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• W2010417785 abstract "MAPK phosphatases (MKPs) are negative regulators of signaling pathways with distinct MAPK substrate specificities. For example, the yeast dual specificity phosphatase Msg5 dephosphorylates the Fus3 and Slt2 MAPKs operating in the mating and cell wall integrity pathways, respectively. Like other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains. These include D-motifs, which contain basic residues that interact with acidic residues in the common docking (CD) domain of MAPKs. Here we show that Msg5 interacts not only with Fus3, Kss1, and Slt2 but also with the pseudokinase Slt2 paralog Mlp1. Using yeast two-hybrid and in vitro interaction assays, we have identified distinct regions within the N-terminal domain of Msg5 that differentially bind either the MAPKs Fus3 and Kss1 or Slt2 and Mlp1. Whereas a canonical D-site within Msg5 mediates interaction with the CD domains of Fus3 and Kss1, a novel motif (102IYT104) within Msg5 is involved in binding to Slt2 and Mlp1. Furthermore, mutation of this site prevents the phosphorylation of Msg5 by Slt2. This motif is conserved in Sdp1, another MKP that dephosphorylates Slt2, as well as in Msg5 orthologs from other yeast species. A region spanning amino acids 274–373 within Slt2 and Mlp1 mediates binding to this Msg5 motif in a CD domain-independent manner. In contrast, Slt2 uses its CD domain to bind to its upstream activator Mkk1. This binding flexibility may allow MAPK pathways to exploit additional regulatory controls in order to provide fine modulation of both pathway activity and specificity. MAPK phosphatases (MKPs) are negative regulators of signaling pathways with distinct MAPK substrate specificities. For example, the yeast dual specificity phosphatase Msg5 dephosphorylates the Fus3 and Slt2 MAPKs operating in the mating and cell wall integrity pathways, respectively. Like other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains. These include D-motifs, which contain basic residues that interact with acidic residues in the common docking (CD) domain of MAPKs. Here we show that Msg5 interacts not only with Fus3, Kss1, and Slt2 but also with the pseudokinase Slt2 paralog Mlp1. Using yeast two-hybrid and in vitro interaction assays, we have identified distinct regions within the N-terminal domain of Msg5 that differentially bind either the MAPKs Fus3 and Kss1 or Slt2 and Mlp1. Whereas a canonical D-site within Msg5 mediates interaction with the CD domains of Fus3 and Kss1, a novel motif (102IYT104) within Msg5 is involved in binding to Slt2 and Mlp1. Furthermore, mutation of this site prevents the phosphorylation of Msg5 by Slt2. This motif is conserved in Sdp1, another MKP that dephosphorylates Slt2, as well as in Msg5 orthologs from other yeast species. A region spanning amino acids 274–373 within Slt2 and Mlp1 mediates binding to this Msg5 motif in a CD domain-independent manner. In contrast, Slt2 uses its CD domain to bind to its upstream activator Mkk1. This binding flexibility may allow MAPK pathways to exploit additional regulatory controls in order to provide fine modulation of both pathway activity and specificity. Mitogen-activated protein kinase (MAPK) pathways constitute very tightly regulated signaling modules that both transduce and convert extracellular stimuli into appropriate cellular responses. Mammalian MAPKs comprise four main subfamilies: the extracellular signal-regulated kinases 1 and 2 (ERK1/2), the c-Jun N-terminal kinases (JNKs), the p38 MAPKs, and ERK5 (1Turjanski A.G. Vaqué J.P. Gutkind J.S. Oncogene. 2007; 26: 3240-3253Crossref PubMed Scopus (352) Google Scholar). These MAPKs contain a highly conserved TXY motif in the kinase activation loop in which both threonine and tyrosine residues must be phosphorylated to achieve kinase activation. In contrast, atypical MAPKs, exemplified by ERK3 and ERK4, contain an SEG motif in which the serine residue is the sole phosphoacceptor (2Coulombe P. Meloche S. Biochim. Biophys. Acta. 2007; 1773: 1376-1387Crossref PubMed Scopus (205) Google Scholar). Several mechanisms exist to ensure high efficiency and fidelity within each specific pathway. Among these, selective recognition by specific protein-protein interactions between the components of a particular pathway is critical for the maintenance of accurate signal transduction (3Tanoue T. Nishida E. Pharmacol. Ther. 2002; 93: 193-202Crossref PubMed Scopus (112) Google Scholar, 4Grewal S. Molina D.M. Bardwell L. Cell. Signal. 