A number of previous works suggested that the MAPKs Fus3 and Kss1 might mediate rapid negative feedback. Our previous study of regulated cell-to-cell variation in system output revealed a Fus3-dependent reduction in variation, suggesting an autoregulatory negative feedback mediated by Fus3 5
. Gartner et al. showed that levels of phosphorylated Fus3 were higher in cells bearing a kinase-dead mutant version of Fus324
. Bhattacharya et al. showed that Ste5 T287A mutant cells, in which the Ste5 carries a lesion in a site of threonine phoshorylated by Fus3 on peptides in vitro
exhibited increased reporter expression 28
, albeit with no change in the EC50 of the dose-response. Finally, phosphoproteomic studies of pheromone response system proteins29
have uncovered numerous sites of phosphorylation on pheromone response system proteins, whose levels change upon pheromone stimulation, many of which lie in consensus MAP kinase target sequences (R. Maxwell and O. Resnekov, personal communication). We therefore hypothesized that the signal decline at different measurement points depends on non-translational, fast-acting negative feedbacks mediated by Fus3 or Kss1.
To test if Fus3 or Kss1 were sources of negative feedback on system activity, we compared the baseline system response, at system points up to and including Fus3 phosphorylation, with system response after selective inhibition of either Fus3 or Kss1 kinase activity. To do this, we first modified reporter strains by replacing either FUS3
with the corresponding purine analog-sensitive allele 30
. We did this by changing the “gatekeeper” residue in each kinase’s ATP binding pocket (Q93 in Fus3, N94 in Kss1) to an alanine. The mutant fus3-as2
kinases were active, as measured by fluorescent protein reporter gene output (Fig. S9a
), and10 μM 1-NM-PP1, a cell-permeable adenosine analogue, inhibited the activity of mutant kinases without inhibiting wild-type kinases (Fig. S9b
). We then quantified Fus3 phosphorylation by quantitative immunoblotting after stimulation with pheromone, either with or without simultaneous inhibition with 1-NM-PP1 (). Fus3 phosphorylation levels did not peak and decline to a plateau when we inhibited Fus3-as2, but, rather, remained high, near peak levels. By contrast, when we inhibited Kss1-as2, Fus3 phosphorylation levels were unaffected (). These results indicated that Fus3 kinase activity mediated one or more negative feedbacks in this system.
We then studied where in the system the Fus3-dependent feedback acted to diminish signal amplitude. Yi et al. showed that the decrease in G-protein FRET within 30 seconds of stimulation depended on Sst2 6
. This finding suggested that the Fus3-dependent negative feedback might upregulate the GTPase-activating protein (GAP) function of Sst2, which would increase G protein reassociation and decrease downstream signal. We tested if Fus3-as2 inhibition affected the observed decline in both G-protein dissociation and Ste5 recruitment. Inhibition of Fus3 activity eliminated the decline in Ste5 translocation (), but surprisingly had no effect on the decline in G-protein dissociation in a G-protein FRET reporter strain carrying fus3-as2
( and Fig. S10
). These results indicated that Fus3-mediated negative feedback acted downstream of mechanisms regulating G-protein association.
To confirm that Fus3 acted downstream of G-protein activation, we measured Ste5 recruitment after deleting SST2. We expected deletion of SST2 to have no effect on Fus3-mediated signal decline, since Sst2 is required for efficient G-protein inactivation and, as we showed above, Fus3-mediated negative feedback does not reduce G-protein dissociation levels. Unexpectedly, when we deleted of SST2, we completely disrupted Fus3-mediated signal decline; unlike SST2+ cells, inhibition of Fus3 did not cause an increase in Ste5 recruitment (). Furthermore, the Ste5 recruitment (with or without Fus3-mediated feedback) peaked and declined, similar to the baseline response of SST2+ cells (compare squares and circles in with circles in ). This finding showed that signal peak-and-decline is the default behavior in the absence of Sst2. Since a sustained non-declining signal is only evident in SST2+ cells in the presence of Fus3-as2 inhibitor, these results also indicate that Sst2 promotes Ste5 membrane recruitment, a hitherto unknown function of the RGS protein family, and that Fus3 negatively regulates this novel signal-promoting function ().
DoRA requires Fus3-mediated negative feedback
We then investigated which portions of the Sst2 protein might be involved in promoting Ste5 membrane recruitment. During analysis of Sst2 point mutants, we found that Ste5 recruitment in a fus3-as2
strain that carried sst2-T134A
instead of wild-type Sst2 peaked-and-declined in the presence and absence of Fus3 inhibitor (), just as observed in Δsst2
cells (). The pheromone-induced growth-inhibition of sst2-T134A
cells reported by halo assays was close to wild-type (Fig. S11a
), and the average number of Sst2-T134A protein molecules per cell was similar to Sst2 abundance in the parent strain, (Fig. S11b
), suggesting that the T134A mutation disrupted a significant fraction of the Fus3-dependent, signal-promoting function of Sst2 without disrupting the bulk of its signal-reducing GAP activity. T134 lies within the N-terminal DEP domains of Sst2, which are required for localization of Sst2 to the membrane by binding the cytosolic tail of Ste2 31
. These results indicate that the DEP domains in Sst2 might aid Ste5 membrane recruitment, perhaps by providing additional membrane-proximal interaction surfaces, and suggest that mechanisms that regulate localization of Sst2 to the membrane, such as disruption of Sst2-Ste2 interactions by Yck1/2-mediated phosphorylation after longer periods of pheromone stimulation 31
, might consequently regulate Ste5 membrane recruitment.