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MAP Kinase Cascades: Scaffolding Signal Specificity
Current Biology, Vol. 12, R53–R55, January 22, 2002, ©2002 Elsevier Science Ltd. All rights reserved.
MAP Kinase Cascades: Scaffolding Signal Specificity
Mitogen-activated protein (MAP) kinases regulate a variety of cellular processes in response to extracellular and intracellular signals. MAP kinases are activated through a protein kinase cascade in which a MAP kinase kinase kinase (or MEKK) activates a MAP kinase kinase (or MEK) that in turn activates the MAP kinase by phosphorylation. These MAP kinase modules are often associated with scaffold proteins, but the molecular roles of these scaffolds has remained elusive [1]. In yeast, distinct MAP kinase pathways regulate mating, invasive growth and the response to high osmolarity (Figure 1) [2]. Because the MEKK Ste11p functions in all three MAP kinase pathways, a problem of specificity arises: how does Ste11p know by which upstream signal it has been activated (referred to as pathway specificity)? Indeed, mutationally activated Ste11p simultaneously induces all three pathways [3,4]. Recent work now demonstrates that pathway specificity is mediated at least in part by scaffold proteins. Scaffolds Localize and Insulate Specific MAP Kinase Pathways In the mating pathway, the scaffold protein Ste5p associates with the MEKK Ste11p, the MEK Ste7p and the MAP kinase Fus3p (Figure 1) [5]. Similarly, the MEK Pbs2p functions as a scaffold, and interacts with Ste11p and the MAP kinase Hog1p in the high osmolarity-glycerol pathway [6]. So far, a scaffold protein has not been identified for the filamentation pathway. In a paper published recently in Current Biology, Harris et al. [7] used a fusion approach to probe the biological function of scaffold proteins. First, they generated fusion proteins between active Ste11p and either of the scaffold proteins Ste5p or Pbs2p. Strikingly, the Ste11p–Ste5p hybrid specifically induces the mating pathway by activating Fus3p, whereas the Ste11p–Pbs2p hybrid induces a high osmolarity response by activating Hog1p. Thus, activated Ste11p becomes confined to a particular pathway when attached to a pathway-specific scaffold, Swiss Institute for Experimental Cancer Research (ISREC), Ch. des Boveresses 155, 1066 Epalinges/VD, Switzerland. 1E-mail: [email protected]; 2E-mail: [email protected]
Dispatch
High osmolarity
Low nitrogen
Ste11p MEKK Ste7p
Fus3p Mating reaction
Pbs2p
Scaffold proteins organize many MAP kinase pathways by interacting with several components of these cascades. Recent studies suggest that scaffold proteins provide local activation platforms that contribute to signal specificity by insulating different MAP kinase pathways.
Pheromone
Ste5p
Frank van Drogen1 and Matthias Peter2
PII S0960-9822(01)00672-8
MEK
Hog1p Glycerol production
Kss1p
MAP kinase
Invasive/pseudohyphal growth Current Biology
Figure 1. Schematic overview of yeast MAP kinase modules that share Ste11p. Extracellular signals such as mating pheromones, osmotic stress and low nitrogen activate specific MAP kinase modules. Ste5p and Pbs2p function as scaffold proteins that organize and insulate the individual components. For further details see the text.
demonstrating that scaffolds actively channel signaling towards the appropriate MAP kinase (Figure 2a). Moreover, these results show that scaffold proteins exclude potential substrates from access to an activated kinase. Harris et al. [7] then analyzed fusions between Ste11p and its downstream kinases Ste7p or Pbs2p. Intriguingly, the Ste11p–Ste7p fusion protein can mimic attachment of Ste11p to the scaffold Ste5p, implying that scaffolds dictate substrate use and promote signaling specificity by presenting a preferred substrate in high concentration. Because scaffolds are able to actively recruit MAP kinase modules to specific subcellular sites [8,9], they also contribute to the spatial organization of MAP kinase signaling. Taken together, these results demonstrate experimentally that scaffolds localize specific MAP kinase modules and insulate signaling networks in vivo. Such pathway insulation is physiologically important, as cells expressing activated Ste11p exhibit a poor mating response because of simultaneous activation of the Hog1p pathway. Finally, although artificial, the fusion strategy used by Harris et al. [7] provides a simple method to force two proteins into a functional complex, which may have broad implications for converting other multifunctional proteins into pathway-specific forms. For example, fusions between kinases with particular substrates or small GTPases with specific effectors may provide useful tools to probe specific cellular functions, or limit their range to specific subcellular compartments. In addition, this approach may allow scientists to screen for molecules that impart specific roles to multifunctional proteins.