2006; 18: 123-134Crossref PubMed Scopus (36) Google Scholar). MAPKs are activated through phosphorylation by MAPK kinases and phosphorylate a wide range of protein substrates, including transcription factors, protein kinases, and other cell response effectors. In turn, MAPK signaling can be down-regulated by dephosphorylation and inactivation by a family of MAPK phosphatases (MKPs). 5The abbreviations used are: MKPMAPK phosphataseKIMkinase interaction motifCDcommon dockingDSPdual specificity phosphataseCWIcell wall integritySDsynthetic dextrose. These phosphorylation/dephosphorylation events require not only a transient enzyme-substrate interaction involving the active sites of these enzymes but also the formation of a complex between the MAPK and its cognate activator, substrate, or inactivator. This is normally achieved through specific docking interactions between conserved regions within both MAPKs and MAPK-interacting proteins (3Tanoue T. Nishida E. Pharmacol. Ther. 2002; 93: 193-202Crossref PubMed Scopus (112) Google Scholar). Such docking sites are located outside the catalytic domains of these proteins and promote binding specificity and high affinity interactions. MAPK phosphatase kinase interaction motif common docking dual specificity phosphatase cell wall integrity synthetic dextrose. One well characterized docking motif found in MAPK-interacting proteins is the D motif, which has also been termed the δ-domain or DEJL (docking site for ERK and JNK, LXL) motif. This is closely related to the conserved kinase interaction motif (KIM) found within MAPK phosphatases and comprises a cluster of positively charged amino acids, normally lysines or arginines, followed by a submotif containing two hydrophobic residues leucine, isoleucine, or valine separated by one residue ((K/R)1–3X2–6ϕXϕ) (5Bardwell L. Shah K. Methods. 2006; 40: 213-223Crossref PubMed Scopus (25) Google Scholar, 6Akella R. Moon T.M. Goldsmith E.J. Biochim. Biophys. Acta. 2008; 1784: 48-55Crossref PubMed Scopus (90) Google Scholar). Swapping one D domain for another can completely change the specificity of protein-protein interactions, indicating that these motifs are essential to maintain binding selectivity among MAPK pathway components (7Mayor Jr., F. Jurado-Pueyo M. Campos P.M. Murga C. Cell Cycle. 2007; 6: 528-533Crossref PubMed Scopus (39) Google Scholar). A second type of docking motif named the DEF motif (for docking site for ERK FXF) has been found only in some subsets of MAPK substrates. This consists of two phenylalanines separated by one amino acid and followed by proline (FXFP) (8Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (443) Google Scholar). Docking sites have been also characterized within MAPKs themselves. The region of the MAPK that interacts with D motifs is complex and is formed by the common docking (CD) domain, the ED (for Glu/Asp-containing) motif, and a hydrophobic docking groove (6Akella R. Moon T.M. Goldsmith E.J. Biochim. Biophys. Acta. 2008; 1784: 48-55Crossref PubMed Scopus (90) Google Scholar). The CD domain is shared by all MAPK subfamilies and is an electrostatic surface depression containing a cluster of negatively charged amino acids (aspartic and glutamic acids) located within the C-terminal domain of the kinase (9Tanoue T. Adachi M. Moriguchi T. Nishida E. Nat. Cell Biol. 2000; 2: 110-116Crossref PubMed Scopus (691) Google Scholar). Substitution of these residues by charge-neutral amino acids completely abrogates docking, indicating the importance of the electrostatic interaction of the CD domain with the positively charged amino acids within the D domains of MAPK-interacting proteins (10Tanoue T. Yamamoto T. Nishida E. J. Biol. Chem. 2002; 277: 22942-22949Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The ED motif is located close to the CD domain in the tertiary structure of MAPKs and has also been reported to contribute to binding specificity (11Tanoue T. Nishida E. Cell. Signal. 