Positions of substrate-binding site mutations and D-motif-binding site mutations within the predicted structure of Mpk1. The structure was modeled against known MAPK structures using the SWISS-MODEL program (http://swissmodel.expasy.org/SWISS-MODEL.html). The two images are rotated 180° with respect to each other. Mutated substrate-binding site residues are indicated in green, and mutated D-motif-binding site residues are indicated in yellow. Residues phosphorylated in the activation loop (T190 and Y192) are indicated in red.

Mutations in the substrate-binding site and the D-motif-binding site of Mpk1 cause differential phenotypes. An mpk1Δ strain (yeast strain DL456) was transformed with centromeric plasmids expressing the indicated alleles of MPK1 or empty vector (pRS315). Transformants were grown overnight to saturation in selective medium (SD lacking Leu), and serial 10-fold dilutions were plated by pin plating from 96-well plates onto YPD alone or YPD plus 8 mM caffeine and incubated at the indicated temperature for 2 days.

Two-hybrid association of Mpk1 with the C-terminal region of Swi4. A Gal4 DNA-binding domain (DBD) fusion to the catalytic domain of Mpk1 (residues 1 to 328; p2266) was tested for interaction with Gal4 activation domain (AD) fusions to full-length Swi4 (residues 1 to 1093; p2352), the N-terminal region (residues 1 to 671; p2717), and the C-terminal region (residues 672 to 1093; p2718) in yeast two-hybrid strain SFY526. Transformants were cultivated in selective medium for 15 h at either 23°C or 39°C. Vector controls (Gal4BDB vector [p1172] and Gal4AD vector [p1173]) are included for each fusion. β-Galactosidase (β-Gal) activity was measured in crude extracts. The specific activity of β-galactosidase (in units) [β-Gal Sp. Act. (U)] is shown on the y axis. Each value represents the mean and standard deviation (error bar) from three independent transformants.

Mpk1 undergoes serine phosphorylation in response to caffeine treatment. (A) Caffeine induces serine phosphorylation of Mpk1. Yeast strain DL456 (mpk1Δ), transformed with a plasmid expressing FLAG-tagged Mpk1 (p2313), was either maintained at 25°C or subjected to cell wall stress (heat shock) by changing the temperature to 39°C (+) or by adding 8 mM caffeine (+) for 2 h. Cell extracts were subjected to immunoprecipitation of Mpk1-FLAG, followed by immunodetection of total Mpk1 with anti-FLAG antibodies, activated Mpk1 with phospho-p42/p44 antibodies (phospho-TEY), and serine phosphorylation of Mpk1 with antiphosphoserine antibodies. (B) Caffeine-induced serine phosphorylation of Mpk1 is independent of Mpk1 activation state or catalytic activity. Yeast strain DL456 (mpk1Δ), transformed with a plasmid expressing FLAG-tagged Mpk1 (p2313), nonactivatable Mpk1-TAYF (p2316), or catalytically inactive Mpk1-K54R (p2317), were subjected to caffeine stress (+) and processed as described above for detection of Mpk1 phosphorylated on serine and total Mpk1.

Dependence of Mpk1 serine phosphorylation on Mec1/Tel1 and Rad53 and its role in FKS2 transcription. (A) Caffeine-induced phosphorylation of Mpk1 Ser423 is blocked in a rad53Δ mutant, and phosphorylation of both Ser423 and Ser428 are blocked in a Mec1/Tel1 mutant. Yeast strains with the sml1Δ gene (wild type [WT] [strain DL3950]), mec1Δ tel1Δ sml1Δ gene (strains DL3955 to DL3957), and rad53Δ sml1Δ gene (strain DL3953) that had been transformed with plasmid expressing Mpk1-FLAG (p2704), Mpk1-S423A-FLAG (p2824), or Mpk1-S428A-FLAG (p2825) were subjected to caffeine stress. The cell extracts were processed for detection of serine-phosphorylated Mpk1 (phospho-Ser) and total Mpk1 (Mpk1-Flag) as described in the legend to Fig. 7. (B) Blocking Ser423 phosphorylation in a rad53Δ mutant allows caffeine-induced cell wall stress to drive FKS2 transcription. An FKS2-lacZ reporter plasmid (p2052) was transformed into yeast strains with the sml1Δ gene (WT; DL3950) and rad53Δ sml1Δ gene (DL3953). Transformants were grown as described in the legend to Fig. 2A and subjected to thermal or caffeine stress or maintained at 23°C. β-Galactosidase activity was measured in crude extracts. β-Galactosidase specific activity (in units) [β-Gal Sp. Act. (U)] is shown on the y axis. Each value represents the mean and standard deviation (error bar) from three independent transformants.

