Differential input by Ste5 scaffold and Msg5 phosphatase route a MAPK cascade to multiple outcomes

Abstract

Pathway specificity is poorly understood for mitogen-activated protein kinase (MAPK) cascades that control different outputs in response to different stimuli. In yeast, it is not known how the same MAPK cascade activates Kss1 MAPK to promote invasive growth (IG) and proliferation, and both Fus3 and Kss1 MAPKs to promote mating. Previous work has suggested that the Kss1 MAPK cascade is activated independently of the mating G protein (Ste4)-scaffold (Ste5) system during IG. Here we demonstrate that Ste4 and Ste5 activate Kss1 during IG and in response to multiple stimuli including butanol. Ste5 activates Kss1 by generating a pool of active MAPKKK (Ste11), whereas additional scaffolding is needed to activate Fus3. Scaffold-independent activation of Kss1 can occur at multiple steps in the pathway, whereas Fus3 is strictly dependent on the scaffold. Pathway specificity is linked to Kss1 immunity to a MAPK phosphatase that constitutively inhibits basal activation of Fus3 and blocks activation of the mating pathway. These findings reveal the versatility of scaffolds and how a single MAPK cascade mediates different outputs.

Figure 1

Activation of Kss1 during vegetative growth is dependent on the mating G protein β subunit (Ste4) and scaffold (Ste5), but not on upstream regulators of the high osmolarity growth (HOG), protein kinase A, protein kinase C, and sucrose nonfermenting pathways. (A) Cartoon of mating response, IG, SVG and HOG pathways in Saccharomyces cerevisiae. (B) FRE(Tec1)-lacZ reporter gene expression. β-Galactosidase assays were performed on mitotically dividing S288c strains harboring a pFRE(TEC1)-lacZ plasmid (Madhani and Fink, 1997a). Standard deviation is shown for triplicate samples. (C) Fre(Tec1)-LacZ activity in S288c strains with mutations in upstream regulators of a variety of protein kinase pathways. (D) Kss1 activity and protein in S288c strains with mating pathway mutations. (E) Kss1 activity and protein in S288c fus3Δ strains with mutations in the mating pathway. Immunoblot analysis was performed on whole-cell extracts prepared from vegetatively dividing cells. (F) Kss1 activity in fus3Δste and ste strains. The active form of Kss1 was detected with an anti-phospho p42/p44 peptide antibody and total Kss1 protein was detected with a polyclonal antibody. The asterisk indicates a protein that crossreacts nonspecifically with the Kss1 antibody. Tcm1 is a ribosomal protein that serves as a normalization control. Similar results were found in W303a. Depending on gel conditions, Kss1 migrates as a doublet when activated for unknown reasons.

Figure 2

Activation of Kss1 during IG is dependent on Ste4 and Ste5. (A, B) IG of S288c strains harboring a FLO8 plasmid requires STE4 and STE5. (C) Enhancement of Kss1 activity as a result of growth in the absence of glucose requires STE4 and STE5. Immunoblot analysis of extracts made from cells grown in liquid medium containing 2% glucose or 2% glycerol/2% ethanol. (D) 1-Butanol activates Kss1 and Fus3 in the presence of Ste4 and Ste5. S288c strains growing in liquid medium containing 2% glucose were induced with 1% 1-butanol for the indicated times.

