Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK

Abstract

Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5(ND)) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or "synthetic," supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling.

FIGURE 1:

Signal inhibition by monophosphorylated Fus3. (A) Transcription reporter (FUS1-lacZ) activity in wild-type (WT), Fus3-deficient, or Fus3 activation loop mutant strains (fus3^T180A, fus3^Y182F, and combined fus3^T180A/Y182F) stimulated with 10 μM α-factor. (B) Same analysis in wild-type cells bearing a single-copy plasmid with no insert (vector), wild-type FUS3, or mutations in the Fus3 activation loop. FUS1-lacZ data are presented as a percentage of maximum activity in wild-type cells (A and B, top) and as a full dose–response curve (B, bottom). Results report ±SEM (n = 3) for each data point, but this is not visible in every case. *Statistically significant Student’s t test of pairwise comparisons for wild type and the individual mutants.

FIGURE 2:

Analysis of differentially phosphorylated forms of Fus3. (A) Wild-type or ste11Δ (MAPKK) mutant cells untreated (0) or treated for 2 or 15 min with 10 μM α-factor were lysed and resolved by immunoblotting after SDS–PAGE and probed with Fus3 antibodies (top) or p44/p42 antibodies (top middle), with Phos-tag reagent and probed with Fus3 antibodies (bottom middle), or with G6PDH load control antibodies (bottom). Bands represent the dual-phosphorylated (ppKss1, ppFus3), monophosphorylated (pFus3), nonphosphorylated (npFus3), and total (Fus3) protein. Except for the topmost panel, this and all subsequent experiments were done using Phos-tag. (B) Wild-type, Fus3 activation loop (T180A, Y182F), and fus3Δ mutants were resolved by Phos-tag SDS–PAGE and immunoblotting with Fus3 antibodies (top), p44/p42 antibodies (middle), or G6PDH load control antibodies (bottom). Arrowheads indicate the bands of interest (i.e., those recognized by p44/42 antibodies). (C) Wild-type, fus3^KR (catalytically inactive), and ste5^ND (nondocking) mutants untreated or treated for 15 min with 10 μM α-factor were resolved by Phos-tag SDS–PAGE and immunoblotted with Fus3 antibodies. Representative data are shown below. Band intensity was quantified as a percentage of total Fus3 in each lane. Results are reported as ±SEM (n ≥ 3). (D) Wild-type, ste7Δ, and phosphatase-deficient ptp2Δ, ptp3Δ, and msg5Δ mutants, alone or in combination, were resolved by Phos-tag SDS–PAGE and immunoblotted with Fus3 antibodies (top) or G6PDH load control antibodies (bottom).

FIGURE 3:

Dynamics of differentially phosphorylated forms of Fus3. (A) Left, wild-type cells were treated for the indicated times with 10 μM α-factor and resolved by Phos-tag SDS–PAGE and immunoblotted with Fus3 antibodies (top) or G6PDH load control antibodies (bottom). Right, dual-phosphorylated (ppFus3), monophosphorylated (pFus3), and nonphosphorylated (npFus3) quantified as a percentage of total Fus3 at 15 min. Results are reported as ±SEM (n ≥ 3). (B and C) Wild-type (WT), ste5^FB (feedback-phosphorylation deficient), ste5^ND (nondocking), ptp2Δ/ptp3Δ, and msg5Δ (phosphatase-deficient) mutants treated and resolved by Phos-tag SDS–PAGE, as described. Dual-phosphorylated Fus3 is quantified as a percentage of total Fus3 at 15 min. Results are reported as ±SEM (n ≥ 3).

FIGURE 4:

Dynamics and mathematical model of the differentially phosphorylated forms of Fus3. (A) Time series for wild-type (top left), ste5^ND (nondocking) mutant (top right), msg5Δ (bottom left), and ptp2Δ/ptp3Δ (bottom right) cells treated with 10 μM α-factor and resolved by Phos-tag SDS–PAGE and immunoblotting with Fus3 antibodies. Dual-phosphorylated (ppFus3), monophosphorylated (pFus3), and nonphosphorylated (npFus3) quantified as a percentage of total Fus3. Results are reported as ±SEM (n ≥ 3). Circles are experimental results. Lines are simulation results of the mathematical model shown in B. (B) Left, diagram of the mathematical models. Black lines represent pathway components present in all models. The dashed line is included in the model in which feedback-phosphorylated Ste5 (pSte5) limits Fus3 phosphorylation (activation), and the red lines are included in the model in which feedback-phosphorylated Ste5 increases Fus3 dephosphorylation (deactivation). The combined model includes both effects. Top right, performance of all three models. This graph shows values for the sum of squared deviations (SSDs) for the 39,000 best parameter sets found by the Monte Carlo algorithm for parameter estimation. Bottom right, model predictions for the combined model and corresponding experimental results for the ste5^ND msg5Δ double mutant.

FIGURE 5:

Synergistic activation of Fus3. (A) Dual-phosphorylated Fus3 in wild type and ste5^ND (nondocking) msg5Δ (phosphatase-deficient) mutants replotted from Figure 3 for comparison with the ste5^ND msg5Δ mutant. Dual-phosphorylated Fus3 is quantified as a percentage of total Fus3 at 15 min. Results are reported as ±SEM (n ≥ 3). (B) Transcription reporter data in the same strains as in A, as a percentage of maximum activity in wild type. Inset, Hill slope and EC₅₀ for each strain. Results are reported as ±SEM (n = 3). (C) Pheromone-induced growth arrest for cells treated with α-factor. Halo diameters are quantified for all strains at 5 μg (left). Results are reported as ±SEM (n = 4). Right, representative halo assays for the wild-type and ste5^ND msg5Δ strains at 1.5, 5, and 15 μg. (D) Polarized growth in cells treated with 300 nM α-factor. Representative images for wild-type, msg5Δ, ste5^ND, and ste5^ND msg5Δ cells, each bearing the integrated polar cap marker Bem1-GFP (arrow), at the indicated times. The path of the polar cap was quantified for 10 cells 45–200 min after pheromone addition. Data are representative of two or more experiments. Scale bar, 10 μM.