Mkp1 of Pneumocystis carinii associates with the yeast transcription factor Rlm1 via a mechanism independent of the activation state

Cellular Signalling 12 (2000) 381–390

http://www.elsevier.com/locate/cellsig

Mkp1 of Pneumocystis carinii associates with the yeast transcriptionfactor Rlm1 via a mechanism independent of the activation state

Deborah Foxa,b, A. George Smulianb,c*aDepartment of Pathology, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA

bInfectious Disease Division, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USAcCincinnati VA Medical Center, Cincinnati, OH 4220, USA

Received 30 October 1999; accepted 1 February 2000

Abstract

The mitogen-activated protein (MAP) kinase Mkp1 of the fungal pathogen Pneumocystis carinii is a functional MAP kinasethat complements the loss of Slt2p, the MAP kinase component of the cell integrity pathway of Saccharomyces cerevisiae, andis activated within P. carinii in response to oxidative stress. Mkp1 displays an unusual feature in that it contains a phosphorylationmotif repeat (TEYMTEY) within the activation loop not present in any other fungal MAPK identified to date. Mutagenesis ofthe T186,Y188 phosphorylation motif within the activation domain of Mkp1 results in the loss of detectable kinase activity butstill retains partial complementation function. In addition to the ability of Mkp1 to restore partial activity to the cell integritypathway in the absence of phosphorylatable residues within the activation loop, the association of Mkp1 with a substrate ofSlt2p, the transcription factor Rlm1p, can also occur in the absence of MAP kinase activation. The results of this study suggestthat the presence of phosphorylatable residues within the activation loop of Mkp1 is not absolutely required for functional(complementation) activity or for the association of Mkp1 with the transcription factor Rlm1p. In contrast, the catalytic lysineof the ATP-binding domain of Mkp1 is necessary for both complementation function and interaction with Rlm1p. 2000Elsevier Science Inc. All rights reserved.

Keywords: Fungal; Mitogen-activated protein kinase; Pneumocystis carinii; Pneumonia

1. Introduction activation loop also influences the recognition of regula-tory subunits and the orientation of residues within the

The regulation of mitogen-activated protein (MAP) catalytic site [5]. The signature threonine and tyrosinekinase activity by phosphorylation of the threonine and residues (TXY) within the MAP kinase activation looptyrosine residues of the TXY motif within the activation have been shown to be necessary for kinase activationloop has been extensively studied. Mitogen-activated and functional activity [6–9].protein kinase, the terminal kinase of the three kinase- Despite the significant advances made in the analysismodule, is activated by the upstream mitogen-activated of MAP kinase activation, many issues regarding MAPprotein kinase kinase (MEK) via a mechanism of dual kinase activity and function remain unresolved, includ-phosphorylation of both the threonine and tyrosine that ing the identification of mechanisms that lead to signal-lie one residue apart within the activation loop, also ling specificity and the regulation of MAP kinase associ-known as the phosphorylation lip [1–4]. The phosphory- ations with their substrates. Unravelling the complex andlation state of the activation loop determines the kinase seemingly contradictory mechanisms that regulate MAPactivity of the protein. Upon activation of the MAP kinase function and specificity will lead to a greater un-kinase via phosphorylation of both threonine and tyro- derstanding of how the cells of both higher and lowersine, the conformation of the activation loop of the eukaryotes respond to their environment. Additionally,kinase is altered to allow access of the substrate to the the identification of crucial signal transduction pathwayactive site [5]. The conformation of the MAP kinase components of pathogenic fungi may be useful for the

development of novel drug targets.* Corresponding author. 231 Bethesda Ave., Cincinnati, OHWe have previously shown that a mitogen-activated45267-0560. Tel.: 513-861-3100 ext. 4425; fax: 513-475-6415.

E-mail address: [email protected] (A.G. Smulian) protein kinase, Mkp1, isolated from the fungal opportu-

0898-6568/00/$ – see front matter 2000 Elsevier Science Inc. All rights reserved.PII: S0898-6568(00)00076-0

382 D. Fox, A.G. Smulian / Cellular Signalling 12 (2000) 381–390

nistic pathogen Pneumocystis carinii, is a functional MAP 2. Materials and methodskinase that complements the loss of Slt2p, the MAP

2.1. Strains, media and general methodskinase component of the cell integrity pathway of Sac-charomyces cerevisiae. Additionally, we have shown that Saccharomyces cerevisiae strains used and generatedMkp1 is activated within P. carinii in response to oxidative in this study are listed in Table 1. Escherichia coli ultra-stress [10]. Mkp1 contains two repeats of a phosphory- competent TOP-10 cells (Invitrogen, Carlsbad, CA)lation motif in the activation loop, an unusual feature were used for DNA isolation. Yeast strain DL456 andthat has not been previously described in fungi. This its derivatives were transformed by electroporation withsame sequence has been found in only one other MAPK the Electroporator II (Invitrogen) at 1500 volts, 200identified to date, ATMPK5 from the plant Arabidopsis ohms, and 50 mF. Yeast strain SFY526 and its derivativesthaliana [11]. The functional significance of this phos- were transformed by a modified lithium acetate methodphorylation motif duplication has not been determined. [12]. Transformants were selected by plating onto syn-

