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1.
Kwan JJ Warner N Maini J Chan Tung KW Zakaria H Pawson T Donaldson LW 《Journal of molecular biology》2006,356(1):142-154
In Saccharomyces cerevisiae, signal transduction through pathways governing mating, osmoregulation, and nitrogen starvation depends upon a direct interaction between the sterile alpha motif (SAM) domains of the Ste11 mitogen-activated protein kinase kinase kinase (MAPKKK) and its regulator Ste50. Previously, we solved the NMR structure of the SAM domain from Ste11 and identified two mutants that diminished binding to the Ste50 SAM domain. Building upon the Ste11 study, we present the NMR structure of the monomeric Ste50 SAM domain and a series of mutants bearing substitutions at surface-exposed hydrophobic amino acid residues. The mid-loop (ML) region of Ste11-SAM, defined by helices H3 and H4 and the end-helix (EH) region of Ste50-SAM, defined by helix H5, were sensitive to substitution, indicating that these two surfaces contribute to the high-affinity interaction. The combination of two mutants, Ste11-SAM-L72R and Ste50-SAM-L69R, formed a high-affinity heterodimer unencumbered by competing homotypic interactions that had prevented earlier NMR studies of the wild-type complex. Yeast bearing mutations that prevented the heterotypic Ste11-Ste50 association in vitro presented signaling defects in the mating and high-osmolarity growth pathways. 相似文献
2.
Ramachander R Kim CA Phillips ML Mackereth CD Thanos CD McIntosh LP Bowie JU 《The Journal of biological chemistry》2002,277(42):39585-39593
SAM (sterile alpha motif) domains are protein-protein interaction modules found in a large number of regulatory proteins. Byr2 and Ste4 are two SAM domain-containing proteins in the mating pheromone response pathway of the fission yeast, Schizosaccharomyces pombe. Byr2 is a mitogen-activated protein kinase kinase kinase that is regulated by Ste4. Tu et al. (Tu, H., Barr, M., Dong, D. L., and Wigler, M. (1997) Mol. Cell. Biol. 17, 5876-5887) showed that the isolated SAM domain of Byr2 binds a fragment of Ste4 that contains both a leucine zipper (Ste4-LZ) domain as well as a SAM domain, suggesting that Byr2-SAM and Ste4-SAM may form a hetero-oligomer. Here, we show that the individual SAM domains of Ste4 and Byr2 are monomeric at low concentrations and bind to each other in a 1:1 stoichiometry with a relatively weak dissociation constant of 56 +/- 3 microm. Inclusion of the Ste4-LZ domain, which determines the oligomeric state of Ste4, has a dramatic effect on binding affinity, however. We find that the Ste4-LZ domain is trimeric and, when included with the Ste4-SAM domain, yields a 3:1 Ste4-LZ-SAM:Byr2-SAM complex with a tight dissociation constant of 19 +/- 4 nm. These results suggest that the Ste4-LZ-SAM protein may recognize multiple binding sites on Byr2-SAM, indicating a new mode of oligomeric organization for SAM domains. The fact that high affinity binding occurs only with the addition of an oligomerization domain suggests that it may be necessary to include ancillary oligomerization modules when searching for binding partners of SAM domains. 相似文献
3.
The mitogen-activated protein kinase (MAPK) Byr2 and its activator Ste4 are involved in the mating pheromone response pathway of Schizosaccharomyces pombe and interact via their SAM domains. SAM domains can self-associate to form higher-order structures, including dimers, polymers and closed oligomers. Ste4-SAM is adjacent to a trimeric leucine zipper domain and we have shown previously that the two domains together (Ste4-LZ-SAM) bind to a monomeric Byr2-SAM with high affinity (Kd approximately 20 nM), forming a 3:1 complex. Here, we map the surfaces of Byr2-SAM and Ste4-SAM that is involved the interaction. A set of 38 mutants of Byr2-SAM and 33 mutants of Ste4-SAM were prepared, covering most of the protein surfaces. These mutants were purified and screened for binding, yielding a map of residues that are required for binding and a complementary map of residues that are not required. We find that the interface maps to regions of the SAM domains that are known to be important for the formation of SAM polymers. These results indicate that SAM domains can create a variety of oligomeric architectures utilizing common binding surfaces. 相似文献
4.
Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae
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Cunle Wu Ekkehard Leberer David Y. Thomas Malcolm Whiteway 《Molecular biology of the cell》1999,10(7):2425-2440
The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as the Schizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity. 相似文献
5.