Dispatch R54
A
B P PP
P PP
Gβγ
C
P PP
Ste5p
Ste11p PP
PP
PP
PP
Ste7p PP
PP
Kss1p Fus3p
PP
Current Biology
MAP Kinases Actively Contribute to Pathway Specificity Although these new results demonstrate that scaffolds localize and channel MAP kinase activation, this cannot be the only mechanism to ensure pathway specificity. O’Rourke and Herskowitz [10] have shown that hog1∆ cells inappropriately activate the mating pathway in response to high extracellular osmolarity, despite the presence of functional scaffolds. Their study suggested that the MAP kinases themselves prevent crosstalk to other pathways. Support for this notion is provided by two recent papers in Current Biology and Molecular Cell, which show that pheromones not only activate the MAP kinase Fus3p, but also Kss1p [11,12]. However, although Kss1p contributes to the expression of pheromone-responsive genes, it is unable to induce filamentation genes in response to α-factor [11,13]. In contrast, filamentation genes are induced in fus3∆ cells, suggesting that Fus3p prevents crosstalk to the filamentation pathway (Figure 2b). While part of this regulation may occur at the level of transcription [14], new results suggest that Fus3p directly interferes with activation of other MAP kinases. Indeed, the extent and duration of Kss1p activity is increased in fus3∆ cells [12], suggesting that activation of Kss1p is inhibited by Fus3p. Although both Kss1p and Fus3p are phosphorylated early after pheromone addition, phosphorylation and activity of Kss1p decreased at later times. Thus, Fus3p may limit activation of Kss1p thereby blocking the filamentation pathway after prolonged α-factor treatment (Figure 2b). This observation resembles the situation for some mammalian MAP kinase pathways, where the magnitude and duration of MAP kinase activation may specify signal identity and determine the physiological outcome. The mechanism behind the interference of Fus3p with the activation of Kss1p is not known at present. It is possible that Fus3p induces or activates a Kss1p-specific phosphatase, or that negative feedback within the kinase cascade interferes with upstream regulators (see below). Negative Feedback Loops Restrict MAP Kinase Cascades Recent work has also highlighted the importance of negative feedback loops operating within MAP kinase
Figure 2. Distinct mechanisms contribute to pathway specificity. (A) Scaffolds interact with components of MAP kinase cascades and channel the activity towards specific MAP kinases. (B) Activated Fus3p interferes with the activation of Kss1p activation after prolonged exposure to α-factor, thereby preventing a filamentous response during mating. (C) Negative feedback loops operate on the upstream kinases Ste7p and Ste11p. These feedback mechanisms may temporally limit MAP kinase activation, and possibly prevent crosstalk between different MAP kinase pathways.