thetic minimal media containing 2% glucose and 0.7%The influence of the phosphorylation motif repeat onyeast nitrogen base without amino acids (Difco). Mediathe function of Mkp1 within the cell integrity pathway ofwas supplemented with amino acids, as necessary. Sorbi-S. cerevisiae was examined by conservative substitutiontol (1 M) was added to solid and liquid media as a cellof the threonine and tyrosine residues of the phosphory-wall integrity osmostabilizer.lation (TEY) motifs within the activation loop. The

phosphorylation motif at T186,Y188 was necessary and2.2. Site-directed mutagenesis of mkp1sufficient for: (1) full restoration of the growth rate; (2)

the maintenance of cell wall integrity upon temperature A PCR-based site-directed mutagenesis method [13]stress; and (3) the ability to phosphorylate exogenous was used to introduce base changes into the activationsubstrate. Mutation of the phosphorylation motif at domain of mkp1, as previously described [10]. A series ofT182,Y184 did not abrogate function. Mutation of the oligonucleotide primers were designed to the activationphosphorylation motif at T186,Y188 (Mkp1T186A,Y188F) or domain region of mkp1 in order to introduce two baseboth phosphorylation motifs (Mkp1T182A,Y184F,T186A,Y188F) re- changes, each at the third position of the codon, to sub-sulted in only a partial reduction of growth rate and stitute both the threonine and tyrosine for alanine andcell wall integrity. However, the loss of the T186,Y188 phenylalanine, respectively [14,15]. The procedure wasphosphorylation sites resulted in an inactive kinase, as performed for each of the two potential phosphorylationneither Mkp1T186A,Y188F nor Mkp1T182A,Y184F,T186A,Y188F were domains, resulting in the following constructs: (1)able to phosphorylate exogenous substrate. In addition pGEMT-mkp1T182A,Y184F containing mutations in the firstto the ability of Mkp1 to restore partial activity to the phosphorylation motif using oligonucleotides Mkp1cell integrity pathway, even in the absence of phosphor- mut TEY#1 (59-GGTGCTGAATTTATGACTGAA-ylation sites within the activation loop, the association TATGTTACC-39) and Mkp1 mut constant (59-TT-of Mkp1 with the substrate of the cell integrity pathway, GACCCTGATTTACAGAGATACCTCTAG-39); (2)Rlm1p can also occur in the absence of phosphorylation- pGEMT-mkp1T186A,Y188F containing mutations in the sec-dependent activation. Taken together, the results of this ond phosphorylation motif using oligonucleotides Mkp1study suggest that the MAP kinase of the cell wall integ- mut TEY#2 (59-GGTACTGAATATATGGCTGAA-rity pathway displays substrate association and regula- TTTGTTACC-39) and Mkp1 mut constant; and (3)tion, even in the absence of phosphorylatable residues pGEMT-mkp1T182A,Y184F,T186A,Y188F containing mutations inwithin the activation domain necessary for phosphoryla- both phosphorylation motifs using oligonucleotides Mkp1

mut dual TEY (59-GGTGCTGAATTCATGGCTGAA-tion-dependent activation of the kinase.

Table 1S. cerevisiae strains

Strain Genotype Source

1788 MATa/MATa leu2-3,112 ura3-52 trp1-1 his4 can1 r LevinDL456 MATa/MATa 1788 mpk1D::TRP1/mpk1D::TRP1 LevinGS456 MATa/MATa DL456 1 pYX213 Fox and SmulianGS457 MATa/MATa DL456 1 pYX[GST:SLT2] Fox and SmulianGS458 MATa/MATa DL456 1 pYX[GST:mkp1] Fox and SmulianGS459 MATa/MATa DL456 1 pYX[GST:mkp1T182A,Y184F] This studyGS460 MATa/MATa DL456 1 pYX[GST:mkp1T186A,Y188F] Fox and SmulianGS461 MATa/MATa DL456 1 pYX[GST:mkp1T182A,Y184F,T186A,Y188F] This studyGS462 MATa/MATa DL456 1 pYX[GST:mkp1K52R] This studySFY526 MATa ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3, Clontech

112 canr gal4-542 gal80-538 URA3::GAL1UAS-GAL1TATA-lac (Harper)

D. Fox, A.G. Smulian / Cellular Signalling 12 (2000) 381–390 383

TTTGTTACC-39) and Mkp1 mut constant. Site-directed BamH1 digested pACT2 (Clontech) to generate pACT-mutagenesis was similarly performed to introduce a [GAD:RLM1]. The transcriptional activation domain ofdead-kinase mutation into mkp1 with oligonucleotides Gal4 (GAD) is fused to the amino terminus of Rlm1Mkp1-DK sense (59-ATCAGATCTATAACAAAT- in the pACT[GAD:RLM1] construct.ATATTTTC-39) and Mkp1-DK antisense (59-AGC-