Figure 3

Kss1 only requires Ste5 to activate Ste11. (A) Kss1 activation is dependent on Ste5 binding to Ste4 and Ste11. Level of Kss1 activity in ste5Δ and ste5Δ fus3Δ strains expressing Ste5, Ste5^C180A or Ste5^L482/485A from centromeric plasmids. The ‘+' indicates 1 h induction with 1% 1-butanol. (B) Ste11-4 activates Kss1 in the absence of Ste5. Levels of active Kss1 and Fus3 and total Kss1 and Fus3 proteins in wild-type and ste5Δ cells expressing either a control vector or Ste11-4 from a centromeric vector. (C) Ste11-4 activates Kss1 and Fus3 in the absence of STE4, STE20, and STE50. (D) Ste5 is needed for Ste11-4 to activate Fus3-101. Active Kss1 and Fus3 in a fus3Δ ste5Δ strain expressing FUS3 or FUS3-101, with or without STE11-4. (E) Ste11ΔN activates Kss1, but not Fus3, in the absence of Ste5. Ste11ΔN was expressed from the GAL1 promoter. Cells were grown in 2% raffinose medium and then in 2% galactose medium for 1 h. The abundance of Kss1 and Fus3 is lower in 2% raffinose medium than in 2% galactose medium, resulting in very low basal activity. (F) Ste5Δ241–336 supports activation of Kss1, but not Fus3, by Ste11-4. Extracts were prepared from the S288c ste5Δ pSTE11-4 strain with CEN vectors expressing Ste5 (lane 1), Ste5Δ241–336 (lane 3) or a control vector (lane 2).

Figure 4

Fus3, but not Kss1, must bind to Ste5 to be activated by Ste11. (A) Overexpression of either Ste11 or Ste7 bypasses the need for Ste5 in basal activation of Kss1. Wild-type and ste5Δ strains harbor a vector expressing STE11 from its native promoter (pSTE11 2 μ) on a multicopy plasmid or STE7 from the GAL1 promoter (GAL-STE7) on a centromeric plasmid. (B) Co-overexpression of Ste7, Ste11, and Kss1 completely bypasses the need for Ste5 to activate Kss1. Relative levels of active Kss1 and total Kss1 protein in wild-type and ste5Δ cells overexpressing Kss1, Ste7, and/or Ste11. The strong intensity of the Kss1 band in strains expressing KSS1 2 μ obscures the weak band of endogenous Kss1. (C) Cdc42^G12V does not stimulate Kss1 activity in a ste5Δ strain. GAL1p-CDC42^G12V was expressed for 1 h in medium containing 2% galactose.

Figure 5

Fus3 is efficiently activated during vegetative growth, but is inhibited by Msg5 MAPK phosphatase. (A) Activation of Kss1 and Fus3 by mating pheromone. Vegetatively growing cells were treated with α factor for 15 min. Parallel immunoblotting showed that the levels of Fus3 and Kss1 remained the same under these conditions (data not shown). (B) Fold effect of activation of Fus3 by α factor is greater than that of Kss1 based on densitometric analysis. The level of active MAPK relative to total MAPK protein was determined by densitometry for each lane and the fold increase as a consequence of α factor addition was plotted. Quantitation was done with the Scion image 1.62c densitometry program of the public domain software NIH image (available at http://rsb.info.nih.gov/nih-image/) using multiple exposures of immunoblots. (C) Effect of increasing Ste5 levels on Fus3 and Kss1 activity. (D) Effect of gain-of-function STE5^hyp1 mutant on Fus3 and Kss1. (E) Level of active Kss1, Fus3, Mpk1, and Hog1 in msg5Δ, ptp2Δ, and ptp3Δ MAPK phosphatase mutants. Kss1, Fus3, and Mpk1 were detected with anti-p42/p44 phosphopeptide antibody. Hog1 with anti-active p38 antibody. The positions of Mpk1 and Hog1 were deduced from mpk1 and hog1 mutants. The level of Kss1 protein did not change in the mutants compared to wild type (data not shown).

Figure 6

Differential regulation of Kss1 and Fus3 by Ste5 and Msg5 is required for pathway specificity. (A) Constitutive mating pathway activation in msg5Δ strain based on FUS1-lacZ, FUS1-GFP, and cell morphology. (B) Decreased FRE(Tec1)-lacZ expression in a msg5Δ strain. (C) Level of active Kss1, Fus3, and Msg5 in STE11-4 strains with ste5Δ and/or msg5Δ mutations. (D) Growth and morphology of STE11-4 strains with ste5Δ and/or msg5Δ mutations. Strains were streaked for single colonies on a SC-selective plate and incubated at room temperature. Nomarski images were taken of the cells. (E) Model for how a single G protein–scaffold and MAPK phosphatase system regulates IG and mating pathways.