CBC208.QXD 02/24/2000 01:55 Page 211 211 Signal transduction: hanging on a scaffold W Richard Burack* and Andrey S Shaw Recent data concerning scaffolding proteins profoundly the MAPK cascade, the use of modern protein interaction challenge our conceptions of multicomponent signal methods have identified a growing list of molecules that transduction systems. Recent studies of the phototransduction can operationally fit this description of an Ste5-like scaf- system in Drosophila suggest two points. First, scaffolding fold (e.g. [9•]). None of these MAPK pathway scaffolds markedly limits the possibilities for signal amplification. shows significant homology to Ste5 or to each other (with Second, the methods generally available to study signal the exceptions of kinase domains in Pbs2p and the MAPK transduction may be too crude to assess the in vivo roles of kinase kinase MEKK1). scaffolds. Studies of the mitogen-activated protein kinase pathway scaffold, Ste5, indicate functions beyond that of a Most investigators ascribe two related functions to scaffolds. passive structural element. Finally, the identification of new First, scaffolds are said to maintain the specificity of the sig- mitogen-activated protein kinase pathway scaffolds suggests naling pathway — a function variously described as the existence of multiple ‘signalosomes’ or ‘transducisomes.’ ‘isolating’ or as ‘stabilizing’ the otherwise weak interactions between the kinases of a single cascade. Second, scaffolds Addresses are said to catalyze the activation of the pathway compo- Department of Pathology, Washington University School of Medicine, nents. MAPK scaffolds may hold the kinases in a manner Box 8118, 660 South Euclid, Saint Louis, MO 63110, USA that directly enhances their mutual interactions, theoretical- *e-mail: rburack@path.wustl.edu ly enhancing the rate of the phosphate transfer. Examples of Current Opinion in Cell Biology 2000, 12:211–216 proteins that could be considered as ‘catalytic scaffolds’ include Ste5, MAPK kinase (MEK) partner 1 (MP1), c-jun 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. N-terminal kinase (JNK) interacting protein (JIP1), JNK/SAPK (stress-activated protein kinase) activating pro- Abbreviations tein 1 (JSAP1) and kinase suppressor of Ras (KSR). ERK extracellular-signal-regulated protein kinase Ina-D inactivation no-after potential However, the premises that scaffolds function to catalyse JIP JNK interacting protein activation and to ensure specificity are largely untested. JNK c-jun N-terminal kinase KSR kinase suppressor of ras Another perspective on scaffold function in non-MAPK- MAPK mitogen-activated protein kinase based signal transduction figures larger in the literature. In MEK MAPK/ERK kinase PLC phospholipase C this model, the scaffold functions to co-localize a group of RKIP Raf kinase inhibitor protein molecules that participate in the same signaling process to a SAPK stress-activated protein kinase specific area of a cell. In contrast to the MAPK pathways, the co-localized proteins do not necessarily directly act on each other, but rather they are all involved in the same sig- Introduction naling pathway. Recent work (described below) suggest that Rather than re-review an already well-reviewed field, we scaffolds enhance the efficiency of signal propagation. will focus on the implications of scaffold proteins on the Examples of such ‘anchoring’ scaffolds include the A-kinase function of signal transduction pathways. Thinking about anchoring proteins (AKAPs) [3], Ina-D (inactivation no-after the function of scaffold proteins raises new questions potential) [10], and Yotiao (an NMDA-receptor associated about the nature of signaling pathways. We will also dis- protein) [11•]. Also included in this category are the tyrosine cuss how technical limitations may impede a greater phosphorylated scaffolds such as the platelet-derived understanding of scaffolds. Recent findings suggest that growth factor (PDGF) receptor cytoplasmic domain, insulin our understanding of scaffolds is likely to take unexpected response substrate-1 (IRS1), and the T cell proteins LAT and surprising turns. For scaffolding novices, any one of (linker for activation of T cells) and SLP76 (Src homology 2 the referenced reviews can provide excellent introductory domain containing leukocyte protein of 76 kDa) [12]. overviews of the field [1–3,4•,5,6•]. Recognizing the different roles of catalytic and anchoring Most of our ideas about scaffolds are influenced by work scaffolds, reviews of MAPK scaffolds (the presumed ‘cat- on the prototype mitogen-activated protein kinase alytic-type’) rarely mention the existence of the other (MAPK) scaffold, Ste5, a yeast protein involved in the ‘anchoring’ class. The two perspectives