2.4. Two-hybrid and b-galactosidase assaysAACTTTGTT ATTATCTTCAAC-39), resulting inthe substitution (K52R) of arginine for the invariant Yeast two-hybrid and b-galactosidase assays werephosphotransfer lysine in conserved subdomain II

performed as described previously [16,17]. The reporter(pGEMT-mkp1K52R).

strain SFY526 was transformed with the Gal4 DNA-binding domain and activation domain plasmids: pAS2.3. Yeast expression constructs[GBD:mkp1], pAS[GBD:mkp1T182A,Y184F], pAS[GBD:

Plasmids of the pYX series direct the expression of mkp1T186A,Y188F], pAS[GBD:mkp1T182A,Y184F,T186A,Y188F], pASwild-type and mutant forms of Mkp1 and are derived [GBD:mkp1K52R], pAS[GBD:SLT2], pACT[GAD:RLM1],from the yeast multicopy expression vector pYX (Inge- pAS2-1 (GBD), pACT2 (GAD), and pCL1 (wild-typenius) modified to contain the constitutive TPI promoter

Gal4 containing both GBD and GAD).from pYX222 and the URA3 selection marker and vec-tor backbone from pYX213. An in-frame amino-termi- 2.5. Antifungal sensitivity assaysnal GST fusion tag from pGEX4T3 was introduced into

The 96-well plate microdilution method was per-the EcoR1 and Nco1 sites upstream and adjacent to theformed as published previously, except that plates werecloning site in the pYX vector series. Plasmid pYX[GST:performed in duplicate with one set incubated at 308Cmkp1] expresses Mkp1 with an amino-terminal gluta-and the other at 378C [18]. The MIC50s of caffeine, ortho-thione-S-transferase tag, pYX[GST:mkp1T182A,Y184F] ex-vanadate, and aluminium were defined as the concentra-presses a version of Mkp1 that lacks the phosphorylat-tion which resulted in 50% growth inhibition, as deter-able residues of the non-conserved TEY motif (TEY#1)

within the activation domain; pYX[GST:mkp1T186A,Y188F] mined by OD 405 nm, compared to control wellsexpresses a version of Mkp1 that lacks the phosphory- receiving no drug. Each concentration value (MIC50)latable residues of the conserved TEY motif (TEY#2), was calculated from quadruplicate samples per assaypYX[GST:mkp1T182A,Y184F,T186A,Y188F] expresses a version of from at least three independent assays.Mkp1 that lacks phosphorylatable residues in either

2.6. Treatment of cultures, affinity purification, andTEY motif (dual TEY); pYX[GST:mkp1K52R] expressesin vitro phosphorylation assaysa version of Mkp1 in which the invariant catalytic lysine

in conserved subdomain II is replaced with arginine To determine the influence of the phosphorylation(dead kinase). The coding region of mkp1 was cloned state of the activation domain of Mkp1 on the growthas a Nco1-Sma1 1200-bp fragment into the Nco1-Sma1 characteristics of the slt2D-deficient strain DL456, pub-gap of pAS2-1 (Clontech) to generate pAS2[GBD:

lished procedures were followed [10,19]. Mid-log phasemkp1]. The mutant versions of mkp1 were also cloned

cultures grown in uracil-deficient synthetic media con-into the Nco1-Sma1 gap of pAS2-1 to generate pAStaining 1 M sorbitol at 248C were inoculated into YEPD[GBD:mkp1T182A,Y184F], pAS[GBD:mkp1T186A,Y188F], pASat a density of 0.25 OD/ml. They were culture density[GBD:mkp1T182A,Y184F,T186A,Y188F], and pAS[GBD:mkp1K52R].monitored over a 40-hour period at 308C. Phosphoryla-In the pAS2 constructs, the DNA-binding domain oftion assays were used to measure activation of Mkp1Gal4 (GBD) is fused to the amino terminus of Mkp1.mutants after exposure to mild temperature changes, asThe coding region of SLT2 was amplified from genomicdescribed previously [10,20]. Transformants were grownDNA isolated from yeast strain 1788 as previously de-to mid-log phase in uracil-deficient synthetic media sup-scribed [10] and cloned into the Nco1-Sal1 gapof pAS2-1plemented with 1 M sorbitol at 248C and inoculated intoand the Nco1-Xho1 gap of pYX as an Nco1-Xho1 frag-YEPD at 0.25 OD/ml. Cultures were incubated at 248Cment, generating pAS[GBD:SLT2] and pYX[GST:for 1 hour prior to temperature shift to 378C, or 248CSLT2], respectively. To obtain the RLM1 coding region,for controls. Yeast from equal volumes of control andgenomic DNA isolated from yeast strain 1788 was usedheat-treated cultures were lysed by bead-beating at 48C.as template for amplification with the 59 RLM1 primerGST-fusion proteins were isolated by incubation of ly-59-GATACAACCCGGGTAGACGCAAG-39, whichsates with glutathione-agarose. The kinase activity ofincorporates a Sma1 site, and the 39 RLM1 primer 59-each purified GST-fusion protein sample was deter-GTAGATCTTTTTGCTTGAATTTTTTTC-39, whichmined by in vitro phosphorylation assay using myelinincorporates a BglII site. The resulting 2100-bp ampli-basic protein (MBP) as the substrate. MBP phosphory-fication product was inserted into pCR-Blunt (In-lation (kinase activity) was visualized on a Storm Phos-vitrogen). The RLM1 coding region was removed as a

2100-bp Sma1-BglII fragment and cloned into Sma1- phorImager (Molecular Dynamics).


