Differential regulation of transcription: repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins - PubMed (original) (raw)

Differential regulation of transcription: repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins

L Bardwell et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Kss1, a yeast mitogen-activated protein kinase (MAPK), in its unphosphorylated (unactivated) state binds directly to and represses Ste12, a transcription factor necessary for expression of genes whose promoters contain filamentous response elements (FREs) and genes whose promoters contain pheromone response elements (PREs). Herein we show that two nuclear proteins, Dig1 and Dig2, are required cofactors in Kss1-imposed repression. Dig1 and Dig2 cooperate with Kss1 to repress Ste12 action at FREs and regulate invasive growth in a naturally invasive strain. Kss1-imposed Dig-dependent repression of Ste12 also occurs at PREs. However, maintenance of repression at PREs is more dependent on Dig1 and/or Dig2 and less dependent on Kss1 than repression at FREs. In addition, derepression at PREs is more dependent on MAPK-mediated phosphorylation than is derepression at FREs. Differential utilization of two types of MAPK-mediated regulation (binding-imposed repression and phosphorylation-dependent activation), in combination with distinct Ste12-containing complexes, contributes to the mechanisms by which separate extracellular stimuli that use the same MAPK cascade can elicit two different transcriptional responses.

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Figures

Figure 1

Figure 1

Dig1 and Dig2 regulate filamentous growth in naturally invasive diploids. (A) Loss of DIG1 and DIG2 enhance diploid pseudohyphal development. Strain JCY102 (MATa/MAT_α DIG+) (a and c) and its otherwise isogenic derivative, YLB503 (MATa/MATα dig1_Δ/dig1_Δ dig2_Δ/dig2_Δ)_ (b and d), were assayed for filament formation on plates containing either low nitrogen (low N) (a and b) or high nitrogen (high N) (c and d). Representative colonies, photographed after 3 days (low N) or 1 day (high N) of growth at 30°C, are shown. (B) Expression of the _FRE_Ty1-lacZ reporter. The strains described in A were transformed with plasmid YEpU-FTyZ and grown on low nitrogen plates for 48 hr at 30°C, and β-galactosidase specific activity (nmol per min per mg of protein) was measured. Values represent the average of measurements, made in duplicate, on protein extracts prepared from at least three transformants of each strain. Error bars indicate the standard deviation. Comparable results were obtained on high nitrogen plates (data not shown).

Figure 2

Figure 2

Dig1 and Dig2 cooperate with Kss1 to inhibit invasive growth. (A) Loss of DIG1 and DIG2 enhance haploid invasive growth. Strain JCY100 (MATa STE+ DIG+) and its otherwise isogenic derivatives, JCY107 (_ste7_Δ), JCY137 (_ste7_Δ _kss1_Δ _fus3_Δ), JCY501 (_dig1_Δ _dig2_Δ), JCY512 (_dig1_Δ _dig2_Δ _ste12_Δ), and YLB507 (_ste7_Δ dig1_Δ dig2_Δ), were streaked onto YPD plates and assayed for surface growth (a), invasive growth (b), and agar penetration (c) after 3 days at 30°C. (B) Effect of Kss1 or Dig1 overproduction on expression of the FRE_Ty1-lacZ reporter: Dig1 and Dig2 are required for Kss1-imposed repression. The strains described in A were transformed with plasmid YEpU-FTyZ or YEpL-FTyZ. JCY137 was also transformed with either YCpLG-KSS1 (p_GAL-KSS1) or p_GAL-DIG1, for overproduction of Kss1 or Dig1, respectively, from the GAL1 promoter. YLB507 was also transformed with YCpLG-KSS1 (p_GAL_-KSS1) or p_DIG1, for overproduction of Kss1 or endogenous-level expression of Dig1, respectively. Strains were grown on plates containing 2% galactose and 0.2% sucrose as the carbon source for 24 hr at 30°C, and β-galactosidase specific activity was measured as in Fig. 1. Values are normalized to that observed for JCY100 (11, 300 nmol per min per mg of protein).

