Emerging Signaling Pathways in Tumor Biology 2010 Editor Pedro A. Lazo IBMCC-Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain Transworld Research Network, T.C. 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India Published by Transworld Research Network 2010; Rights Reserved Transworld Research Network T.C. 37/661(2), Fort P.O., Trivandrum-695 023, Kerala, India Editor Pedro A. Lazo Managing Editor S.G. Pandalai Publication Manager A. Gayathri Transworld Research Network and the Editor assume no responsibility for the opinions and statements advanced by contributors ISBN: 978-81-7895-477-6 Preface The specificity of biological effects in different cells types is determined by the composition of their proteome, the physical and functional interaction between different signaling pathways and theirs spatial and temporal organization in signaling complexes. Although the implication of some specific pathways with major phenotypes, such as proliferation, apoptosis or differentiation is well known, the final effect varies depending on cell type, which might even be the opposite. These phenotypes play an essential role, and are frequently deregulated in tumor cells, which make some of them potential targets for development of new drugs. In this context, protein kinases represent the main regulatory family, which can be activated and deactivated by reversible phosphorylation, or by integration in spatially organized signaling complexes with scaffold proteins. The human kinome is composed of 518 kinases and for many of them their characterization from the point of view of regulation and substrate specificity, as well as their integration in novel signaling pathways, or their effect on other better know pathways such as MAPK, are not yet understood. In this book we have focused the attention on some of the less well known kinases such as Cot1/Tpl2, LKB1, WNK, GSK3, VRK, and PLK3. Also there is a chapter on development of specific inhibitors for mitotic kinases. There are two additional chapters. One focused on the KSR scaffold protein, an organizer of signaling complexes and intracellular signal compartmentalization, as well as another on DUSP phosphatases, the key deactivator of regulated kinases, which comparatively receive little attention, but are an essential component in regulatory mechanisms. Pedro A. Lazo Contents Chapter 1 Modulation of Ras signaling by the KSR family of molecular scaffolds 1 Luís G. Pérez-Rivas, Stephanie Prieto and José Lozano Chapter 2 The biological function of the proto-oncogene Cot/tpl-2 (MAP kinase kinase kinase 8) 25 Margarita Fernández, Irene Soria, Marta López-Peláez Antonio Martín-Duce and Susana Alemany Chapter 3 WNK kinase signalling in cancer biology 43 Sónia Moniz and Peter Jordan Chapter 4 The tumour suppressor role of LKB1 in human cancer 71 Paola A. Marignani and Montse Sanchez-Cespedes Chapter 5 The role of glycogen synthase kinase-3 in the decision between cell survival and cell death 95 Roberta Venè and Francesca Tosetti Chapter 6 Polo-like kinase 3 in normal and tumor biology 117 Dazhong Xu and Wei Dai Chapter 7 Vaccinia-related kinase (VRK) signaling in cell and tumor biology 135 Marta Sanz-García, Alberto Valbuena, Inmaculada López-Sánchez Sandra Blanco, Isabel F. Fernández, Marta Vázquez-Cedeira and Pedro A. Lazo Chapter 8 Targeting mitotic kinases for anti-cancer treatment 157 Hae-ock Lee, Yoo-Kyung Lee, and Hyunsook Lee Chapter 9 Atypical DUSPs: 19 phosphatases in search of a role 185 Yolanda Bayón and Andrés Alonso Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India Emerging Signaling Pathways in Tumor Biology, 2010: 1-23 ISBN: 978-81-7895-477-6 Editor: Pedro A. Lazo 1. Modulation of Ras signaling by the KSR family of molecular scaffolds Luís G. Pérez-Rivas, Stephanie Prieto and José Lozano Dpto. Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain Abstract. KSR proteins were initially identified in C. elegans and D. melanogaster as positive regulators of Ras signaling. Murine and human homologs were subsequently isolated, allowing for their characterization at the molecular level. The mammalian homologs are functionally classified as pseudokinases due to the lack of a catalytic lysine involved in the phosphotransfer reaction and found in most active kinases. Over the past 10 years, work done in several groups has allowed a detailed molecular characterization of KSR proteins, mainly at the functional level. In the near future, a more complete picture of the mode of action of KSR proteins will require additional studies at the structural level. However, the wealth of data currently known about KSR clearly indicates that it is a critical player in Ras signal transduction, functioning as a molecular scaffold that complexes the individual components of the Raf/MEK/ERK cascade into multiprotein signaling platforms (signalosomes). Here, we will review some of the well-established concepts regarding the regulation of Ras signaling by KSR proteins, in both normal and malignant cells. We will also include