Fig. 1. Suppression of the lytic defect by Mkp1 is not abolished by mutation of phosphorylation sites within the activation domain. (a) Diagrammaticrepresentation of the protein structure of Mkp1 of P. carinii. The kinase catalytic core containing 11 conserved subdomains is centrally locatedwithin the protein. The amino and carboxyl ends of the protein (dark shading) are non-conserved. Amino acid sequence identity comparisonof the subdomain VII to VIII region (containing the activation loop) for Mkp1 of P. carinii (AF077677), ATMPK5 of A. thaliana (Q39025),and Slt2p of S. cerevisiae (Q00772) is shown as an expanded view. The atypical TEY repeat region is underlined. The locations of introducedmutations used in this study are denoted with asterisks. (b) Mutagenesis of the conserved phosphotransfer lysine or phosphorylation sites in theactivation domain of Mkp1 results in only partial loss of complementation activity. Diploid yeast strains were streaked onto uracil-deficientsynthetic media with or without 1 M sorbitol and incubated for 3 days at the indicated temperature. Strains are (clockwise from top) slt2D

containing the vector pYX[GST:mkp1T186A,Y188F] (GS460), slt2D containing the vector pYX[GST:mkp1T182A,Y184F] (GS459), slt2D containing the vectorpYX[GST:mkp1] (GS458), slt2D containing the control vector pYX213 (GS456), slt2D containing the vector pYX[GST:mkp1K52R] (GS462), slt2D

containing the vector pYX[GST:mkp1T182A,Y184F,T186A,Y188F] (GS461).

2.7. Nucleotide sequence accession numbers protocols available for P. carinii, the analysis of thephosphorylation motif repeat of Mkp1 was performed

These sequences are available under the GenBank ac-in the closely-related yeast, S. cerevisiae The conse-

cession numbers AF077677 (mkp1 cDNA) and AF084383quence of each of the phosphorylation site mutations

(mkp1 gene).on the cell integrity pathway of S. cerevisiae was assessedby genetic complementation of the S. cerevisiae slt2Dmutation. In the absence of an osmotic stabilizer, the3. Resultsslt2D defect results in phenotypes including slow growth,

3.1. Analysis of the functional significance of the temperature sensitivity, and increased sensitivity to sub-TEY motif repeat of Mkp1 stances such as aluminium, orthovanadate, and caffeine

[19,21–23]. Over-expression of mkp1T182A,Y184F, contain-Mkp1 contains an atypical phosphorylation motifing non-phosphorylatable substitutions in the first TEYrepeat (TEYMTEY) within the activation loop (sub-motif, displayed the same growth and temperature sen-domain VII–VIII) that is present in only one othersitivity phenotype as the wild-type Mkp1 with full com-MAPK identified to date, ATMPK5 of Arabidopsisplementation at 308C and partial complementation atthaliana (Fig. 1a). To determine the significance of378C (Fig. 1b), and exhibited the same antifungal resis-the TEY motif repeat on the function of Mkp1, wetance phenotype as Mkp1 in the presence of aluminium,designed a series of expression plasmids containing site-caffeine, or orthovanadate (Table 2). Unexpectedly, over-directed mutations of Mkp1 in the first TEY motifexpression of mkp1T186A,Y188F or mkp1T182A,Y184F,T186A,Y188F,(mkp1T182A,Y184F), the second TEY motif (mkp1T186A,Y188F),which lack phosphorylatable residues in the second mo-or both TEY motifs (mkp1T182A,Y184F,T186A,Y188F). Due to the

limitations of the culture system and transformation tif or both motifs, respectively, displayed complemen-


Table 2Restoration of antifungal resistance phenotype

Strain MIC50

Caffeinea Aluminumb Vanadatea

308C 378C 308C 378C 30/378C

GS456 (slt2D) 1.7 0.9 130 60 0.40GS457 [GST:SLT2] 13.3 8.9 1300 970 1.5GS458 [GST:mkp1] 9.4 3.8 1200 920 0.76GS459 [GST:mkp1T182A,Y184F] 7.9 4.4 1500 680 0.71GS460 [GST:mkp1T186A,Y188F] 3.1 0.9 70 65 0.28GS461 [GST:mkp1T182A,Y184F,T186A,Y] 3.2 1.5 180 60 0.30GS462 [GST:mkp1K52R] 1.6 0.8 60 65 0.28

a Concentration in mM.b Concentration in mM.