6.
Bhattacharjya S Xu P Gingras R Shaykhutdinov R Wu C Whiteway M Ni F 《Journal of molecular biology》2004,344(4):1071-1087
Ste11, a homologue of mammalian MAPKKKs, together with its binding partner Ste50 works in a number of MAPK signaling pathways of Saccharomyces cerevisiae. Ste11/Ste50 binding is mediated by their sterile alpha motifs or SAM domains, of which homologues are also found in many other intracellular signaling and regulatory proteins. Here, we present the solution structure of the SAM domain or residues D37-R104 of Ste11 and its interactions with the cognate SAM domain-containing region of Ste50, residues M27-Q131. NMR pulse-field-gradient (PFG) and rotational correlation time measurements (tauc) establish that the Ste11 SAM domain exists predominantly as a symmetric dimer in solution. The solution structure of the dimeric Ste11 SAM domain consists of five well-defined helices per monomer packed into a compact globular structure. The dimeric structure of the SAM domain is maintained by a novel dimer interface involving interactions between a number of hydrophobic residues situated on helix 4 and at the beginning of the C-terminal long helix (helix 5). The dimer structure may also be stabilized by potential salt bridge interactions across the interface. NMR H/2H exchange experiments showed that binding of the Ste50 SAM to the Ste11 SAM very likely involves the positively charged extreme C-terminal region as well as exposed hydrophobic patches of the dimeric Ste11 SAM domain. The dimeric structure of the Ste11 SAM and its interactions with the Ste50 SAM may have important roles in the regulation and activation of the Ste11 kinase and signal transmission and amplifications through the Ste50-Ste11 complex. 相似文献
7.
8.
M. Ramezani Rad G. Jansen F. Bühring C. P. Hollenberg 《Molecular genetics and genomics : MGG》1998,259(1):29-38
STE50 is required to sustain pheromone-induced signal transduction in?S. cerevisiae. Here we report that Ste50p is involved in regulating pseudohyphal development. Both of these processes are also dependent on Ste11p. Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p. Overexpression of STE11 also suppresses the mating defects of ste50 mutants. We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various conditions. A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated, as well as from normally growing cells. Formation of the Ste50p-Ste11p complex does not require Gα, Gβ, Ste20p or Ste5p. Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and occurs in the absence of Ste50p. We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation programmes: mating and filamentous growth. 相似文献
9.
Grimshaw SJ Mott HR Stott KM Nielsen PR Evetts KA Hopkins LJ Nietlispach D Owen D 《The Journal of biological chemistry》2004,279(3):2192-2201
10.
M. Ramezani Rad G. Jansen F. Bühring C. P. Hollenberg 《Molecular & general genetics : MGG》1998,259(1):29-38
STE50 is required to sustain pheromone-induced signal transduction in S. cerevisiae. Here we report that Ste50p is involved in regulating pseudohyphal development. Both of these processes are also dependent
on Ste11p. Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p. Overexpression of STE11 also suppresses the mating defects of ste50 mutants. We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various
conditions. A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated,
as well as from normally growing cells. Formation of the Ste50p-Ste11p complex does not require Gα, Gβ, Ste20p or Ste5p. Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and
occurs in the absence of Ste50p. We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation
programmes: mating and filamentous growth.
Received: 20 February 1998 / Accepted: 17 March 1998 相似文献
11.
The RA domain of Ste50 adaptor protein is required for delivery of Ste11 to the plasma membrane in the filamentous growth signaling pathway of the yeast Saccharomyces cerevisiae 总被引:1,自引:0,他引:1
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In Saccharomyces cerevisiae, pheromone response requires Ste5 scaffold protein, which ensures efficient G-protein-dependent recruitment of mitogen-activated protein kinase (MAPK) cascade components Ste11 (MAPK kinase kinase), Ste7 (MAPK kinase), and Fus3 (MAPK) to the plasma membrane for activation by Ste20 protein kinase. Ste20, which phosphorylates Ste11 to initiate signaling, is activated by binding to Cdc42 GTPase (membrane anchored via its C-terminal geranylgeranylation). Less clear is how activated and membrane-localized Ste20 contacts Ste11 to trigger invasive growth signaling, which also requires Ste7 and the MAPK Kss1, but not Ste5. Ste50 protein associates constitutively via an N-terminal sterile-alpha motif domain with Ste11, and this interaction is required for optimal invasive growth and hyperosmotic stress (high-osmolarity glycerol [HOG]) signaling but has a lesser role in pheromone response. We show that a conserved C-terminal, so-called "Ras association" (RA) domain in Ste50 is also essential for invasive growth and HOG signaling in vivo. In vitro the Ste50 RA domain is not able to associate with Ras2, but it does associate with Cdc42 and binds to a different face than does Ste20. RA domain function can be replaced by the nine C-terminal, plasma membrane-targeting residues (KKSKKCAIL) of Cdc42, and membrane-targeted Ste50 also suppresses the signaling deficiency of cdc42 alleles specifically defective in invasive growth. Thus, Ste50 serves as an adaptor to tether Ste11 to the plasma membrane and can do so via association with Cdc42, thereby permitting the encounter of Ste11 with activated Ste20. 相似文献
12.