2003; 15: 455-462Crossref PubMed Scopus (282) Google Scholar). Close to the CD/ED regions resides a hydrophobic docking groove that is involved in binding to the hydrophobic submotif of the D site of MAPK partners (10Tanoue T. Yamamoto T. Nishida E. J. Biol. Chem. 2002; 277: 22942-22949Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In contrast, the DEF motif binds to a separate hydrophobic pocket that only becomes accessible upon MAPK activation (12Lee T. Hoofnagle A.N. Kabuyama Y. Stroud J. Min X. Goldsmith E.J. Chen L. Resing K.A. Ahn N.G. Mol. Cell. 2004; 14: 43-55Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). In addition, a novel interaction motif (FRIEDE) within the atypical MAPKs ERK3 and ERK4 has been shown to mediate binding to their substrate MAPKAP kinase MK5 (13Aberg E. Torgersen K.M. Johansen B. Keyse S.M. Perander M. Seternes O.M. J. Biol. Chem. 2009; 284: 19392-19401Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Comparison of the MAPK docking interactions from yeast to humans reveals the existence of conserved molecular mechanisms underlying the specificity of these interactions (11Tanoue T. Nishida E. Cell. Signal. 2003; 15: 455-462Crossref PubMed Scopus (282) Google Scholar, 15Reményi A. Good M.C. Bhattacharyya R.P. Lim W.A. Mol. Cell. 2005; 20: 951-962Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Dual specificity phosphatases (DSPs) are able to dephosphorylate both threonine and tyrosine residues in their target proteins. A subfamily of these DSPs specifically targets MAPKs. These enzymes are designated as dual specificity MKPs and play an important role in the regulation of MAPK signaling. All MKPs share a common structure comprising an N-terminal non-catalytic regulatory domain, which contains the sequences necessary for MAPK docking, and a C-terminal catalytic domain (16Owens D.M. Keyse S.M. Oncogene. 2007; 26: 3203-3213Crossref PubMed Scopus (642) Google Scholar). Saccharomyces cerevisiae has five MAPK signaling pathways involved in mating, pseudohyphal/invasive growth, cell wall integrity (CWI), osmoregulation, and ascospore formation, mediated by Fus3, Kss1, Slt2, Hog1, and Smk1, respectively (17Chen R.E. Thorner J. Biochim. Biophys. Acta. 2007; 1773: 1311-1340Crossref PubMed Scopus (463) Google Scholar). Fus3 and Kss1 are orthologous to ERK1/2, and Hog1 is orthologous to p38 (18Caffrey D.R. O'Neill L.A. Shields D.C. J. Mol. Evol. 1999; 49: 567-582Crossref PubMed Scopus (106) Google Scholar). The closest mammalian MAPK to Slt2 is ERK5 (19Truman A.W. Millson S.H. Nuttall J.M. King V. Mollapour M. Prodromou C. Pearl L.H. Piper P.W. Eukaryot. Cell. 2006; 5: 1914-1924Crossref PubMed Scopus (48) Google Scholar). The CWI-related protein Mlp1 is a pseudokinase paralog of Slt2 that lacks catalytic residues critical for protein kinase activity as well as the conserved Thr residue within the MAPK activation loop dual phosphorylation site. Whereas an individual MAPK can be inactivated by more than one protein phosphatase, it is also the case that a single protein phosphatase is able to interact with and inactivate several MAPKs (20Martín H. Flández M. Nombela C. Molina M. Mol. Microbiol. 2005; 58: 6-16Crossref PubMed Scopus (120) Google Scholar). For example, Msg5 has been shown to dephosphorylate Fus3 and Slt2, affecting both basal and inducible kinase activities (21Doi K. Gartner A. Ammerer G. Errede B. Shinkawa H. Sugimoto K. Matsumoto K. EMBO J. 1994; 13: 61-70Crossref PubMed Scopus (205) Google Scholar, 22Zhan X.L. Deschenes R.J. Guan K.L. Genes Dev. 1997; 11: 1690-1702Crossref PubMed Scopus (126) Google Scholar, 23Flández M. Cosano I.C. Nombela C. Martín H. Molina M. J. Biol. Chem. 2004; 279: 11027-11034Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 24Andersson J. Simpson D.M. Qi M. Wang Y. Elion E.A. EMBO J. 2004; 23: 2564-2576Crossref PubMed Scopus (60) Google Scholar). Msg5 is expressed in yeast cells as two isoforms that differ in the first 45 amino acids due to the use of alternative translation initiation sites (23Flández M. Cosano I.C. Nombela C. Martín H. Molina M. J. Biol. Chem. 2004; 279: 11027-11034Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We have recently shown that this MKP is able to interact not only with Fus3 and Slt2 but also with Kss1, a MAPK whose phosphorylation is not down-regulated by Msg5 (25Marín M.J. Flández M. Bermejo C. Arroyo J. Martín H. Molina M. Mol. Genet. Genomics. 