tation ability, albeit at low levels, as measured by the and Y188 residues within the activation loop. We havedegree of suppression of the lytic defect and caffeine previously shown that Mkp1 is activated by exposuresensitivity phenotype, resulting in values higher than to temperature stress when expressed in S. cerevisiaethat of the inactive kinase, mkp1K52R (Fig. 1b and [10]. To determine the phosphorylation motif usage ofTable 2). Additionally, over-expression of the Mkp1 following temperature stimulation, the specificmkp1T186A,Y188F or mkp1T182A,Y184F,T186A,Y188F constructs in liq- activity (kinase activity per unit of protein) of each ofuid culture resulted in partial restoration of the normal the Mkp1 variants was examined. The specific activity ofgrowth rate at 308C that was less than either wild-type each construct was measured by immune-complex kinaseMkp1 or Mkp1T182A,Y184F, but appreciably higher than the assay with MBP as substrate following a 2.5-hour temper-mkp1K52R dead kinase control (Fig. 2). ature shift from 248C to 378C. The specific activity of

Mkp1T182A,Y184F, containing mutations in the initial phos-3.2. Second TEY motif (T186,Y188) is sufficient andphorylation motif, was identical to that of wild-typenecessary for Mkp1 kinase activity in response toMkp1 (Fig. 3a). However, mutagenesis of the secondmoderate heat shockmotif at T186 and Y188 within the activation domain

The kinase activity of Mkp1 is dependent upon acti- of Mkp1 resulted in loss of activity. Additionally,vation by the presence of the phosphorylatable T186 Mkp1T182A,Y184F,Y186A,Y188F, which lacks any phosphorylata-

ble residues, was unable to phosphorylate MBP. Thepresence of the phosphotransfer lysine of subdomain IIwas also necessary for kinase activity, as the Mkp1K52R

protein displayed no significant activity. To confirm thatthe loss of kinase activity observed was due to the ab-sence of phosphorylatable residues within the activationdomain and not the result of mutagenesis-induced pro-tein instability, the expression of each fusion proteinwas confirmed (Fig 3b). The amount of fusion proteinobtained from cultures exposed to elevated tempera-tures was less than that obtained from those incubated atthe permissive control temperature due to lysis resultingfrom partial restoration of the temperature lysis pheno-type (Fig. 3b). However, the significant increase in ki-nase activity despite the decrease in units of fusion pro-tein demonstrates the substantial increase in specificactivity of Mkp1 and Mkp1T182A,Y184F upon heat exposure.

Fig. 2. Mutation of the phosphorylation sites within the activationdomain of Mkp1 does not prevent the partial restoration of growth 3.3. Mkp1 interacts with Rlm1p in the absence ofat 308C. Transformants grown to mid-log phase in uracil-deficient MAP kinase activationsynthetic media containing 1 M sorbitol at 288C were inoculated intoYEPD at 0.25 OD/ml. Cultures were incubated at 308C and growth, Mkp1 of P. carinii has been shown to complement theas documented by absorbance (OD600) readings, was observed over slt2D–defect of the cell integrity pathway of S. cerevisiaea 40-hour period. Cultures are as follows: GS458 [GST:mkp1] (h), [10]. Because the transcription factor Rlm1p is a down-GS457 [GST:SLT2] (m), GS459 [GST:mkp1T182A,Y184F] (d), GS460

stream component of the cell integrity pathway and has[GST:mkp1T186A,Y188F] (j), GS461 [GST:mkp1T182A,Y184F,T186A,Y188F] (n) andGS462 [GST:mkp1K52R] (s). been shown to interact with Slt2p, the influence of the


to the DNA-binding domain of Gal4 (GBD), resulting inthe plasmids pAS[GBD:mkp1], pAS[GBD:mkp1T182A,Y184F],pAS[GBD:mkp1T182A,Y184F,T186A,Y188F], and pAS[GBD:SLT2],respectively. Rlm1p was fused to the transcriptional acti-vation domain of Gal4 (GAD), resulting in the plasmidpACT[GAD:RLM1]. The fusion proteins were exam-ined for their ability to drive transcription of the lacZreporter gene when expressed singly or in combination(Table 3). In the absence of either GAD or the GAD:Rlm1p fusion, expression of the GBD:Slt2p fusion re-sulted in a high level of transcription from the lacZpromoter. This activity has been attributed to the pres-ence of a poly-glutamine tract within the carboxyl-termi-nus of Slt2p, which functions as a cryptic transcriptionalactivation domain [25]. This poly-glutamine tract is notpresent in Mkp1. The lack of a similar cryptic transcrip-tional activation function of Mkp1 was demonstratedby the inability of the GBD:Mkp1 fusion to activatetranscription when expressed alone or with the GADcontrol vector. However, when the GBD:Mkp1 andGAD:Rlm1p fusions were co-expressed, the level ofstimulation was increased 29-fold over background lev-Fig. 3. Removal of phosphorylation sites within the activation domain

of Mkp1 results in loss of kinase activity following shift to elevated els, suggesting that Mkp1 and Rlm1p can interact intemperature. (a) GST-fusion proteins were isolated with glutathione- vivo (Table 3). To determine the dependence of theagarose from yeast transformant cultures inoculated with an equal phosphorylation state and/or activation state of Mkp1number of cells. Cultures were incubated at 248C and shifted to 248C