modules (Figure 2c). For example, Fus3p is able to phosphorylate its activator Ste7p [15], although the functional significance of this phosphorylation has not been investigated. Interestingly, a kinase-inactive Fus3p mutant shows increased phosphorylation on its activating residues, consistent with enhanced activity of one or both of the upstream kinases. Negative feedback in response to Fus3p activation also targets Ste11p. Using a reconstituted Fus3p kinase module, Breitkreuz et al. [11] show that Ste11p ‘disappears’ from the Ste5p scaffold in a Fus3p-dependent manner, suggesting that the complex is either disassembled after activation, or that Ste11p is degraded. Indeed, in contrast to Ste7p and Fus3p, Ste11p cannot be detected at mating projections (shmoo tips) [9], although its binding to Ste5p is required for signaling through the mating pathway. Recent results demonstrate that active Ste11p is indeed unstable in vivo and degraded in a MAP-kinase-dependent manner (our unpublished results). Thus, we speculate that downstream MAP kinases degrade activated Ste11p to prevent activated Ste11p from ‘spilling over’ into other pathways. Alternatively, downregulation of the upstream regulators Ste7p and Ste11p by activated Fus3p may limit its activation, thereby ensuring a transient biological response. MAP Kinases Possess Intrinsic Substrate Specificity While scaffold molecules and feedback loops ensure specific MAP kinase activation, they are unlikely to directly contribute to substrate recognition of MAP kinases. Photobleaching experiments revealed that activation of Fus3p triggers its dissociation from Ste5p [9]. In turn, activated Fus3p rapidly translocates into the nucleus in a scaffold-independent manner, implying that substrate specificity must be contained within the MAP kinases themselves. In contrast, Ste5p remains stably bound at sites of activation, suggesting that it may activate many Fus3p molecules and thus amplify the pheromone signal. Nuclear substrates of Fus3p include the cyclin-dependent kinase inhibitor Far1p [16] and the transcriptional repressors Dig1p and Dig2p [17,18]. Interestingly, Fus3p but not Kss1p is able to phosphorylate Far1p in vivo and in vitro, while both Kss1p and Fus3p phosphorylate the Dig
Current Biology R55
proteins [11]. As all MAP kinases phosphorylate very similar motifs with the minimal consensus sequence Ser/Thr–Pro [19], it is important to provide further specificity determinants. Indeed, many substrates interact with MAP kinases through conserved docking regions [20], which contribute to specificity by recruiting the kinases to the correct substrates and enhance their fidelity and efficiency of action. Little is known about these domains in yeast MAP kinases, and it will be important to identify mutants that specifically alter their substrate specificity. In summary, these new results reveal intriguing mechanisms, which together ensure MAP kinase pathway specificity in vivo (Figure 2). The papers highlight the particular importance of scaffold molecules and suggest that MAP kinases and feedback loops contribute to activation kinetics and pathway insulation. A future challenge will be to determine the MAP kinase substrates that are involved in pathway specificity, and to identify specific substrate docking sites on MAP kinases. Given the remarkable conservation of MAP kinase modules, these mechanisms are likely to serve as paradigms for the regulation of mammalian MAP kinase pathways. References 1. Whitmarsh, A.J. and Davis, R.J. (1998). Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals. Trends Biochem. Sci. 23, 481–485. 2. Gustin, M.C., Albertyn, J., Alexander, M., and Davenport, K. (1998). MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62, 1264–1300. 3. Stevenson, B.J., Rhodes, N., Errede, B., and Sprague, G.J. (1992). Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. Genes Dev. 6, 1293–1304. 4. van Drogen, F., O’Rourke, S., Stucke, V., and Peter, M. (2000). Phosphorylation of the MEKK Ste11p by the PAKlike kinase Ste20p is required for MAP kinase signaling in vivo. Curr. Biol. 10, 630–639. 5. Elion, E.A. (2000). Pheromone response, mating and cell biology. Curr. Opin. Microbiol. 3, 573–581. 6. Posas, F., and Saito, H. (1997). Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science 276, 1702–1705. 7. Harris, K., Lamson, R.E., Nelson, B., Marton, M.J., Roberts, C.J., Boone, C., and Pryciak, P.M. (2001) Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins. Curr. Biol. 11, 1815–1824. 8. Pryciak, P.M., and Huntress, F.A. (1998). Membrane recruitment of the kinase cascade scaffold protein Ste5 by the G beta gamma complex underlies activation of the yeast pheromone response pathway. Genes Dev. 12, 2684–2697. 9. van Drogen, F., Stucke, V., Jorritsma, G., and Peter, M. (2001) MAP kinase dynamics in response to pheromones in budding yeast. Nat. Cell Biol. 3, 1051–1059. 10. O’Rourke, S.M., and Herskowitz, I. (1998). The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev. 12, 2874–2886. 11. Breitkreutz, A., Boucher, L., and Tyers, M. (2001). MAPK specificity in the yeast pheromone response independent of transcriptional activation. Curr. Biol. 11, 1266–1271.