Mutations in the substrate-binding site and the D-motif-binding site of Mpk1 cause differential effects on cell wall stress induction of FKS2 and PRM5 expression. (A) Cell wall stress-induced PRM5 transcription. A PRM5-lacZ reporter plasmid (p1366) was cotransformed with centromeric plasmids expressing MPK1-FLAG (p2704), plasmids with the indicated point mutants in MPK1, or empty vector (pRS315) into an mpk1Δ mutant (yeast strain DL3195). Transformants were grown to saturation at 23°C in SD medium with 10% sorbitol lacking Ura and Leu. Cultures were diluted into 100 ml of medium so that subsequent incubation at 23°C, 39°C, or 23°C with 8 mM caffeine for 2 h resulted in mid-log-phase cultures (A₆₀₀ of 1.0). β-Galactosidase activity was measured in crude extracts. The specific activity of β-galactosidase (in units) [β-Gal Sp. Act. (U)] is shown on the y axis. Each value represents the mean ± standard deviation (error bar) from three independent transformants. (B) Cell wall stress-induced FKS2 transcription. An FKS2-lacZ reporter plasmid (p2052) was cotransformed with centromeric plasmids expressing MPK1 (p2704), plasmids with the indicated point mutations in MPK1, or empty vector (pRS315) into an mpk1Δ mlp1Δ mutant (DL3196). The mpk1Δ mlp1Δ mutant was used for this experiment because the Mlp1 pseudokinase contributes to FKS2 expression. Transformants were grown as described above and diluted into 100 ml of medium so that subsequent incubation at 23°C, 39°C, or 23°C with 8 mM caffeine (MPK1 only) for 15 h resulted in mid-log-phase cultures (A₆₀₀ of 1.0 to 1.5). (C) Point mutations in Mpk1 are not compromised for phosphorylation by their activating protein kinases (Mkk1/2). Protein extracts from cells treated as in panel A (left untreated at 23°C [lanes U], heated to 39°C [lanes H], or treated with 8 mM caffeine [lanes C]) were subjected to immunoprecipitation of Mpk1-FLAG with anti-FLAG antibodies followed by SDS-PAGE. Dual phosphorylation of Mpk1 was detected with anti-phospho-p44/p42 MAPK antibodies; total Mpk1 was detected with anti-FLAG antibodies.

Coimmunoprecipitation of Swi4 with mutant forms of Mpk1 in response to heat shock and caffeine treatment. Yeast strain DL456 (mpk1Δ) was cotransformed with plasmids bearing the indicated FLAG-tagged MPK1 allele (wild-type MPK1 [p2704], mpk1-R196A [p2706], and mpk1-K83A [p2709]) and HIS-tagged SWI4 (p2418). Transformants were cultivated to mid-log phase in selective medium lacking methionine (to induce expression of Swi4-6HIS) and either maintained at 25°C or subjected to cell wall stress by changing the temperature to 39°C (+) or by adding 8 mM caffeine for 2 h (+). Cell extracts (lysate) and immunoprecipitates (IP) with anti-FLAG M2 affinity gel were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibodies to detect Mpk1 or with anti-HIS antibodies to detect Swi4.

Behavior of a strain with a mutation in the Mpk1-binding site of Swi4. (A) Cell wall stress-induced FKS2 transcription is impaired in the swi4-IAIA mutant. An FKS2-lacZ reporter plasmid (p2052) was cotransformed with centromeric plasmids expressing SWI4 (p2713), plasmids with the indicated point mutations in SWI4 (I913A/I915A [Swi4-IAIA] [p2714] or L966A/L968A [Swi4-LALA] [p2715]), or empty vector (pRS315) into a swi4Δ mutant (yeast strain DL3145). Transformants were grown as described in the legend to Fig. 2A and subjected to cell wall stress at 39°C or maintained at 23°C. β-Galactosidase activity was measured in crude extracts. The specific activity of β-galactosidase (in units) [β-Gal Sp. Act. (U)] is shown on the y axis. Each value represents the mean and standard deviation (error bar) from three independent transformants. (B) The Swi4-IAIA mutant protein fails to coimmunoprecipitate with activated Mpk1. A swi4Δ mutant (DL3145) was cotransformed with plasmids bearing FLAG-tagged MPK1 (p2704) and a HIS-tagged SWI4 allele (wild-type SWI4 [p2418] or swi4-IAIA [p2716]). Transformants were cultivated to mid-log phase in selective medium lacking methionine (to induce expression of Swi4-6HIS) and either maintained at 25°C or subjected to cell wall stress by heat shock (changing the temperature to 39°C) for 2 h (+). Cell extracts (lysate) and immunoprecipitates (IP) with anti-FLAG M2 affinity gel were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG antibodies to detect Mpk1 or anti-HIS antibodies to detect Swi4. No Ab, no antibody. (C) CLN2 transcription is not impaired in the swi4-IAIA mutant. A CLN2-lacZ reporter plasmid (p2066) was cotransformed with centromeric plasmids expressing SWI4 (p2713), the swi4-IAIA mutant (p2714), or empty vector (pRS315) into a swi4Δ mutant (DL3145). Transformants were grown as described above but were maintained at 23°C. (D) The Swi4-IAIA mutant is not impaired for Swi4 binding. A swi4Δ mutant (DL3145) was transformed with a plasmid bearing a HIS-tagged SWI4 allele (wild-type SWI4 [p2418] or swi4-IAIA [p2716]). Transformants were cultivated to mid-log phase at 30°C in selective medium lacking methionine (to induce expression of Swi4-6HIS). Cell extracts (lysate) and immunoprecipitates (IP) with Ni-nitrilotriacetic acid beads were subjected to SDS-PAGE and analyzed by immunoblotting with anti-HIS antibodies to detect Swi4 or anti-Swi6 antibodies to detect coprecipitated endogenous Swi6. Ab, antibody. (E) The Swi4-IAIA mutant is defective for FKS2, but not CLN2, promoter occupancy. Transformants from panel D and a vector control (p2415) were cultivated in YPD medium or subjected to heat shock at 39°C for 1 h prior to ChIP analysis using primers designed to detect the endogenous FKS2 or CLN2 promoter. PCRs of whole-cell extract (WCE) from the unstressed wild-type Swi4 sample is also shown.