Jansen G Bühring F Hollenberg CP Ramezani Rad M 《Molecular genetics and genomics : MGG》2001,265(1):102-117
In Saccharomyces cerevisiae, the MAPKKK Ste11p is involved in three mitogen-activated protein kinase (MAPK) pathways required for mating, filamentous growth and the SHO1-dependent response to hyperosmolarity. All three pathways are also dependent on Ste50p. Ste50p and Ste11p interact constitutively via their N-terminal regions, which include putative SAM domains. Here we show that the interaction of Ste50p and Ste11p is differentially required for modulation of Ste11p function during mating, filamentous growth and the SHO1-dependent response to hyperosmolarity. Two derivatives of Ste50p with mutations in the SAM domain were isolated and characterised. The mutant Ste50 proteins showed reduced binding to Ste11p and a tendency to form homodimers in two-hybrid and in vitro binding assays. Interestingly, these two Ste50p-SAM mutants were associated with increased activation of the mating and filamentous-growth pathways, but a reduction in the SHO1-dependent growth response to hyperosmolarity, relative to the wild-type Ste50p. Moreover, when exposed to hyperosmolarity, these Ste50p-SAM mutants activate genes in the mating (FUS1) and filamentous-growth (FLO11) pathways to higher levels than does the wild type. Thus the Ste50p-Ste11p interaction may differentially modulate the flow of information through the various MAPK-mediated pathways. 相似文献
13.
14.
Gang Xu Gregor Jansen David Y. Thomas Cornelis P. Hollenberg Massoud Ramezani Rad 《Molecular microbiology》1996,20(4):773-783
In the yeast Saccharomyces cerevisiae, the hetero-trimeric G protein transduces the mating pheromone signal from a cell-surface receptor. Free Gβγ then activates a mitogen-activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal-transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone-induced signal which leads cells to hormone-induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone-induced signal transduction, and overexpression of STE50 intensifies the pheromone-induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the α-pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two-hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a new component, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP kinase complex in S. cerevisiae. 相似文献
15.
Jung KW Kim SY Okagaki LH Nielsen K Bahn YS 《Fungal genetics and biology : FG & B》2011,48(2):154-165
The mitogen-activated protein kinase (MAPK) pathways control diverse cellular functions in pathogenic fungi, including sexual differentiation, stress response, and maintenance of cell wall integrity. Here we characterized a Cryptococcus neoformans gene, which is homologous to the yeast Ste50 that is known to play an important role in mating pheromone response and stress response as an adaptor protein to the Ste11 MAPK kinase kinase in Saccharomyces cerevisiae. The C. neoformans Ste50 was not involved in any of the stress responses or virulence factor production (capsule and melanin) that are controlled by the HOG and Ras/cAMP signaling pathways. However, Ste50 was required for mating in both serotype A and serotype D C. neoformans strains. The ste50Δ mutant was completely defective in cell-cell fusion and mating pheromone production. Double mutation of the STE50 gene blocked increased production of pheromone and the hyper-filamentation phenotype of cells deleted of the CRG1 gene, which encodes the RGS protein that negatively regulates pheromone responsive G-protein signaling via the MAPK pathway. Regardless of the presence of the basidiomycota-specific SH3 domains of Ste50 that are known to be required for full virulence of Ustilago maydis, Ste50 was dispensable for virulence of C. neoformans in a murine model of cryptococcosis. In conclusion, the Ste50 adaptor protein controls sexual differentiation of C. neoformans via the pheromone-responsive MAPK pathway but is not required for virulence. 相似文献
16.
The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated G beta gamma complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway. 相似文献
17.