2009; 281: 345-359Crossref PubMed Scopus (21) Google Scholar). Moreover, whereas both Msg5 isoforms interact similarly with Slt2, the longer form binds both Fus3 and Kss1 with higher affinity than the shorter isoform. In a study of the docking interactions within and between the Fus3- or Kss1-mediated pathways, Reményi et al. (15Reményi A. Good M.C. Bhattacharyya R.P. Lim W.A. Mol. Cell. 2005; 20: 951-962Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) found docking motifs in different proteins known to physically interact with Fus3, such as Ste7, Msg5, and Far1. Mutation of a D domain within Msg5 partially reduced the ability of this MKP to decrease the elevated mating pathway output displayed by an msg5-null mutant, indicating a contribution of the docking interaction mediated by this motif to the ability of Msg5 to inactivate Fus3. But the incomplete effect of D domain mutation also suggests that additional Fus3-docking motifs may be present in Msg5 (15Reményi A. Good M.C. Bhattacharyya R.P. Lim W.A. Mol. Cell. 2005; 20: 951-962Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The interaction with Slt2 has been shown to be mediated by the N-terminal domain of Msg5 (23Flández M. Cosano I.C. Nombela C. Martín H. Molina M. J. Biol. Chem. 2004; 279: 11027-11034Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Here we report the characterization of the interactions between Msg5 and a number of yeast MAPKs, showing the involvement of distinct regions located at the N terminus of Msg5 in MAPK binding. D domain-dependent mechanisms participate in the binding to mating (Fus3 and Kss1) MAPKs. In contrast, a novel motif of Msg5 is involved in the interaction with the cell wall integrity (Slt2 and Mlp1) kinases. We also provide evidence indicating that, as is the case in other MAPK pathways, the CD domain of Slt2 mediates binding to its activator Mkk1, whereas a CD-proximal region containing the conserved MAPK L16 loop is involved in binding to the negative regulator Msg5 in a CD domain-independent fashion. The S. cerevisiae strains PJ69-4A (MATa, trp1-901, leu2-3112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1-HIS3, GAL2-ADE2, met2::GAL7-lacZ) and PJ69-4α (MATα, trp1-901, leu2-3112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1-HIS3, GAL2-ADE2, met2::GAL7-lacZ), from Clontech, were used for two-hybrid assays. Strains DD1-2D (msg5-1::LEU2) (21Doi K. Gartner A. Ammerer G. Errede B. Shinkawa H. Sugimoto K. Matsumoto K. EMBO J. 1994; 13: 61-70Crossref PubMed Scopus (205) Google Scholar), YMF1 (MATa, ura3-52, his4, trp1-1, leu2-3, 112, canR, MSG5::6MYC::LEU2) (23Flández M. Cosano I.C. Nombela C. Martín H. Molina M. J. Biol. Chem. 2004; 279: 11027-11034Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0), and Y07373 (BY4741 isogenic, msg5Δ::KanMX4) from Euroscarf (Frankfurt, Germany) were used for Western blotting analysis experiments. Standard procedures were employed for yeast genetic manipulations (26Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2545) Google Scholar). Yeast cultures were performed as reported previously (27Martín H. Rodríguez-Pachón J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). When necessary, Congo red (Sigma) or α-factor (Sigma) was added at the indicated concentrations. General DNA methods were performed using standard techniques. Oligonucleotide primers used for DNA amplification and directed mutagenesis by PCR are indicated in supplemental Tables I and II. Vectors pGBKT7 (bearing the Gal4 DNA binding domain) and pGADT7 (bearing the Gal4 activation domain) were obtained from Clontech. Yeast MAPK-containing constructs pGADT7-Kss1, pGADT7-Fus3, pGADT7-Smk1, pGADT7-Slt2, and pGADT7-Hog1 were previously described (28Collister M. Didmon M.P. MacIsaac F. Stark M.J. MacDonald N.Q. Keyse S.M. FEBS Lett. 2002; 527: 186-192Crossref PubMed Scopus (30) Google Scholar), apart from pGADT7-Mlp1, which was constructed by subcloning the PCR-amplified MLP1 coding sequence from S. cerevisiae genomic DNA into pGADT7 as an NdeI-XhoI fragment. Mutated