Journal of Molecular Biology
Solution Structure of the Dimeric SAM Domain of MAPKKK Ste11 and its Interactions with the Adaptor Protein Ste50 from the Budding Yeast: Implications for Ste11 Activation and Signal Transmission Through the Ste50–Ste11 Complex
Introduction
The mitogen-activated protein kinase (MAPK) cascades are well-conserved signaling modules found in all eukaroytic cells.1 MAPK-mediated signal transduction networks have been shown to transmit many extracellular stimuli from the cell surface to the nucleus and to elicit a wide variety of cellular responses including protein synthesis, cell proliferation, differentiation and changes in morphology. The MAPK modules typically consist of three kinases; a MAPK, a MAPKK (also known as ERK or MEK), and a MAPKKK (or MEKK). In response to plasma membrane originated signals, components of these kinase modules are activated sequentially through phosphorylations. Initially, an upstream and membrane-associated kinase, also called MAPKKKK, activates a downstream kinase, the MAPKKK. This activated MAPKKK phosphorylates the downstream MAPKK (a kinase with dual serine/threonine and tyrosine specificity) that in turn activates the MAPK.2, 3 Once activated, the MAPK phosphorylates a number of transcription regulators leading to transcriptional activation of stimuli-specific genes. Therefore, regulation of these kinase modules is critical for signal transmission, pathway specificity and kinetics of signaling responses. Classically, such regulation is often imparted by protein interaction domains that may either work in cis (part of the same kinase) or in trans (mediated by other proteins). These protein interaction domains function through their unique abilities to recognize a variety of binding partners, effectively controlling the signaling cascades.4, 5
The budding yeast Saccharomyces cerevisiae has been used extensively as a model organism for functional analyses of MAPK signal transduction cascades. These studies revealed that there are up to six MAPK modules in yeast, regulating pheromone responses, filamentation, high-osmolarity growth (HOG), cell wall integrity and sporulation.6, 7 Among the three kinase components of the MAPK modules, the MAPKKK Ste11 is critical, since it is shared by almost all the pathways and activates many other downstream kinases.6 Previous studies using the yeast two-hybrid system and analyses of interaction domains have demonstrated that Ste11 has a modular architecture composed of several functional domains. The N-terminal half of the protein is involved predominantly in regulatory interactions and is implicated in binding to an adaptor protein Ste508, 9 and to a scaffolding protein Ste5.10, 11 The interaction between Ste11 and Ste50 is required for the activation of the Ste11 kinase.12, 13, 14 Sequence alignments and in-depth genetic and biochemical characterization have established that relatively short N-terminal segments containing SAM (sterile alpha motif) domains are responsible for the interactions between Ste11 and Ste50.14, 15, 16
The SAM domain has now been established as a widespread “protein interaction” domain of ∼70 residues involved in diverse functions ranging from signal transduction to transciptional repression.4, 17 Extensive structural analyses of a number of SAM domains have demonstrated that these domains can mediate homo or hetero oligomerizations using different modes of association.18, 19, 20 Extensive homo-oligomerization of SAM domains leads to formation of helical filaments from the polycomb group of proteins and the Ets family of transcriptional repressor proteins (TEL),21, 22 while monomeric solution structures have been deduced for the SAM domains from proteins involved in transcriptional regulation, i.e. Ets-1, GABPα, Erg,23, 24 and from the tumor suppressor protein p73.25 Very recently, a monomeric solution structure of the Ste50 SAM domain consisting of residues 27–108 has been reported.26 SAM domains have also been shown to interact with other protein motifs, such as PDZ domains,27 SH2 domains,28 Cdc2 family of kinases29 and the MAP kinase, Erk 2.30 Apart from protein–protein interactions, SAM domains of the post-transcriptional regulator Smg protein from Drosophila melanogaster have now been shown to even bind RNA.31, 32
Here, we have determined the three-dimensional solution structure of the dimeric SAM domain of Ste11 by use of NMR spectroscopy. To our knowledge, this is the first report on the solution structure of a dimeric SAM domain. The interactions between the SAM domains of Ste11 and Ste50 were further studied by native polyacrylamide gel electrophoresis, glutathione-S-transferase (GST)-pull down and NMR H/2H exchange experiments. Our results suggest that the dimerization of the Ste11 kinase may be nucleated by the unique dimeric structure of the Ste11 SAM domain and the dimeric Ste11 SAM may further associate with Ste50 to initiate signaling events through the yeast MAPK cascades.
Section snippets
Resonance assignments of the SAM domain of Ste11
Figure 1 shows a heteronuclear single quantum coherence (HSQC) spectrum of the D37-R104 (labeled as from residue 1–68) domain of Ste11, indicating backbone HN and 15N cross-peaks of individual residues. A single peak for each residue in the well-dispersed HSQC spectrum suggests a uniquely folded structure for the SAM domain of Ste11. Assignments for the backbone HN, 15N, 13Cα, 13Cβ and 13C′ resonances were achieved by a combined analysis of the triple-resonance HNCA, HN(CO)CA, CBCA(CO)NH and
A novel dimeric structure of the Ste11 SAM domain
In the past few years, several three-dimensional structures of the SAM domains have been determined, which include the monomeric solution structures of the Ste50 SAM domain,26 the EphB2 SAM domain,19 the p73 SAM domain25 and the Ets SAM domain,23 a closed dimeric structure of the EphA4 receptor SAM domain20 and the open polymeric structures of SAM domains from transcriptional repressor proteins TEL21 and ph.22 Each subunit of the dimeric Ste11 SAM domain has a tertiary fold very similar to
Expression and purification of the Ste11 and Ste50 SAM domains
DNA sequences corresponding to the SAM domains of Ste11 (D37–R104, numbering according to Swiss-Prot accession number P23561) and Ste50 (M27–Q131, numbering according to Swiss-Prot accession number P25344) were amplified by PCR from the genomic DNA of the budding yeast and cloned into pET14b and pGEX-2T vectors with a thrombin-cleavable N-terminal His6-tag (Qiagen) and GST-tag (Pharmacia), respectively. Both the fragments were over-expressed in Escherichia coli BL21 (DE3) pLysS, induced at an A
Acknowledgements
This work was supported by the Genome and Health Initiative of the National Research Council of Canada (NRCC publication No. 46226), sponsored by the Government of Canada.
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