Pheromone induction promotes Ste11 degradation through a MAPK feedback and ubiquitin-dependent mechanism - PubMed (original) (raw)

Pheromone induction promotes Ste11 degradation through a MAPK feedback and ubiquitin-dependent mechanism

R K Esch et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Ste11 is the mitogen-activated protein kinase (MAPK) kinase kinase in the MAPK cascades that mediate mating, high osmolarity glycerol, and filamentous growth responses in Saccharomyces cerevisiae. We show stimulation of the mating pathway by pheromone promotes an accelerated turnover of Ste11 through a MAPK feedback and ubiquitin-dependent mechanism. This degradation is pathway specific, because Ste11 is stable during activation of the high osmolarity glycerol pathway. Because the steady-state amount of Ste11 does not change significantly during pheromone induction, we infer that maintenance of MAPK activation involves repeated cycles in which naive Ste11 is activated and then targeted for degradation. This model predicts that elimination of active Ste11 would rapidly curtail MAPK activation upon attenuation of the upstream signal. This prediction is confirmed by the finding that blocking ubiquitin-dependent Ste11 degradation during pheromone induction abolishes the characteristic attenuation profile for MAPK activation.

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Figures

Figure 1

Figure 1

Pheromone induction accelerates degradation of Ste11. Representative immune blots showing the time-dependent amount of Ste11M (A_–_C Upper) from extracts of a wild-type strain (C699–15 pNC734) without induction (A) and with pheromone induction (B and_C_). Extracts from cultures switched to dextrose show the persistence of pre-existing Ste11M (A and_B_) whereas those from cultures maintained in galactose show the steady-state amount of Ste11M (C). The steady-state amount of tubulin (Tub1) in each sample serves as an internal reference (A_–_C Lower). (D) Plots of the relative amount of Ste11M (Ste11M/Tub1) vs. time from no induction dextrose (□), pheromone induction dextrose (■), and pheromone induction galactose (⧫) cultures. The amount of Ste11M at each time is the average density of the Ste11M signal divided by the average density of the Tub1 signal. Relative amounts at different times are normalized to the amount of Ste11M at_t_ = 0 for the corresponding time course. Values shown on the plots are the average from three or more independent experiments. Bars show the average deviation for each point.

Figure 2

Figure 2

Pheromone-induced degradation of Ste11 occurs through a MAPK feedback and ubiquitin-dependent mechanism. Representative immune blots showing the time-dependent decrease in pre-existing Ste11M (A_–_C Upper) from extracts of a wild-type strain (C699–15 pNC245) without pheromone induction (C), the same strain overexpressing the dominant negative doa4-C 571 S_allele (C699–15 pNC245 YEp_doa4C571S) during pheromone induction (A) and an isogenic_fus3_Δ_kss1Δ_ strain (C699–75 pNC245) during pheromone induction (B). The steady-state amount of tubulin (Tub1) in each sample serves as an internal reference (A_–_C Lower). (D) Plots of the relative amount of Ste11M (Ste11M/Tub1) vs. time from wild type no pheromone (□),doa4-C 571 S with pheromone (▴), and_fus3_Δ_kss1Δ_ with pheromone (●) strains. Determination of the relative amount of Ste11 at each time point is as described in Fig. 1. Values are the average from three independent experiments. Bars show the average deviation for each point.

Figure 3

Figure 3

Osmotic induction does not accelerate Ste11 degradation. Representative immune blots showing the time-dependent decrease in pre-existing Ste11M (A and B Upper) from extracts of a ssk2_Δ ssk22Δ strain (FP50 pNC734) without (A) or with osmotic induction (B). The steady-state amount of Tub1 in each sample serves as an internal reference (A and_B). (C) Plots of the relative amount of Ste11M (Ste11M/Tub1) vs. time from the no induction (open symbols) and 0.4 M NaCl induction (closed symbols) time courses. Determination of the relative amount of Ste11 at each time point is as described in Fig. 1. Values are the average from three independent experiments. Bars show the average deviation for each point.

Figure 4

Figure 4

Ubiquitin-dependent degradation is required for down-regulation of MAPK activity during the adaptive response to pheromone. Representative PhosphorImage of 32P incorporation into MBP (A and B Upper) from Fus3M immune complex kinase assays and immune blots of Fus3M (A and B Lower) by using extracts from (A) a_DOA4_ strain (E929–6C pGA1903) and (B) a_doa4C_ 571 S (E929–6C pGA1903 YEp_doa4C571S_) strain during a pheromone induction time course. (C) Plots of pheromone-induced Fus3 activation in DOA4 (●) and_doa4C_ 571 S(▴) strains. Activities are determined from PhosphorImage quantification of 32P incorporation into MBP. Fus3 activation is in arbitrary units relative to the average value obtained for Fus3 immune complex assays using extracts isolated from the respective DOA4 and_doa4C_ 571 S strain extracts at t = 0 min. Plot shows the average from four assays using extracts from three independent time courses. Bars show the average deviation for each point.

Figure 5

Figure 5

Model for the cycle of Ste11 synthesis, activation, and degradation during pheromone-induced stimulation of the mating pathway MAPK activation cascade. See text for explanation.

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