some recent and provocative data that sheds new light on the mechanism of KSR function. Correspondence/Reprint request: Dr. José Lozano, Dpto. Biología Molecular y Bioquímica, Universidad de Málaga. Campus de Teatinos s/n. 29071 Málaga, Spain. Phone: (+34) 95 213 6661, E-mail: [email protected] 2 Luís G. Pérez-Rivas et al. 1. Introduction The kinase suppressor of Ras (KSR) family of proteins comprise an evolutionary conserved group of molecular scaffolds that function as modulators of Ras signaling by bringing together the different components of the Raf/MEK/ERK cascade [1, 2]. KSR1, the founding member of the KSR family of proteins, was identified in 1995 by means of genetic screens in D. melanogaster and C. elegans [3-5]. At that time, a murine ksr1 gene was also cloned by Therrien et al. [5]. The corresponding mKSR1 protein has been, since then, the most studied member of the KSR family and currently serves as a model to explain the biological function of these proteins. To date, the family includes one gene in Drosophila (ksr), two genes in mammalian cells (KSR1 and KSR2) and two genes in C. elegans (ksr-1 and ksr-2) [4-8]. All KSR proteins identified so far have a C-terminal sequence which includes the 11 highly conserved subdomains found in most eukaryotic kinases and, therefore, they were initially named as if they were true kinases. However, the failure to find a KSR-specific direct substrate and, more importantly, the lack of an invariant catalytic residue (K) in subdomain II of mammalian KSR isoforms has led to the classification of these proteins in the pseudokinase subgroup of the human kinome, together with other proteins also containing degenerated kinase domains [9]. Currently, KSR is considered to function mainly as a molecular scaffold that facilitates the assembling of the individual components of the Raf/MEK/ERK signaling cascade [10-13]. Mammalian KSR proteins are expressed in most adult tissues, although their levels are usually very low and, frecuently hard to detect by immunoblotting [10, 14, 15]. The highest expression level has been reported for a brain-specific isoform (B-KSR1) [16]. Titration experiments, showed that, in fact, the in vivo KSR levels need to be carefully regulated and kept below a certain limit in order for KSR to properly function as a molecular scaffold [17]. This also applies to other known scaffolds, such as JNK-interacting protein 1 (JIP1) [18] and the molecular explanation for these requirement is summarized in Figure 1. Essentially, the concentration of a scaffold protein cannot be increased above its optimal level (defined by the stoichiometry of the reaction) to avoid chelation of its ligands into non- competent complexes [1]. For this reason, some of the early works aimed to define the role of KSR1 in Ras signaling resulted in contradictory outcomes, with some investigators reporting a negative role in ERK activation and Ras transformation and others reporting just the opposite [17, 19-24]. KSR1 function was mainly investigated by overexpressing mKSR1 in cultured cells and, therefore, the amount of ectopic mKSR1 was usually way above the endogenous (suboptimal) level, resulting in inhibition of Ras signaling. KSR proteins in Ras signaling 3 Under circumstances of low transfection efficiency, the ectopically expressed mKSR1 helped to reach the optimal scaffold concentration, enhancing signal transduction. To avoid this problem, which is inherent to the plasmid transfection methods, Morrison and coworkers developed an assay to measure the Ras-dependent ERK scaffolding activity of KSR in Xenopus laevis oocytes. The system allows for a careful titration of the amount of KSR expressed in each oocyte an scores their meiotic maturation (germinal vesicle breakdown, or GVBD) as the biological readout [1, 17]. More recently, the availability of immortalized KSR1-deficient cells has greatly facilitated the study of KSR function since variable amounts of mKSR1 proteins (wild-type or mutant) can be reintroduced in these cells by retroviral infection and subsequent sorting of cell populations with different KSR1 expression levels [25-27]. The following sections are a summary of the current molecular knowledge regarding KSR structure and function. Figure 1. The effective concentration of KSR1 is critical for an optimal scaffolding activity. This is achieved when the scaffold (KSR1, in red) and its ligands (C-Raf, MEK and ERK, in yellow, green and orange, respectively) are in a concentration near the stoichiometry of the reaction (B). Below such levels, a local increase in the effective concentration of KSR1 results in an increased signaling (A). However, an excess of the scaffold (i.e., a concentration higher than the optimal) results in the formation of non-productive complexes that block signaling (C). 4 Luís G. Pérez-Rivas et al. 2. Domain organization and function KSR proteins are similar to members of the Raf family of S/T kinases [4- 7], and as such are included in the tyrosine-like kinase (TLK) group of kinases [9].