12. Sabbagh, W., Flatauer, L.J., Bardwell, A.J., and Bardwell, L. (2001) Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation. Mol. Cell 8, 683–691. 13. Roberts, C.J., Nelson, B., Marton, M.J., Stoughton, R., Meyer, M.R., Bennett, H.A., He, Y.D., Dai, H., Walker, W.L., et al. (2000). Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287, 873–880. 14. Madhani, H.D., Styles, C.A., and Fink, G.R. (1997). MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation. Cell 91, 673–684. 15. Zhou, Z., Gartner, A., Cade, R., Ammerer, G., and Errede, B. (1993). Pheromone-induced signal transduction in Saccharomyces cerevisiae requires the sequential function of three protein kinases. Mol. Cell. Biol. 13, 2069–2080. 16. Peter, M., Gartner, A., Horecka, J., Ammerer, G., and Herskowitz, I. (1993). FAR1 links the signal transduction pathway to the cell cycle machinery in yeast. Cell 73, 747–760. 17. Cook, J.G., Bardwell, L., Kron, S.J., and Thorner, J. (1996). Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae. Genes Dev. 10, 2831–2848. 18. Tedford, K., Kim, S., Sa, D., Stevens, K., and Tyers, M. (1997). Regulation of the mating pheromone and invasive growth responses in yeast by two MAP kinase substrates. Curr. Biol. 7, 228–238. 19. Countaway, J.L., Northwood, I.C., Davis, R.J. (1989). Mechanism of phosphorylation of the epidermal growth factor receptor at threonine 669. J. Biol. Chem. 264, 10828–10835. 20. Sharrocks, A.D., Yang, S.H., and Galanis, A. (2002). Docking domains and substrate-specificity determination for MAP kinases. Trends Biochem. Sci. 25, 448–453.
MAP Kinase Cascades: Scaffolding Signal Specificity
Mitogen-activated protein (MAP) kinases regulate a variety of cellular processes in response to extracellular and intracellular signals. MAP kinases are activated through a protein kinase cascade in which a MAP kinase kinase kinase (or MEKK) activates a MAP kinase kinase (or MEK) that in turn activates the MAP kinase by phosphorylation. These MAP kinase modules are often associated with scaffold proteins, but the molecular roles of these scaffolds has remained elusive [1]. In yeast, distinct MAP kinase pathways regulate mating, invasive growth and the response to high osmolarity (Figure 1) [2]. Because the MEKK Ste11p functions in all three MAP kinase pathways, a problem of specificity arises: how does Ste11p know by which upstream signal it has been activated (referred to as pathway specificity)? Indeed, mutationally activated Ste11p simultaneously induces all three pathways [3,4]. Recent work now demonstrates that pathway specificity is mediated at least in part by scaffold proteins. Scaffolds Localize and Insulate Specific MAP Kinase Pathways In the mating pathway, the scaffold protein Ste5p associates with the MEKK Ste11p, the MEK Ste7p and the MAP kinase Fus3p (Figure 1) [5]. Similarly, the MEK Pbs2p functions as a scaffold, and interacts with Ste11p and the MAP kinase Hog1p in the high osmolarity-glycerol pathway [6]. So far, a scaffold protein has not been identified for the filamentation pathway. In a paper published recently in Current Biology, Harris et al. [7] used a fusion approach to probe the biological function of scaffold proteins. First, they generated fusion proteins between active Ste11p and either of the scaffold proteins Ste5p or Pbs2p. Strikingly, the Ste11p–Ste5p hybrid specifically induces the mating pathway by activating Fus3p, whereas the Ste11p–Pbs2p hybrid induces a high osmolarity response by activating Hog1p. Thus, activated Ste11p becomes confined to a particular pathway when attached to a pathway-specific scaffold, Swiss Institute for Experimental Cancer Research (ISREC), Ch. des Boveresses 155, 1066 Epalinges/VD, Switzerland. 1E-mail: [email protected]; 2E-mail: [email protected]
Dispatch
High osmolarity
Low nitrogen
Ste11p MEKK Ste7p
Fus3p Mating reaction
Pbs2p
Scaffold proteins organize many MAP kinase pathways by interacting with several components of these cascades. Recent studies suggest that scaffold proteins provide local activation platforms that contribute to signal specificity by insulating different MAP kinase pathways.
