Human homologue of<i>Drosophila</i>CNK interacts with Ras effector proteins Raf and Rlf<sup>1</sup>

Lanigan, Thomas M.; Liu, Albert; Huang, Yang; Mei, Lin; Margolis, Ben; Guan, Kun Liang

doi:10.1096/fj.02-1096com

Cited by 53 publications

(56 citation statements)

References 42 publications

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“…Another explanation for the crucial role of Rgl2 in PDAC transformation is isoform-specific Ral-independent functions, because Rgl2 has been shown to interact with the Ras scaffolding protein CNK (43), whereas RalGDS interacts with PDK1 to promote AKT activation (20,52). In line with this hypothesis is our observations that Rgl2 knockdown and RalA31N expression strongly suppressed anchorage-independent growth in MIA PaCa-2 without suppression of RalA activity and that in MIA PaCa-2 cells, activated RalA was not able to rescue the anchorage-independent growth phenotype of Rgl2 knockdown.…”

Section: Discussionmentioning

confidence: 99%

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Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Vigil

Martin

Williams

et al. 2010

Journal of Biological Chemistry

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Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Rasmediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Rasmediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.Mutational activation of the KRAS oncogene is the most prevalent genetic alteration associated with essentially 100% of pancreatic ductal adenocarcinomas (PDACs) 3 (1). Therefore, considerable effort has been made to develop inhibitors of K-Ras for PDAC treatment. Currently, most of these efforts are focused on inhibitors of K-Ras downstream effector signaling, with numerous inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)-AKT pathway currently under clinical evaluation (2). However, recent studies suggest that additional Ras effector pathways may also play significant roles in Ras-mediated oncogenesis. In particular, there is increasing evidence for the importance of Ral guanine nucleotide exchange factors (RalGEFs), activators of the Ral (Ras-like) small GTPases (3), in cancer growth (4).Ral GTPases function as GDP/GTP-regulated binary switches that regulate extracellular stimulus-regulated signaling networks that control a diversity of cellular processes, including exocytosis, endocytosis, activation of transcription factors, and actin cytoskeletal reorganization (4). Surprisingly, despite their significant sequence identity (82%) and interaction with a common set of effectors, the related RalA and RalB isoforms serve very distinct functions in oncogenesis. Importantly, Chien and White (5) found that RalA is necessary for tumor cell anchorage-independent growth, whereas RalB is necessary for tumor but no...

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Section: Discussionmentioning

confidence: 99%

“…One possible mechanism is suggested by Rgl2 association with CNK, a scaffold protein that also interacts with Raf and may facilitate ERK activation (43). Thus, depletion of Rgl2 may disrupt the ability of CNK to promote Raf signaling.…”

Section: Rgl2 Overexpression In Pdac Tumors-thementioning

confidence: 99%

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Vigil

Martin

Williams

et al. 2010

Journal of Biological Chemistry

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“…CNK2A, the homologue of Drosophila CNK, interacts with the Ras effector proteins Raf and Rlf and supports neuronal differentiation (31)(32)(33). Whereas the expression of CNK2A and CNK2B is restricted to brain tissue, CNK1 is expressed ubiquitously.…”

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confidence: 99%

CNK1 Is a Scaffold Protein That Regulates Src-mediated Raf-1 Activation

Ziogas

Moelling

Radziwill

2005

Journal of Biological Chemistry

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Raf-1 is a regulator of cellular proliferation, differentiation, and apoptosis. Activation of the Raf-1 kinase activity is tightly regulated and involves targeting to the membrane by Ras and phosphorylation by various kinases, including the tyrosine kinase Src. Here we demonstrate that the connector enhancer of Ksr1, CNK1, mediates Src-dependent tyrosine phosphorylation and activation of Raf-1. CNK1 binds preactivated Raf-1 and activated Src and forms a trimeric complex. CNK1 regulates the activation of Raf-1 by Src in a concentrationdependent manner typical for a scaffold protein. Downregulation of endogenously expressed CNK1 by small inhibitory RNA interferes with Src-dependent activation of ERK. Thus, CNK1 allows cross-talk between Src and Raf-1 and is essential for the full activation of Raf-1.

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“…It is possible that alternative roles for cnk-1 may be detectable only in the right genetically sensitized background. Notably, however, both Drosophila and C. elegans CNK proteins are most similar to mammalian CNK2, which has been found to affect Ras signaling (24,26) and less similar to mammalian CNK1, which has been found to affect Rho signaling (25,27,28). It is possible that the multiple mammalian CNK proteins have evolved to serve different GTPase signaling pathways.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, the mechanism by which CNK promotes Raf activity is unclear. Overexpression studies of mammalian CNK1 and CNK2 suggest that CNK may have a more widespread role in signal transduction, including roles in the Rho and Ral GTPase signaling pathways (23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 99%

Caenorhabditis elegans CNK-1 promotes Raf activation but is not essential for Ras/Raf signaling

Rocheleau

Rönnlund²,

Tuck³

et al. 2005

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

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Connector enhancer of Ksr (CNK) is a conserved multidomain protein essential for Ras signaling in Drosophila melanogaster and thought to be involved in Raf kinase activation. However, the precise role of CNK in Ras signaling is not known, and mammalian CNKs are proposed to have distinct functions. Caenorhabditis elegans has a single CNK homologue, cnk-1. Here, we describe the role of cnk-1 in C. elegans Ras signaling and its requirements for LIN-45 Raf activation. We find that cnk-1 positively regulates multiple Ras signaling events during development, but, unlike Drosophila CNK, cnk-1 does not appear to be essential for signaling. cnk-1 mutants appear to be normal but show cell-type-specific genetic interactions with mutations in two other Ras pathway scaffolds͞adaptors ksr-1 and sur-8. Genetic epistasis using various activated LIN-45 Raf transgenes shows that CNK-1 promotes LIN-45 Raf activation at a step between the dephosphorylation of inhibitory sites in the regulatory domain and activating phosphorylation in the kinase domain. Our data are consistent with a model in which CNK promotes Raf phosphorylation͞activation through membrane localization, oligomerization, or association with an activating kinase.scaffold ͉ vulva ͉ lin-45

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Human homologue ofDrosophilaCNK interacts with Ras effector proteins Raf and Rlf¹

Cited by 53 publications

References 42 publications

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

CNK1 Is a Scaffold Protein That Regulates Src-mediated Raf-1 Activation

Caenorhabditis elegans CNK-1 promotes Raf activation but is not essential for Ras/Raf signaling

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Human homologue ofDrosophilaCNK interacts with Ras effector proteins Raf and Rlf1

Cited by 53 publications

References 42 publications

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

CNK1 Is a Scaffold Protein That Regulates Src-mediated Raf-1 Activation

Caenorhabditis elegans CNK-1 promotes Raf activation but is not essential for Ras/Raf signaling

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Human homologue ofDrosophilaCNK interacts with Ras effector proteins Raf and Rlf¹