Figure 3

Figure 3

Unphosphorylated Kss1 represses Ste12 action at a pheromone-inducible promoter. Strain YPH499 (MATa) and its otherwise isogenic derivatives, YDM600 (_kss1_Δ) and YDM200 (_fus3_Δ), were transformed with plasmid YEpU-FUS1Z, grown to midexponential phase, and incubated in the absence (−) and presence (+) of 1 μM α-factor mating pheromone (αF) for 90 min, and β-galactosidase specific activity was measured. Also, strain YDM600 was cotransformed with YEpT-FUS1Z and empty vector YCpU (−), YCpU-KSS1 (w.t.), YCpU-kss1(AEF), YCpU-kss1(Y24F), YCpU-kss1(Δloop), or YCpU-kss1(Y231C); streaked on plates selective for plasmid maintenance; and grown for 48 hr at 30°C. β-Galactosidase-specific activity was then measured. Values are normalized to that observed for YPH499 treated with pheromone (250 nmol per min per mg of protein).

Figure 4

Figure 4

Kss1 overproduction inhibits pheromone-induced transcription and Dig1 and Dig2 are required for this Kss1-imposed repression. (A) Strain YDM200 (MATa _fus3_Δ) was cotransformed with plasmid pJD11 containing a PRE-driven lacZ reporter and an empty vector, YEpU (bars 1 and 2), a multicopy plasmid, YEp-KSS1 ( bars 3 and 4), or a _GAL_-driven multicopy plasmid, YEp_GAL_-KSS1 (bars 5 and 6); grown to midexponential phase in medium containing 2% galactose and 0.2% sucrose; and incubated in the absence (−) and presence (+) of 6 μM α-factor mating pheromone (αF) for 2 hr. β-Galactosidase specific activity was then measured. Values are normalized to that observed for the pheromone-induced control cells (point 2; 4,500 nmol per min per mg of protein). (B) The strains described in A were labeled with either 35S (Upper) or 32P (Lower), incubated with or without 6 μM α-factor for 15 min, lysed, subjected to immunoprecipitation with polyclonal anti-Kss1 antiserum, resolved by SDS/PAGE, and analyzed by fluorography, all as described elsewhere (23). (C) Strain YPH499 (MATa DIG+) and its otherwise isogenic derivative, JCY5 (_dig1_Δ _dig2_Δ), were cotransformed with YEpU-FUS1Z and either an empty vector YCpLG (−) or YCpLG-KSS1 (GAL-KSS1) (+), grown to midexponential phase in medium containing 2% galactose and 0.2% sucrose, and incubated in the absence (−) and presence (+) of 1 μM α-factor for 90 min. β-Galactosidase specific activity was then measured. The values for vector-containing YPH499 not treated with pheromone (1.5 relative units for both points), which should lie below the abscissa, are shown as a bar just above the line to increase the clarity of presentation. Values are normalized to that observed for vector-containing YPH499 treated with pheromone (198 nmol per min per mg of protein). Standard deviations (data not shown) were less than 10% of the mean in all cases.

Figure 5

Figure 5

Model for differential control of FREs and PREs by MAPK- and Dig1/2-mediated regulation. (A) At both elements, unphosphorylated MAPK [principally Kss1 (–27)] binds directly to Ste12 and to Dig1 (and Dig2), thereby stabilizing Dig1/2–Ste12 complexes and potentiating Dig-mediated repression of Ste12. (B) At FREs, phosphorylation of Kss1 by Ste7, in response to upstream signals, weakens its association with Ste12, consequently promoting dissociation of Dig proteins from Ste12–Tec1 complexes, permitting substantial derepression (27). Ste7-dependent phosphorylation of Kss1 may also attenuate its binding to Ste12 at pheromone-inducible promoters, but this event is not sufficient for effective derepression, presumably because Dig proteins are bound more stably to Ste12–Ste12 homooligomers. (C) At FREs, phosphorylated (activated) Kss1 reinforces the transition, presumably by phosphorylating Dig1/2 (12, 13) and/or Ste12 and/or Tec1 (25, 27). Phosphorylation of these targets may prevent their reassociation, stimulate the transactivator activity of Ste12 and/or Tec1, or both. However, the level of Kss1 activation that results from the signals promoting invasive growth are insufficient to achieve derepression at PRE-bound complexes. In contrast, when more fully activated by pheromone, the MAPKs [principally Fus3 (25, 48)] derepress pheromone-inducible promoters, again presumably by phosphorylating Dig1/2 and Ste12 (11, 12, 13, 24). FRE-bound complexes are apparently insensitive to Fus3 action, perhaps because Fus3 cannot gain access to these complexes or phosphorylate them appropriately.

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