derivatives of these reading frames (pGADT7-Kss1CD, pGADT7-Fus3CD, pGADT7-Slt2CD, pGADT7-Slt2CD3, and pGADT7-Mlp1CD) were modified by overlap extension PCR-mediated site-directed mutagenesis and subcloned as described for the wild-type construct (28Collister M. Didmon M.P. MacIsaac F. Stark M.J. MacDonald N.Q. Keyse S.M. FEBS Lett. 2002; 527: 186-192Crossref PubMed Scopus (30) Google Scholar). The MSG5 reading frame was amplified from S. cerevisiae genomic DNA; wild-type, mutant, and truncated reading frames derived from this template were subcloned as NdeI-XhoI fragments into the NdeI/SalI sites in pGBKT7: pGBKT7-Msg5, pGBKT7-Msg5(1–245), pGBKT7-Msg5(246–489), pGBKT7-Msg5(126–489), pGBKT7-Msg5(1–123), pGBKT7-Msg5(90–489), pGBKT7-Msg5(1–45), pGBKT7-Msg5(46–489), pGBKT7-Msg5MD1, pGBKT7-Msg5MD2, pGBKT7-Msg5MD1/2, pGBKT7-Msg5MD3, pGBKT7-Msg5(1–123)MD1/2, and pGBKT7-Msg5(1–123)MD3. For pGBKT7-Msg5MD1/2 and pGBKT7-Msg5(1–123)MD1/2, PCR was performed as for single point mutations but using mutant half-product templates to generate compound double and truncated mutant reading frames following the second round of amplification. YCplac22MSG5MD1, YCplac22MSG5MD2, and YCplac22MSG5MD3 plasmids were generated by site-directed mutagenesis of YCplac22MSG5m (23Flández M. Cosano I.C. Nombela C. Martín H. Molina M. J. Biol. Chem. 2004; 279: 11027-11034Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) following the method described previously (23Flández M. Cosano I.C. Nombela C. Martín H. Molina M. J. Biol. Chem. 2004; 279: 11027-11034Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). YCplac111-Slt2(1–373) was constructed by cloning a ∼3.2-kb EcoRI-EcoRI fragment containing SLT2 into YCpLac111 (CEN4, LEU2) (23Flández M. Cosano I.C. Nombela C. Martín H. Molina M. J. Biol. Chem. 2004; 279: 11027-11034Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and then introducing a consecutive pair of stop codons in the corresponding protein positions 374 and 375 by site-directed mutagenesis of the obtained plasmid. In order to express Msg5 fused to GST in yeast, the previously described plasmid pEG(KG)-MSG5 was used (23Flández M. Cosano I.C. Nombela C. Martín H. Molina M. J. Biol. Chem. 2004; 279: 11027-11034Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). pEG(KG)-Slt2(274–373) was constructed by PCR-amplifying the corresponding SLT2 region using YCplac111-Slt2(1–373) as template and then subcloning the BamHI- digested PCR product into pEG-KG (2 μm, GAL1–10UAS-CYC1). To obtain recombinant GST-Mkk1, plasmid pGEX-MKK1 was used (29Jiménez-Sánchez M. Cid V.J. Molina M. J. Biol. Chem. 2007; 282: 31174-31185Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Recombinant Msg5-GST, Msg5MD3-GST, and Msg5(1–123)MD3-GST were produced from plasmids constructed by subcloning PCR products featuring NdeI and XhoI sites at the 5′- and 3′-ends into a modified pGEX-P1 vector (gift of Dr. B. McStay). Recombinant Slt2, Mlp1, and Mkk1 His-tagged proteins or protein fragments were produced from pET15B-derived plasmids after cloning the corresponding PCR-amplified SLT2 and MLP1 gene fragments in NdeI and XhoI sites. Assays were performed according to the manufacturer's instructions (Matchmaker 3 kit, Clontech). The vector pGBKT7 or its derivatives were transformed into PJ69-4A cells and mated with PJ69-4α cells transformed with plasmid pGADT7 or its derivatives. Yeast diploids carrying both types of plasmids were selected on synthetic dextrose (SD) medium deficient for leucine and tryptophan. In order to perform a qualitative analysis of the two-hybrid interactions, representative colonies from the different diploid strains were cultivated overnight in liquid YPD, and the corresponding cellular suspensions were prepared. 