Pheromone
Ste5p
Frank van Drogen1 and Matthias Peter2
PII S0960-9822(01)00672-8
MEK
Hog1p Glycerol production
Kss1p
MAP kinase
Invasive/pseudohyphal growth Current Biology
Figure 1. Schematic overview of yeast MAP kinase modules that share Ste11p. Extracellular signals such as mating pheromones, osmotic stress and low nitrogen activate specific MAP kinase modules. Ste5p and Pbs2p function as scaffold proteins that organize and insulate the individual components. For further details see the text.
demonstrating that scaffolds actively channel signaling towards the appropriate MAP kinase (Figure 2a). Moreover, these results show that scaffold proteins exclude potential substrates from access to an activated kinase. Harris et al. [7] then analyzed fusions between Ste11p and its downstream kinases Ste7p or Pbs2p. Intriguingly, the Ste11p–Ste7p fusion protein can mimic attachment of Ste11p to the scaffold Ste5p, implying that scaffolds dictate substrate use and promote signaling specificity by presenting a preferred substrate in high concentration. Because scaffolds are able to actively recruit MAP kinase modules to specific subcellular sites [8,9], they also contribute to the spatial organization of MAP kinase signaling. Taken together, these results demonstrate experimentally that scaffolds localize specific MAP kinase modules and insulate signaling networks in vivo. Such pathway insulation is physiologically important, as cells expressing activated Ste11p exhibit a poor mating response because of simultaneous activation of the Hog1p pathway. Finally, although artificial, the fusion strategy used by Harris et al. [7] provides a simple method to force two proteins into a functional complex, which may have broad implications for converting other multifunctional proteins into pathway-specific forms. For example, fusions between kinases with particular substrates or small GTPases with specific effectors may provide useful tools to probe specific cellular functions, or limit their range to specific subcellular compartments. In addition, this approach may allow scientists to screen for molecules that impart specific roles to multifunctional proteins.
Dispatch R54
A
B P PP
P PP
Gβγ
C
P PP
Ste5p
Ste11p PP
PP
PP
PP
Ste7p PP
PP
Kss1p Fus3p
PP
Current Biology
MAP Kinases Actively Contribute to Pathway Specificity Although these new results demonstrate that scaffolds localize and channel MAP kinase activation, this cannot be the only mechanism to ensure pathway specificity. O’Rourke and Herskowitz [10] have shown that hog1∆ cells inappropriately activate the mating pathway in response to high extracellular osmolarity, despite the presence of functional scaffolds. Their study suggested that the MAP kinases themselves prevent crosstalk to other pathways. Support for this notion is provided by two recent papers in Current Biology and Molecular Cell, which show that pheromones not only activate the MAP kinase Fus3p, but also Kss1p [11,12]. However, although Kss1p contributes to the expression of pheromone-responsive genes, it is unable to induce filamentation genes in response to α-factor [11,13]. In contrast, filamentation genes are induced in fus3∆ cells, suggesting that Fus3p prevents crosstalk to the filamentation pathway (Figure 2b). While part of this regulation may occur at the level of transcription [14], new results suggest that Fus3p directly interferes with activation of other MAP kinases. Indeed, the extent and duration of Kss1p activity is increased in fus3∆ cells [12], suggesting that activation of Kss1p is inhibited by Fus3p. Although both Kss1p and Fus3p are phosphorylated early after pheromone addition, phosphorylation and activity of Kss1p decreased at later times. Thus, Fus3p may limit activation of Kss1p thereby blocking the filamentation pathway after prolonged α-factor treatment (Figure 2b). This observation resembles the situation for some mammalian MAP kinase pathways, where the magnitude and duration of MAP kinase activation may specify signal identity and determine the physiological outcome. The mechanism behind the interference of Fus3p with the activation of Kss1p is not known at present. It is possible that Fus3p induces or activates a Kss1p-specific phosphatase, or that negative feedback within the kinase cascade interferes with upstream regulators (see below). Negative Feedback Loops Restrict MAP Kinase Cascades Recent work has also highlighted the importance of negative feedback loops operating within MAP kinase
Figure 2. Distinct mechanisms contribute to pathway specificity. (A) Scaffolds interact with components of MAP kinase cascades and channel the activity towards specific MAP kinases. (B) Activated Fus3p interferes with the activation of Kss1p activation after prolonged exposure to α-factor, thereby preventing a filamentous response during mating. (C) Negative feedback loops operate on the upstream kinases Ste7p and Ste11p. These feedback mechanisms may temporally limit MAP kinase activation, and possibly prevent crosstalk between different MAP kinase pathways.