on scaffold function Fus3 MAPK pathway [7,8]. Given the remarkable conser- are not mutually exclusive: catalytic scaffolds also co-localize vation of the MAPK pathways in eukaryotic cells, many components and co-localization is a means to enhance investigators believe that Ste5 equivalents must exist in mutual interactions and thus signaling efficiency. Given that mammalian cells. To date, no protein homologs of Ste5 the idea that scaffolds perform a catalytic function is largely have been identified, however. Rather, working on the untested, it seems very possible that the distinction premise that Ste5 binds to all three kinase components of between these two perspectives will continue to blur. CBC208.QXD 02/24/2000 01:55 Page 212 212 Cell regulation Signals and signal amplification Scaffolds and ideas about ‘switch-like’ It is widely believed that multi-component signaling path- signaling ways function to amplify signals. In a multicomponent If not for amplification, why have multicomponent signal kinase cascade, if each kinase can phosphorylate and acti- transduction systems? Ferrell and co-workers [14••,15] vate many downstream kinases, the net result of a have suggested that the multistep nature of MAPK path- three-step kinase pathway is to geometrically amplify the ways allows for ‘switch-like’ responses that effectively initial signal. The strict utilization of scaffolds in a multi- eliminate ‘noise’. The model that represents switch-like kinase cascade, however, is likely to severely limit the signaling (known as the ‘ultrasensitive’ model) originating amount of signal amplification. Furthermore, the aggregate within the MAPK pathway is based on the multicompo- of components binding to a particular scaffold suggests the nent nature of the pathway and the requirement for two possibility of a discrete cellular complex that mediates a distinct phosphorylation events to activate both MAPK specific signal. Zuker and co-workers, in recent studies of kinase (MAPKK or MEK) and MAPK. The model requires the Drosophila phototransduction pathway, addressed two related assumptions: first, that all the enzymes these issues [10,13••]. (MAPKK kinase [MAPKKK], MAPKK, and MAPK) freely diffuse with respect to each other (i.e. there is no Ina-D scaffolds this system via its five PDZ (post-synaptic transducisome); second, that the activating dual phospho- densitiy/disc-large/ZO1 domains. The known Ina-D lig- rylations of MAPKK and MAPK occur nonprocessively. ands include phospholipase C (PLC), protein kinase C and (Nonprocessive describes a reaction in which the kinase the channel protein, TRP (transient receptor potential). dissociates from its substrate protein after the first phos- Acting as an anchoring scaffold, Ina-D functions by assem- phate is transferred. The second phosphorylation event bling the relevant signaling molecules at a specific requires a second binding of kinase to its substrate protein.) subcellular location in the Drosophila photoreceptor cell. Photoreceptor signal transduction begins with photon acti- Contrary to the expectation that the more efficient proces- vation of rhodopsin, which in turn activates a sive mechanism would be used, in vitro studies using G-protein-coupled PLC leading to the opening of an ion purified enzymes show that MAPK (or ERK [extracellular- channel. Ina-D mutants that cannot bind all components signal-regulated kinase] activation by MEK is demonstrate defective signaling. These findings lead nonprocessive [16,17], which seems to validate one of the Zuker and co-workers to suggest that Ina-D functions to necessary assumptions underlying this model of ‘switch- assemble a signaling complex, a ‘transducisome’, as a like’ signaling. However, current models of scaffold quantal unit of signal transduction. function are incompatible with the quantitative model of ‘switch-like’ signaling because scaffold functions violate Exploiting mutants that express various amounts of the critical assumptions necessary for the model [15]. First, α subunit of G protein (Gα) and PLC, Scott and Zuker scaffolds will limit mutual diffusion of the pathway com- [13••] have shown that signal amplification is not a feature ponents. Second, by holding the kinase and its substrate in of this scaffolded signal transduction process. In a mutant proximity to each other, the scaffold would convert a non- in which signaling inactivation is defective, the activation processive reaction to a processive one. Therefore, in of a single rhodopsin molecule by a single photon molecule addition to limiting amplification, scaffold function may results in a continuous signal. Single photon responses also preclude the recently proposed model in which the were measured by whole cell patch clamp techniques.