10-μl volumes of inocula (normalized by optical density at 600 nm) were spotted onto two types of media: SD medium lacking leucine and tryptophan and SD medium lacking leucine, tryptophan, histidine, and adenine. Cell growth on the latter medium provides evidence for the occurrence of protein-protein interaction. For semiquantitative analysis of the two-hybrid interactions, exponentially growing diploid cells bearing the relevant plasmids were collected, and the β-galactosidase activity was determined using o-nitrophenyl-β-galactoside as a substrate, according to the manufacturer's instructions (Clontech). Recombinant GST- or histidine-tagged proteins were expressed in Escherichia coli strain Rosetta DE3 (Novagen). Cells were collected and lysed by sonication in PBS buffer containing 2 mm PMSF, 1 mm DTT, 1 mm EDTA, and 1 mg/ml lysozyme in the presence of protease inhibitor mixture (Roche Applied Science). Extracts were clarified by centrifugation and then stored at −80 °C. For obtaining yeast extracts, the procedures employed have been described previously (27Martín H. Rodríguez-Pachón J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). For in vivo binding assays, yeast cells bearing pEG(KG)-derived plasmids were grown in complete synthetic medium lacking uracil with 2% galactose at 24 °C. Cells were collected and lysed as above in lysis buffer lacking SDS and Nonidet P-40. Yeast lysates were incubated with glutathione-Sepharose beads (Amersham Biosciences) for 2 h. Beads were washed extensively with the same buffer and resuspended in SDS loading buffer, and proteins were analyzed by SDS-PAGE and immunoblotting as described (23Flández M. Cosano I.C. Nombela C. Martín H. Molina M. J. Biol. Chem. 2004; 279: 11027-11034Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In vitro binding assays were performed by mixing E. coli extracts containing GST or GST-fused proteins with E. coli extracts bearing the corresponding His-tagged proteins and then processed as above. Immunodetection of Myc- and HA- tagged proteins was carried out using either monoclonal 9E10 (Covance) or monoclonal 4A6 (Millipore) and monoclonal HA.11 (Covance) antibodies, respectively. Actin was immunodetected by using monoclonal C4 antibodies (MP Biomedicals). Polyclonal anti-phospho-p44/p42 MAPK (Thr-202/Tyr-204) (Cell Signaling), anti-GST (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and monoclonal anti-His (Sigma) antibodies were also used as described previously (27Martín H. Rodríguez-Pachón J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Immunodetection of Gal4BD fusion proteins was carried out using monoclonal GAL4DNA-BD antibody (Clontech). The primary antibodies were detected using either a horseradish peroxidase-conjugated secondary antibody with the ECL detection system (Amersham Biosciences) or a fluorescently conjugated secondary antibody with an Odyssey Infrared Imaging System (LI-COR Biosciences). Real-time quantitative PCR assays were performed as described previously (30García R. Bermejo C. Grau C. Pérez R. Rodríguez-Peña J.M. Francois J. Nombela C. Arroyo J. J. Biol. Chem. 2004; 279: 15183-15195Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), using an ABI 7700 instrument (Applied Biosystems). Each cDNA was assayed in at least duplicate PCR reactions. For quantification, the abundance of each gene was determined relative to the standard transcript of ACT1, and the final data of relative gene expression between the two conditions tested were calculated following the 2−ΔΔCt method, as described (31Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (127155) Google Scholar). The primer sequences are available upon request. In order to gain insight into the mechanisms that regulate the interaction of Msg5 with its substrate MAPKs, we first wanted to define the MAPKs that physically interact with this phosphatase. To this end, the yeast two-hybrid assay was used. Msg5 was fused to the Gal4-DNA binding domain (Gal4BD), and the five MAPKs (Kss1, Fus3, Smk1, Slt2, and Hog1) as well as the pseudokinase Slt2 paralog Mlp1 (32Kim K.Y. Truman A.W. Levin D.E. Mol. Cell Biol. 2008; 28: 2579-2589Crossref PubMed Scopus (97) Google Scholar) were fused to the Gal4 activation domain (Gal4AD). Expression of all of these hybrid proteins was analyzed by Western blotting (supplemental Fig. S1). Formation of a complex between Msg5 and the corresponding MAPK results in reconstitution of the Gal4 transcription factor and expression of the Gal4-dependent reporter genes HIS3, ADE2, and lacZ. These interactions can then be monitored by either growth of the diploid strains on selective medium lacking adenine and histidine or semiquantitative assays of β-galactosidase activity. As shown in Fig. 