Ongay-Larios L Saviñón-Tejeda AL Williamson MJ Durán-Avelar Md Coria R 《FEBS letters》2000,467(1):22-26
In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered alpha pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the beta propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and alpha cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone-stimulated receptor and that this domain could be an important point for physical interaction between the Gbeta and the Galpha subunits. 相似文献
18.
The sterile alpha motif or SAM domain is one of the most frequently present protein interaction modules with diverse functional attributions. SAM domain of the Ste11 protein of budding yeast plays important roles in mitogen‐activated protein kinase cascades. In the current study, urea‐induced, at subdenaturing concentrations, structural, and dynamical changes in the Ste11 SAM domain have been investigated by nuclear magnetic resonance spectroscopy. Our study revealed that a number of residues from Helix 1 and Helix 5 of the Ste11 SAM domain display plausible alternate conformational states and largest chemical shift perturbations at low urea concentrations. Amide proton (H/D) exchange experiments indicated that Helix 1, loop, and Helix 5 become more susceptible to solvent exchange with increased concentrations of urea. Notably, Helix 1 and Helix 5 are directly involved in binding interactions of the Ste11 SAM domain. Our data further demonstrate that the existence of alternate conformational states around the regions involved in dimeric interactions in native or near native conditions. Proteins 2014; 82:2957–2969. © 2014 Wiley Periodicals, Inc. 相似文献
19.
Genetic analysis of the interface between Cdc42p and the CRIB domain of Ste20p in Saccharomyces cerevisiae 总被引:2,自引:0,他引:2
Mutagenesis was used to probe the interface between the small GTPase Cdc42p and the CRIB domain motif of Ste20p. Members of a cluster of hydrophobic residues of Cdc42p were changed to alanine and/or arginine. The interaction of the wild-type and mutant proteins was measured using the two-hybrid assay; many, but not all, changes reduced interaction between Cdc42p and the target CRIB domain. Mutations in conserved residues in the CRIB domain were also tested for their importance in the association with Cdc42p. Two conserved CRIB domain histidines were changed to aspartic acid. These mutants reduced mating, as well as responsiveness to pheromone-induced gene expression and cell cycle arrest, but did not reduce in vitro the kinase activity of Ste20p. GFP-tagged mutant proteins were unable to localize to sites of polarized growth. In addition, these point mutants were synthetically lethal with disruption of CLA4 and blocked the Ste20p-Cdc42p two-hybrid interaction. Compensatory mutations in Cdc42p that reestablished the two-hybrid association with the mutant Ste20p CRIB domain baits were identified. These mutations improved the pheromone responsiveness of cells containing the CRIB mutations, but did not rescue the lethality associated with the CRIB mutant CLA4 deletion interaction. These results suggest that the Ste20p-Cdc42p interaction plays a direct role in Ste20p kinase function and that this interaction is required for efficient activity of the pheromone response pathway. 相似文献
20.
SAM domain-based protein oligomerization observed by live-cell fluorescence fluctuation spectroscopy
Sterile-alpha-motif (SAM) domains are common protein interaction motifs observed in organisms as diverse as yeast and human. They play a role in protein homo- and hetero-interactions in processes ranging from signal transduction to RNA binding. In addition, mutations in SAM domain and SAM-mediated oligomers have been linked to several diseases. To date, the observation of heterogeneous SAM-mediated oligomers in vivo has been elusive, which represents a common challenge in dissecting cellular biochemistry in live-cell systems. In this study, we report the oligomerization and binding stoichiometry of high-order, multi-component complexes of (SAM) domain proteins Ste11 and Ste50 in live yeast cells using fluorescence fluctuation methods. Fluorescence cross-correlation spectroscopy (FCCS) and 1-dimensional photon counting histogram (1dPCH) confirm the SAM-mediated interaction and oligomerization of Ste11 and Ste50. Two-dimensional PCH (2dPCH), with endogenously expressed proteins tagged with GFP or mCherry, uniquely indicates that Ste11 and Ste50 form a heterogeneous complex in the yeast cytosol comprised of a dimer of Ste11 and a monomer of Ste50. In addition, Ste50 also exists as a high order oligomer that does not interact with Ste11, and the size of this oligomer decreases in response to signals that activate the MAP kinase cascade. Surprisingly, a SAM domain mutant of Ste50 disrupted not only the Ste50 oligomers but also Ste11 dimerization. These results establish an in vivo model of Ste50 and Ste11 homo- and hetero-oligomerization and highlight the usefulness of 2dPCH for quantitative dissection of complex molecular interactions in genetic model organisms such as yeast. 相似文献