on its association with Rlm1p, a series of GBD:Mkp1or 378C for 2.5 hours. One-half of each purified fusion protein prepara-fusion constructs containing mtations in the activationtion was incubated with [g-32P]ATP in the presence of 10 mg MBP

for 20 min at 308C and fractionated on a 16% Tris-glycine gel to domain or ATP-binding site of Mkp1 were tested fordetermine kinase activity. (b) The remaining portions were fraction- their ability to interact with Rlm1p (Table 3). The rela-ated on a 10% Tris-glycine gel and immunoblotted with anti-GST tive strength of association with Rlm1p, expressed asantiserum for fusion protein quantitation.

percent interaction relative to the b-galactosidase activityvalue of the wild-type Mkp1-Rlm1p interaction (Fig. 4),of each of the GBD constructs are as follows: Mkp1phosphorylation state of the activation loop of Mkp1(100%), Mkp1T182A,Y184F (32.5%), Mkp1T182A,Y184F,T186A,Y188Fon its predicted association with Rlm1p was examined(17.1%), Mkp1K52R (4.7%), and Mkp1 in the absence ofusing the yeast two-hybrid system [24]. Fusions of Mkp1,

both wild-type and mutant versions, and Slt2p were made Rlm1p (3.3%). Removal of the non-conserved T182,

Table 3Two-hybrid interaction between Mkp1 and Rlm1p

DNA-binding protein Activating protein Filter assaya ONPG assayb

Gal4 wt (pCL1) Gal4 wt (pCL1) 1 519.24DBD-Slt2p – 1 69.83– GAD-Rlm1p 2 1.37GBD GAD-Rlm1p 2 1.0GBD-Mkp1 GAD 2 1.49GBD-Mkp1 GAD-Rlm1p 1 58.38GBD-Mkp1K52R GAD 2 1.25GBD-Mkp1K52R GAD-Rlm1p 6 2.73GBD-Mkp1T182A,Y184F GAD 2 1.45GBD-Mkp1T182A,Y184F GAD-Rlm1p 1 19.0GBD-Mkp1T186A,Y188F GAD 2 1.93GBD-Mkp1T186A,Y188F GAD-Rlm1p 1 NDGBD-Mkp1T182A,Y184F,T186A,Y188F GAD 2 1.47GBD-Mkp1T182A,Y184F,T186A,Y188F GAD-Rlm1p 1 10.0

a b-galactosidase activity determined by filter assay for the yeast strain SFY526 containing the indicated constructs. b-galactosidase activityis indicated by: 1 for strong positive (blue), 6 for weak positive (pale blue/white), and – for non-positive (white). Filter assays were performedfor at least 10 independent transformants per construct.

b b-galactosidase activity determined by ONPG assay for the yeast strain SFY526 containing the indicated constructs. The mean value isexpressed in Millar units and represents values obtained from at least 4 independent transformants per construct assayed in duplicate.


Fig. 4. Interaction of Mkp1 with Rlm1 is partially dependent on phosphorylation state of activation loop. The SFY526 lacZ reporter strain wastransformed with GAD:RLM1 and/or the various GBD:mkp1 constructs as described in Materials and Methods. Interaction values are shownas relative to the interaction between Mkp1 and Rlm1p and are based on mean b-galactosidase units shown in Table 3. The relative percentagevalues shown are: Mkp and Rlm1p (100%), Mkp1T182A,Y184F and Rlm1p (32.5%), Mkp1T182A,Y184F,T186A,Y188F and Rlm1p (17.1%), Mkp1K52R and Rlm1p(4.7%) and Mkp1 in the absence of Rlm1p (3.3%).

Y184 phosphorylation motif reduced the association ofMkp1 with Rlm1p by nearly 68%. Interestingly, removalof both phosphorylation motifs reduced the associationby only 82.9%, compared to the 95.3% reduction ob-tained with the kinase inactive mutant, Mkp1K52R. Toconfirm that differences in the interaction strengths ofeach of the Mkp1 variants with Rlm1p were not theresult of mutagenesis-induced protein instability, theexpression of each fusion protein was confirmed (datanot shown).

3.4. Predicted structure of the activation loop of Mkp1

The predicted three-dimensional structure of Mkp1was determined by comparative protein modelling [26].Overall, the predicted structure of Mkp1 was found tobe very similar to the solved three-dimensional structureof ERK2 (Fig. 5) [1,27]. The position and conformationof the activation loop of Mkp1 was not significantlydifferent from that of ERK2, despite the presence ofan additional TEY [1]. The Tyr-188 residue of the con-served phosphorylation motif (T186,Y188) of the non-phosphorylated form of Mkp1 is buried within the activesite, as is the case with its counterpart in ERK2. How-ever, the Tyr-184 residue of the non-conserved motif(T182,Y184) is not buried within the catalytic site of

