FUNCTIONS AND MODULATION OF MAP KINASE PATHWAYS 1,25,43,46,56,57,68,71- Gray Pearson and Melanie Cobb isoforms and ERKs 5 and 7. 73,108,109,126 Department of Pharmacology, University of ERK3 was found as a cDNA library 11 Texas Southwestern Medical Center, Dallas, clone. A summary of the cellular processes these Texas 75390, USA. MAP kinases are involved in is shown in Table 1. Gray Pearson’s research focuses on studying the Upstream regulation of ERK1/2 regulation of MEK5 and ERK5 and Melanie The collaborative findings from a number of Cobb’s laboratory studies various aspects of MAP laboratories led to the connection of ERK1/2 to their kinase signaling. upstream regulators MEK1 and 2, the identification of Raf-1 as the upstream activator of these MEKs, and the observation that Raf-1 is an effector of the proto- oncogene Ras.17,67,69,101,119 The linear connection of Ras to ERK1/2 suggested a function for ERK1/2 in Background proliferation and oncogenic growth.76 This conclusion was later supported by the observation that an The transmission of extracellular signals into activated mutant of MEK1 can transform cells.85 intracellular responses is a complex process which Subsequently, through the use of dominant interfering often involves the activity of mitogen-activated protein mutants and the pharmacological inhibitors of (MAP) kinases.96 The activation of a MAP kinase MEK1/2, these ubiquitous kinases have been shown involves a three kinase cascade consisting of a MAP to be intimately involved in processes including kinase kinase (MAPKKK or MEKK) which activates a embryogenesis, cell differentiation, glucose sensing MAP/ERK kinase (MAPKK or MEK), which then and synaptic plasticity.32,40,63,98 stimulates a phosphorylation-dependent increase in the activity of the MAP kinase. Upon activation, MAP MEKs kinases can phosphorylate a variety of intracellular MEK1 was purified as a biological activator of targets including transcription factors, transcriptional ERK1/2.4,106 The identification of MEKs 2-7 employed adaptor proteins, membrane and cytoplasmic DNA-based molecular as opposed to protein substrates, and other protein kinases. purification techniques.26,33,49,52,79,107,113,125,126 These kinases are distinct from other cascade components ERK1/2 in that they are dual-specificity kinases, which means The MAP kinases extracellular signal-regulated they can phosphorylate tyrosine and serine/threonine protein kinases 1 and 2 (ERK1,2) were first identified residues. Unlike MAP kinases, which phosphorylate a as mitogen-stimulated phosphoproteins in the early wide range of proteins, MEKs appear to be largely 1980s, and later as insulin and nerve growth factor dedicated to the activation of MAP kinases. There is (NGF)-stimulated activities that retained the ability to also a great deal of specificity in which MAP kinases phosphorylate the model substrates microtubule- are phosphorylated by the MEKs. Our understanding associated protein-2 (MAP2) and myelin basic protein of how MEKs integrate signals from multiple (MBP).2,9,99,100 The physiological function of ERK1/2 regulatory inputs or serve as points of signal proteins was unknown. Early on, however, these integration is limited, therefore, MEKs will not be activities were shown to reactivate phosphatase- discussed in further detail. Instead a schematic treated ribosomal protein S6 kinase.10,15,103 representing the circuitry of MAP kinases best conveys the relevant information about MEK signaling Discovery of additional MAP kinases and will follow a brief discussion of MEKKs. In the following years, the MAP kinase family was discovered to include three c-Jun N-terminal kinase MEKKs (JNK) and four p38 isoforms, ERK3 isoforms, ERK5 The most readily identifiable feature of MAP kinase and ERK7. The first JNK family members were signaling is the three kinase cascade consisting of a independently identified as cycloheximide-activated MEKK, a MEK and a MAP kinase.31,50,96 The three- MBP kinases and also purified due to their ability to kinase organization of this cascade is identical to that interact with the N-terminus of the transcription factor of the three-kinase cascade of Ste11p-Ste7p- c-Jun.51,70 p38a was identified as an inflammatory Fus3p/Kss1p in the yeast pheromone response cytokine-stimulated tyrosine phosphoprotein, a target pathway.96 MEK1/2 and ERK1/2 are clearly related in of an inhibitor of tumor necrosis factoraa (TNF ) sequence to Ste7p and Fus3p. However, Raf-1 is the production, and a re-activating kinase for MAP unique member of this signaling cascade in that there kinase-activated protein kinase-2 (MAPKAP2).48,75,104 is no identified yeast homolog. Considering the PCR-based cloning strategies and a two-hybrid parallels between yeast and mammalian signaling, it screen led to the discovery of additional JNK and p38 was assumed that one or more Ste11p