Behavior of Mpk1 mutants that block serine phosphorylation induced by caffeine. (A) Ser423 and Ser428 of Mpk1 are phosphorylated in response to caffeine. Yeast strain DL456 (mpk1Δ), transformed with plasmids expressing FLAG-tagged Mpk1 (p2313) or Ser-to-Ala mutant forms (S423A [p2824], S428A [p2825], or S423A S428A [p2826]) were subjected to caffeine stress (+), and extracts were processed for detection of serine-phosphorylated Mpk1 (phospho-Ser), activated Mpk1 (phospho-TEY), and total Mpk1 (Mpk1-Flag) as described in the legend to Fig. 7. (B) Blocking phosphorylation of Mpk1 at Ser423 allows caffeine-induced cell wall stress to drive FKS2 transcription. An FKS2-lacZ reporter plasmid (p2052) was transformed into yeast strains with the indicated MPK1 alleles integrated into the genome (MPK1 [DL3929], mpk1-S423A [DL3930], mpk1-S428A [DL3931], and mpk1-S423A S428A [DL3932]). Transformants were grown as described in the legend to Fig. 2A and subjected to thermal or caffeine stress or maintained at 23°C. β-Galactosidase activity was measured in crude extracts. β-Galactosidase specific activity (in units) [β-Gal Sp. Act. (U)] is shown on the y axis. Each value represents the mean and standard deviation (error bar) from three independent transformants. (C) Blocking phosphorylation of Mpk1 on Ser423 allows caffeine-activated Mpk1 to bind Swi4. Yeast strain DL456 (mpk1Δ), cotransformed with plasmids expressing FLAG-tagged Mpk1 (p2313) or Ser-to-Ala mutant forms (Mpk1-S423A [p2824] or Mpk1-S428A [p2825]) and Swi4-6HIS (p2418) were subjected to heat shock or caffeine stress (+), and extracts were processed for coimmunoprecipitation of Swi6 with Mpk1 as described in the legend to Fig. 6B.

Model for interaction of Mpk1 with Swi4. Mpk1 activated by cell wall stressors other than caffeine is capable of activating Swi4-dependent transcription. Swi4 binds Mpk1 through the D motif near the C terminus of Swi4, which allows Swi4 to bind the FKS2 promoter. This interaction is proposed to relieve the autoinhibitory interaction of the Swi4 C terminus that prevents DNA binding. In this regard, Mpk1 serves the same function as Swi6 in allowing Swi4 to bind DNA. Swi6 is recruited to the Mpk1-Swi4 complex and binds to Swi4 through their respective C termini (not shown), in a manner similar to their interaction on G₁-induced cell cycle genes (e.g., CLN2). Mpk1 activated by caffeine is not capable of Swi4-driven transcription. Under these conditions, activated Mpk1 is incapable of interacting with Swi4. Additional phosphorylations on Mpk1 Ser423 and Ser428 are provoked by caffeine treatment through the DNA damage checkpoint kinases Mec1/Tel1 and Rad53. Phosphorylation of Ser423, which is dependent on Rad53, specifically prevents Mpk1 binding to Swi4. DB, D-motif-binding site; SB, substrate-binding site; p, phosphate group; CFW, calcofluor white.