Fig. 5. Ribbon diagram of the predicted structure of Mkp1 in non-the enzyme, but instead extends away from the catalyticphosphorylated form. The activation loop is indicated on the right (ar-site and toward the outer surface of the protein. Therow). Side-chains of residues T182 and Y184 are highlighted in yellow.substitution of phenylalanine for tyrosine at residues Side chains of residues T186 and Y188 are shown in red. The catalytic

184 or 188 did not significantly alter the structure of loop is indicated (labelled Cat loop) [1]. The image was drawn withSwiss-PdbViewer version 3.1 and rendered with Quickdraw3D [26,40].the non-phosphorylated activation loop. Likewise, the


replacement of threonine with alanine at residues 182 of Mkp1T182A,Y184F to interact with Rlm1p could be dueor 186 did not result in a significant change in the confor- to mutagenesis-induced destabilization of the activationmation of the activation loop of Mkp1 (data not shown). loop of the kinase. However, this is probably not the

case because Mkp1T182A,Y184F is a functional kinase, asdemonstrated by its ability to restore the cell integrity

4. Discussion pathway and to phosphorylate an exogenous substratefollowing activation. Additionally, analysis of the struc-The goal of this study was to determine the signifi-ture of the activation loop following the introductioncance of the phosphorylation motif repeat on the func-of non-phosphorylatable substitutions did not appear totion of Mkp1 within the cell wall integrity pathway ofalter loop conformation. Therefore, the non-conservedS. cerevisiae and to apply this knowledge to the under-TEY motif may play some role in the regulation ofstanding of the regulation of the associations of MAPsubstrate interaction. The relevance of this to the associ-kinases with their substrates. When expressed in a S.ation of Rlm1p with the endogenous cell wall integritycerevisiae strain containing a defect in the cell wall integ-MAP kinase Slt2p is not known since Slt2p containsrity pathway, the conserved second TEY (T186,Y188)only the conserved TEY motif [15].of the activation domain of Mkp1 is the preferred site

Rlm1p is involved in the regulation of the transcrip-of phosphorylation in response to temperature stress.tion of several genes involved in cell wall biosynthesisExpression of the mkp1T182A,Y184F mutant containing non-[17,28,29]. The transcriptional activity of Rlm1p hasphosphorylatable substitutions in the non-conserved ini-been shown to be dependent upon Slt2p, although thetial TEY (T182,Y184) motif results in a fully functionalimportance of the phosphorylation state of Slt2p on itsMAP kinase with levels of complementation and kinaseability to interact with Rlm1p has not been fully exam-activity equivalent to those observed with the wild-typeined [17,30]. Conflicting results exist as to whether orMkp1. Interestingly, overexpression of an mkp1 mutantnot Slt2p has the ability to phosphorylate Rlm1p inwith non-phosphorylatable residues in only the con-vivo [17,30]. In light of the available data, it has beenserved second TEY (mkp1T186A,Y188F) motif, or in bothsuggested that Slt2p and Rlm1p may associate as partTEY motifs (mkp1T182A,Y184F,T186A,Y188F), displayed low lev-of a larger complex which interacts with another proteinels of complementation ability, including partial restora-that is responsible for the phosphorylation of Rlm1ption of caffeine resistance, whereas the inactive kinase[17]. Therefore, it is possible that the cell wall integritycontrol mutant, mkp1K52R, had no detectable comple-MAP kinase may associate with Rlm1p in the absencementation function. This low level of complementationof phosphorylation, leading to the formation of an acti-ability appears to be independent of Mkp1 kinase activ-vation complex that regulates the DNA-binding activityity as neither mutant was able to phosphorylate MBPand/or subcellular localization of the transcription fac-in vitro. In addition to the ability of Mkp1 to restoretor. We have determined that Mkp1 is able to associatepartial activity to the cell integrity pathway in the ab-with Rlm1p in a manner independent of kinase activity.sence of phosphorylation of the activation loop, theHowever, the dependence of the transcriptional activityassociation of Mkp1 with a known Slt2p substrate, theof Rlm1p on the activation state of Mkp1 is not known.transcription factor Rlm1p, can also occur in the absenceOver-expression of RLM1 has been shown to suppressof phosphorylation. The association of Mkp1 withpartially the sensitivity of cell wall integrity mutants toRlm1p was strongest with the wild-type MAP kinase,caffeine, a presumed cell wall biosynthesis inhibitor [30].but mutation of the phosphorylation sites within theTherefore, the ability of kinase-inactive Mkp1 to restoreactivation domain did not completely abolish the inter-partially caffeine resistance to S. cerevisiae with the slt2Daction of the kinase with Rlm1p. However, mutation ofdefect suggests that activation of the MAP kinase bythe catalytic lysine of subdoman II effectively abolishedphosphorylation is not absolutely required for the acti-any significant interaction. These results suggest that,vation and/or regulation of pathways known to bealthough the catalytic lysine of the ATP-binding domainRlm1p-dependent.is necessary for interaction with Rlm1p, the presence