1A, only the yeast cells expressing Msg5 together with Kss1, Fus3, Slt2, or Mlp1 were able to grow on selective medium lacking adenine and histidine. In addition, expression of lacZ was only observed in these same cells (Fig. 1B). The strongest interaction with Msg5, in terms of increased β-galactosidase activity, was observed with Mlp1. Interactions with Slt2 and Kss1 were also significant, whereas the lowest strength interaction was observed with Fus3. Western blotting analysis revealed that this was not due to low level expression of Gal4AD-Fus3 (supplemental Fig. S1). Despite being expressed to a similar extent as the other kinases (supplemental Fig. S1), neither Hog1 nor Smk1 was able to interact with Msg5 in the two-hybrid assay (Figs. 1, A and B). A catalytically inactive mutant of Msg5C319S (21Doi K. Gartner A. Ammerer G. Errede B. Shinkawa H. Sugimoto K. Matsumoto K. EMBO J. 1994; 13: 61-70Crossref PubMed Scopus (205) Google Scholar) also failed to interact with either Hog1 or Smk1 (data not shown), ruling out the possibility that the absence of binding was due to dephosphorylation of these kinases by Msg5. Msg5 is composed of an N-terminal domain (approximately residues 1–245) and a C-terminal catalytic domain (approximately residues 246–489). To determine the involvement of these two regions in interactions with distinct MAPKs, we extended our two-hybrid analysis by using constructs containing either the isolated N-terminal or C-terminal domains of Msg5. As observed in Fig. 1C, the N-terminal domain of Msg5 interacted with all four of the MAPKs that bind full-length Msg5. Although the β-galactosidase levels were significantly reduced when compared with those measured between full-length Msg5 and these MAPKs, the strongest interaction was again observed with Mlp1 (Fig. 1D). The C-terminal domain of Msg5 fused to Gal4BD did not show interaction with any of the MAPKs (Fig. 1D), although it was highly expressed (supplemental Fig. S1). These results indicate that the N-terminal domain of Msg5 is both necessary and sufficient to mediate protein-protein interactions with MAPK substrates. In order to further elucidate the region of Msg5 that is responsible for the interactions with Fus3, Kss1, Slt2, and Mlp1, we next tested a set of Msg5 truncations fused to Gal4BD for binding to the distinct Gal4AD-MAPK fusions. The expression of these hybrid Msg5 proteins was also monitored by Western blotting (supplemental Fig. S1). As shown in Fig. 2, A and B, the region encompassing the first 123 amino acids of Msg5 (Msg5(1–123)) is necessary and sufficient for the interaction with all four MAPKs. In contrast, although highly expressed (supplemental Fig. S1), a construct lacking the first 123 residues (Msg5(124–489)) failed to interact with all four MAPKs. However, although an Msg5 construct lacking the first 45 amino acids interacted with both Slt2 and Mlp1, it could no longer bind to either Fus3 or Kss1 (Fig. 2, A and C). Consistent with these data, an Msg5 fragment containing only the first 45 amino acids interacted with Fus3 and Kss1 but not with Mlp1 and Slt2 (Fig. 2, A and B). These two-hybrid results are also in agreement with our previously reported data showing that the short Msg5 isoform, which lacks the first 45 amino acids, displays a much lower affinity for Fus3 and Kss1 than the full-length Msg5 isoform in co-purification assays, whereas both isoforms bind Slt2 with the same affinity (25Marín M.J. Flández M. Bermejo C. Arroyo J. Martín H. Molina M. Mol. Genet. Genomics. 2009; 281: 345-359Crossref PubMed Scopus (21) Google Scholar). Finally, a mutant of Msg5 lacking the first 89 amino acids did not interact with Fus3 and Kss1 but was still able to interact with Slt2 and Mlp1 (Fig. 2, A and C). In summary, these two-hybrid data show that different regions of Msg5 me" @default.
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• W2010417785 date "2011-12-01" @default.
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• W2010417785 title "Distinct Docking Mechanisms Mediate Interactions between the Msg5 Phosphatase and Mating or Cell Integrity Mitogen-activated Protein Kinases (MAPKs) in Saccharomyces cerevisiae" @default.
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