homologs would exist in mammals. Tocris Cookson Ltd., UK Tocris Cookson Inc., USA Tel: + 44 (0)117 982 6551 Tel: (800) 421-3701 Fax: + 44 (0)117 982 6552 www.tocris.com Fax: (800) 483-1993 e-mail: [email protected] [email protected] e-mail: [email protected] Table 1. Stimuli and nuclear substrates of MAP kinases MAP Kinase Stimuli Nuclear Substrates ERK1/2 Growth factors Elk1, c-Myc, SAPs, c-Jun, Serum NeuroD1, PDX-1, STAT3, Hormones RSKs, Mnks, MSK, etc. Cytokines Small molecules p38 isoforms Hormones ATF-2, MEF2, SAPs, STAT3, Cytokines MAPKAPs, Mnks, MSK Osmotic stress Heat shock JNK isoforms Hormones c-Jun, Elk-1, STAT3 Cytokines Inhibitors of DNA and protein synthesis Osmotic stress ERK5 Growth factors MEF2, RSKs Serum Hormones Osmotic stress ERK3 and ERK7 none identified none identified MEKK1 was the first of the Ste11p homologs MEK1/2-ERK1/2 activation step introduces a identified in mammals. MEKK1, a protein of 195 kDa, threshold for activation.37 Protein kinases contain a displayed the ability to phosphorylate MEKs 1, 2, 3, 4, poorly conserved loop just C-terminal to the catalytic 6, and 7in vitro .122 Although early evidence residues, referred to as the activation loop. In the suggested that MEKK1 was a regulator of MEK1/2, MAP kinases, this loop contains a TXY motif except later results indicated a better capacity to ERK3. Phosphorylation of both the threonine and phosphorylate MEKs 3, 4 and 6.87,121 Consistent with tyrosine residues of this motif is required to activate the ability to phosphorylate these MEKs, MEKK1 ERK1/2 and other MAP kinases.95 The non- most likely coordinates downstream signaling through processive phosphorylation of ERK2 on tyrosine activation of MEKs 4 and 7 and the JNK before threonine in cells and in vitro allows for the pathway.120,123 Later cloning efforts have led to the introduction of an activation threshold.38,102 discovery of MEKKs 2-6.8,42,55,116 Comparison of their primary sequences reveals MEKK1 as being most The existence of three proteins in series provides for similar to MEKK4, MEKK2 as being closely related to multiple points of regulatory input. For example, Raf-1 MEKK3, and MEKK5, (aka apoptosis-stimulating contains multiple sites of phosphorylation, which are kinase (ASK1)), and MEKK6, (aka ASK2), being most targeted by a number of different protein kinases.90 like each other. Other non-Ste11p homologs, such as Raf-1 also interacts with a variety of adaptor the Ste 20 homologs TAO1 and TAO2 (also known as proteins.91 The various combinations of PSK1), the mixed-lineage kinases (MLKs), tumor phosphorylations and protein-protein interactions can progression locus-2 (Tpl-2/Cot) and transforming influence both the activity state of Raf-1 and its ability growth factor-bb (TGF- )-activated kinase (TAK1) to interact with MEK1/2 and ERK1/2.91 MEK1/2 are discovered in mammals have also shown MEK kinase also targets of phosphorylation, which disrupts their activity and the ability to activate MAP kinases in ability to interact with Raf-1.19,41 Therefore the fidelity cells.13,14,35,93,105 Figure 1 depicts the complexity of of signaling to ERK1/2 is dictated by the integration of the organization of MAP kinase cascades. Of note are a broad collection of signals. the number of MEK-MAPK combinations a given MEKK can regulate and the resulting points of cross- Scaffolding proteins regulate MAP kinase talk. How the organization of MAP kinases cascades cascades affects their function will be discussed next. In signaling from Raf-1 to MEK1/2 and ERK1/2, the presence of a scaffolding protein, such as kinase Properties of MAP kinase cascades suppressor of Ras (KSR), connector enhancer of KSR (CNK) and Sur-8, can influence the amplitude Our generalized understanding of the features of and duration of a signal through an individual MAP MAP kinase cascades has been largely inferred from kinase pathway.77,110,111 Studies in yeast have shown features of signaling from Raf-1-MEK1/2-ERK1/2 as that scaffolding proteins are paramount for achieving summarized next. It is believed that similar specificity in that they control which MAP kinase mechanisms of regulation exist in other MAP kinase pathway is activated as well. The yeast MEKK, cascades. Also, some ideas concerning why MAP Ste11p can activate Ste7p, the MEK for Kss1p and kinase cascades may require additional modes of Fus3p, in response to pheromone, or Pbs2p, the MEK regulation in addition to those in place for Raf-1- for Hog1p (yeast p38), in response to osmotic MEK1/2-ERK1/2 will be discussed. stress.29,97 Which signal activates Ste11p, and which MEK is targeted for activation by Ste11p is dictated by The activation of MEK1/2 by Raf-1 may represent an scaffolding proteins. In the pheromone response, amplification of signal, due to the greater abundance Ste5p scaffolds the interaction of Ste11p with Ste7p, of MEK1/2.