Another putative MAP kinase of the cell integrityof phosphorylatable residues within the activation do-pathway has been identified in S. cerevisiae. Mlp1p ismain of Mkp1 is not absolutely required for associationan unusual MAP kinase that contains a lysine in placeof Mkp1 with Rlm1p. The second conserved TEYof the conserved threonine residue of the activation(T186,Y188) of the activation domain of Mkp1 appearsdomain and an arginine in place of the invariant catalyticto be the preferred site of phosphorylation by the celllysine in subdomain II [30]. Although such substitutionsintegrity pathway, but the possibility remains that thehave been made in other MAP kinases to render themnon-conserved phosphorylation motif may also be uti-inactive, Mlp1p is able to reverse the caffeine sensitivitylized. This conclusion is based on the observation thatphenotype when over-expressed in a cell integrity path-mutation of the non-conserved TEY motif results in away defective strain and has been shown to interact withreduction in the ability of Mkp1 to interact with Rlm1p

as compared to the wild-type kinase. The reduced ability Rlm1p, the downstream target of Slt2p [30]. Interest-


ingly, mutation of the tyrosine residue within the activa- sponse and the effectors involved. The identificationand characterization of substrates and additional com-tion domain of Mlp1p results in a loss of complementa-ponents of the cell integrity pathway in P. carinii willtion function, but the effect of the mutation on theallow a more detailed analysis of the function and regu-ability of Mlp1p to associate with Rlm1p has not beenlation of Mkp1. The effect of a hypothetical simultane-reported [30]. In addition, the mammalian MAP kinasesous phosphorylation of both repeats on the stabilityERK2 and ERK7 display functions that are independentand positioning of the activation loop and substrateof their kinase activity [31,32]. Therefore, it is possiblespecificity region is unknown. Steric hindrance may neg-that Mkp1 could also have some function in the inactiveatively influence the likelihood of such an event. Thestate that is independent of its ability to phosphorylatemost plausible hypothesis is that only one motif is phos-substrate. Mutation of the conserved lysine residue inphorylated at any given time and that the phosphoryla-subdomain II (K52) has been shown to create a non-tion of one motif or the other by an upstream MEKproductive binding mode for ATP which alters the pep-would influence the substrate choice of the kinase sincetide substrate binding ability of the MAP kinase [5,33].the substrate specificity region could have different on-The means by which the catalytic lysine mutation influ-formations depending on which TEY motif was phos-ences MAP kinase peptide substrate binding is not clear.phorylated [1]. This hypothesis also suggests that Mkp1However, the loss of the catalytic lysine residue in sub-could be differentially regulated, depending on the typedomain II does not prevent the phosphorylation of theof stimulus. Given the small genome size of P. cariniithreonine and tyrosine residues within the activation(8 3 106 bp), the organism may conserve gene densitydomain by the upstream MEK, nor does it prevent nu-if the TEY repeat allows a single MAP kinase to func-clear translocation of MAP kinase in the phosphory-tion in more than one pathway to ensure responsivenesslated or non-phosphorylated state [34,35]. Therefore,of the organism to the extracellular environment.the proper alignment of ATP by the catalytic lysine in

subdomain II may be more important in the regulationof the substrate binding specificity of Mkp1 than the

Acknowledgmentsphosphorylation state of the activation loop. However,this may not e true for all MAP kinases because the We thank J.C. Edman for the P. carinii cDNA librarykinase-independent activation of topoisomerase II by and D. Levin for the S. cerevisiae strains. George Smu-ERK2 is dependent not on the catalytic activity of the lian is a recipient of a Burroughs Wellcome Fund NewMAP kinase, but rather on the phosphorylation state Investigator Award in Pathogenic Mycology. This workof the activation loop [32]. Additionally, the non-phos- was supported by the Office of Research and Develop-phorylated form of the MAP kinase Kss1p of the yeast ment, Medical Research Service, Department of Veter-filamentation pathway has been shown to exert an inhib- ans Affairs and grant AI 34759 from the National Insti-itory function by binding the transcription factor, Ste12p tutes of Health.[36–39].

Despite the low level of complementation ability ofmutants of mkp1 lacking the conserved T186,Y188 Referencesphosphorylation motif, the non-conserved TEY motif

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[8] Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J,cerevisiae and P. carinii are both ascomycetes, the useShabanowitz J, Hunt DF, Weber MJ, Sturgill TW. EMBO J 1991;of S. cerevisiae as a model organism for P. carinii may10:885–92.not be appropriate in all cases. The stimuli that activate [9] Ray LB, Sturgill TW. Proc Natl Acad Sci USA 1988;85:3753–7.

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MH, Goldsmith EJ. Biochemistry 1996;35:5641–6.[22] Schott EJ, Gardner RC. Mol Gen Genet 1997;254:63–72. [34] Fukuda M, Gotoh Y, Nishida E. EMBO J 1997;16:1901–8.[23] Torres L, Martin H, Garcia-Saez MI, Arroyo J, Molina M, San- [35] Kosako H, Nishida E, Gotoh Y. EMBO J 1993;12:787–94.

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Documents

Mkp1 of Pneumocystis carinii associates with the yeast transcription factor Rlm1 via a mechanism independent of the activation state