma and the promising outcome of clinical trials evaluating the Raf inhibitor Nexavar 1 (Sorafenib, BAY 43-9006) have sparked a broad interest in the scientific community. After a short historical detour and an introduction into Raf kinase signaling, we are going to discuss here recent outcomes of Raf kinase research with respect to tumor formation and give an overview on current efforts to develop anticancer therapies interfering with aberrant Raf kinase signaling. ' 2006 Wiley-Liss, Inc.Key words: cancer; clinical trials; transformation; drug delivery; signal transduction; mitogenic cascade; Raf kinases Raf kinases: The historyIn 1983, the cloning of the acutely transforming replication-defective mouse type C virus 3611-MSV and characterization of its acquired oncogene was reported. 1 Since 3611-MSV induces rapidly growing f ibrosarcoma in mice, the transduced oncogene was called v-raf, whereas its cellular homologue was named c-raf. 1 Shortly thereafter, Bister and coworkers described the cloning of the avian acute leukemia retrovirus Mil Hill No. 2 (MH2). 2 MH2 was found to carry a second potential oncogene in addition to vmyc, which was termed v-mil, whereas its cellular counterpart was called c-mil. 3 Already a hybridization analysis and the gross comparison of restriction maps of v-raf and v-mil pointed at sequence homologies between these 2 genes. 4 By direct sequencing of both genes it was finally proven that 3611-MSV and MH2 have integrated orthologues of the same gene into their genomes. 5 In the following we are going to use the nomenclature for Raf kinases as proposed in a recent review from Wellbrock et al. 6 In contrast to all other oncogene kinases known at that time, no tyrosine kinase activity was detected in C-Raf. 7 Indeed, it was found that C-Raf is a serine/threonine kinase. 8,9 This and the observation that Raf coexists with the Myc oncogene in retroviruses 10 led to two novel concepts: (i) There is a tyrosine to serine phosphorylation switch as the growth factor signal enters the cell, and (ii) the mechanistic basis for cooperation between nuclear and cytoplasmic oncogenes as well as for signal transduction from cell surface receptors to the nucleus is the phosphorylation of transcription factor class oncogenes such as Myc. 11-13 Growth factor abrogation and oncogene cooperation studies were performed to corroborate this signaling scheme. 14-17 Additional important early findings were (i) the cooperation between Raf and Myc in growth factor independent proliferation, immortalization and tumor induction, 14,18,19 leading to the Raf-Myc balance model, 20 and (ii) the cell lineage-switch ...

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“…De plus, des travaux récents ont montré que SHP-2 entre en jeu dans la déphosphorylation d'un résidu phosphotyrosine de SPRY nécessaire pour ses effets inhibiteurs. Ceci suggère donc un autre mode par lequel le complexe GAB/SHP-2 modulerait la signalisation [24]. Des études sur des souris chez lesquelles le gène KSR1 fut inactivé ont confirmé son rôle dans la signalisation RAS/MAPK et ont montré que l'inactivation du gène provoque une perturbation du développement de tumeurs mammaires ou de papillomes RAS dépendants [25,26].…”

Section: Apport Des Modèles Génétiquesunclassified

“…Des travaux récents de notre laboratoire ont montré que l'activité de CNK est régie par la tyrosine kinase Src42A (famille des kinases Src) [29]. Quoique le recrutement de RAF à CNK semble direct, celui de KSR/MEK à CNK fait intervenir une autre protéine appelée HYP (Hyphen ou AVE, Aveugle) [24]. Cette dernière n'est constituée que d'un domaine SAM (sterile alpha-motif) qui hétérodimérise avec le domaine SAM de CNK [30].…”

Section: Apport Des Modèles Génétiquesunclassified

“…La formation d'un complexe comprenant RAF, KSR, MEK, CNK et HYP s'avère donc critique pour la transmission de signaux au sein du module RAS/ERK chez la drosophile. Non seulement la formation d'un tel complexe permet le recrutement de MEK à RAF, mais il participe directement au mécanisme d'activation de RAF où KSR joue un rôle essentiel indépendamment de sa propriété de protéine d'échafaudage [24]. En effet, la formation du dimère KSR-RAF induit l'adoption d'une conformation active par RAF [31,32].…”

Section: Apport Des Modèles Génétiquesunclassified

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