modules (Figure 2c). For example, Fus3p is able to phosphorylate its activator Ste7p [15], although the functional significance of this phosphorylation has not been investigated. Interestingly, a kinase-inactive Fus3p mutant shows increased phosphorylation on its activating residues, consistent with enhanced activity of one or both of the upstream kinases. Negative feedback in response to Fus3p activation also targets Ste11p. Using a reconstituted Fus3p kinase module, Breitkreuz et al. [11] show that Ste11p ‘disappears’ from the Ste5p scaffold in a Fus3p-dependent manner, suggesting that the complex is either disassembled after activation, or that Ste11p is degraded. Indeed, in contrast to Ste7p and Fus3p, Ste11p cannot be detected at mating projections (shmoo tips) [9], although its binding to Ste5p is required for signaling through the mating pathway. Recent results demonstrate that active Ste11p is indeed unstable in vivo and degraded in a MAP-kinase-dependent manner (our unpublished results). Thus, we speculate that downstream MAP kinases degrade activated Ste11p to prevent activated Ste11p from ‘spilling over’ into other pathways. Alternatively, downregulation of the upstream regulators Ste7p and Ste11p by activated Fus3p may limit its activation, thereby ensuring a transient biological response. MAP Kinases Possess Intrinsic Substrate Specificity While scaffold molecules and feedback loops ensure specific MAP kinase activation, they are unlikely to directly contribute to substrate recognition of MAP kinases. Photobleaching experiments revealed that activation of Fus3p triggers its dissociation from Ste5p [9]. In turn, activated Fus3p rapidly translocates into the nucleus in a scaffold-independent manner, implying that substrate specificity must be contained within the MAP kinases themselves. In contrast, Ste5p remains stably bound at sites of activation, suggesting that it may activate many Fus3p molecules and thus amplify the pheromone signal. Nuclear substrates of Fus3p include the cyclin-dependent kinase inhibitor Far1p [16] and the transcriptional repressors Dig1p and Dig2p [17,18]. Interestingly, Fus3p but not Kss1p is able to phosphorylate Far1p in vivo and in vitro, while both Kss1p and Fus3p phosphorylate the Dig
Current Biology R55
proteins [11]. As all MAP kinases phosphorylate very similar motifs with the minimal consensus sequence Ser/Thr–Pro [19], it is important to provide further specificity determinants. Indeed, many substrates interact with MAP kinases through conserved docking regions [20], which contribute to specificity by recruiting the kinases to the correct substrates and enhance their fidelity and efficiency of action. Little is known about these domains in yeast MAP kinases, and it will be important to identify mutants that specifically alter their substrate specificity. In summary, these new results reveal intriguing mechanisms, which together ensure MAP kinase pathway specificity in vivo (Figure 2). The papers highlight the particular importance of scaffold molecules and suggest that MAP kinases and feedback loops contribute to activation kinetics and pathway insulation. A future challenge will be to determine the MAP kinase substrates that are involved in pathway specificity, and to identify specific substrate docking sites on MAP kinases. Given the remarkable conservation of MAP kinase modules, these mechanisms are likely to serve as paradigms for the regulation of mammalian MAP kinase pathways. References 1. Whitmarsh, A.J. and Davis, R.J. (1998). Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals. Trends Biochem. Sci. 23, 481–485. 2. Gustin, M.C., Albertyn, J., Alexander, M., and Davenport, K. (1998). MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62, 1264–1300. 3. Stevenson, B.J., Rhodes, N., Errede, B., and Sprague, G.J. (1992). Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. Genes Dev. 6, 1293–1304. 4. van Drogen, F., O’Rourke, S., Stucke, V., and Peter, M. (2000). Phosphorylation of the MEKK Ste11p by the PAKlike kinase Ste20p is required for MAP kinase signaling in vivo. Curr. Biol. 10, 630–639. 5. Elion, E.A. (2000). Pheromone response, mating and cell biology. Curr. Opin. Microbiol. 3, 573–581. 6. Posas, F., and Saito, H. (1997). Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science 276, 1702–1705. 7. Harris, K., Lamson, R.E., Nelson, B., Marton, M.J., Roberts, C.J., Boone, C., and Pryciak, P.M. (2001) Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins. Curr. Biol. 11, 1815–1824. 8. Pryciak, P.M., and Huntress, F.A. (1998). Membrane recruitment of the kinase cascade scaffold protein Ste5 by the G beta gamma complex underlies activation of the yeast pheromone response pathway. Genes Dev. 12, 2684–2697. 9. van Drogen, F., Stucke, V., Jorritsma, G., and Peter, M. (2001) MAP kinase dynamics in response to pheromones in budding yeast. Nat. Cell Biol. 3, 1051–1059. 10. O’Rourke, S.M., and Herskowitz, I. (1998). The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev. 12, 2874–2886. 11. Breitkreutz, A., Boucher, L., and Tyers, M. (2001). MAPK specificity in the yeast pheromone response independent of transcriptional activation. Curr. Biol. 11, 1266–1271.
12. Sabbagh, W., Flatauer, L.J., Bardwell, A.J., and Bardwell, L. (2001) Specificity of MAP kinase signaling in yeast differentiation involves transient versus sustained MAPK activation. Mol. Cell 8, 683–691. 13. Roberts, C.J., Nelson, B., Marton, M.J., Stoughton, R., Meyer, M.R., Bennett, H.A., He, Y.D., Dai, H., Walker, W.L., et al. (2000). Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287, 873–880. 14. Madhani, H.D., Styles, C.A., and Fink, G.R. (1997). MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation. Cell 91, 673–684. 15. Zhou, Z., Gartner, A., Cade, R., Ammerer, G., and Errede, B. (1993). Pheromone-induced signal transduction in Saccharomyces cerevisiae requires the sequential function of three protein kinases. Mol. Cell. Biol. 13, 2069–2080. 16. Peter, M., Gartner, A., Horecka, J., Ammerer, G., and Herskowitz, I. (1993). FAR1 links the signal transduction pathway to the cell cycle machinery in yeast. Cell 73, 747–760. 17. Cook, J.G., Bardwell, L., Kron, S.J., and Thorner, J. (1996). Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae. Genes Dev. 10, 2831–2848. 18. Tedford, K., Kim, S., Sa, D., Stevens, K., and Tyers, M. (1997). Regulation of the mating pheromone and invasive growth responses in yeast by two MAP kinase substrates. Curr. Biol. 7, 228–238. 19. Countaway, J.L., Northwood, I.C., Davis, R.J. (1989). Mechanism of phosphorylation of the epidermal growth factor receptor at threonine 669. J. Biol. Chem. 264, 10828–10835. 20. Sharrocks, A.D., Yang, S.H., and Galanis, A. (2002). Docking domains and substrate-specificity determination for MAP kinases. Trends Biochem. Sci. 25, 448–453.