5-FOA Sensitive
0 15 120 0 15 120 Ste7-Wt Ste7-L 194 S
min 100 80 60 40 20 0 120 Mpk1 Kss1 Fus3 Mpk1 Kss1 Fus3 Ste7-Wt Ste7-L194S min 0 15 120 0 15 120 0 15 120 0 15 120 Ste7-Wt Ste7-L194S min 2.5 2.0 1.5 1.0 0.5 0
Figure 3.6: Relative stability of Tec1 in yeast expressing wild-type Ste7 versus Ste7- L194S at basal levels and after exposure to mating pheromone. (A) Representative blots showing the relative amount of Tec1 in yeast strains expressing wild-type Ste7 and Ste7- L194S without mating pheromone (uninduced) and with mating pheromone (induced with 50nM α-factor). Tec1 was tagged with a 13x-myc tag and detected with polyclonal anti- myc antibodies. Tubulin was used as a loading control and detected with anti-tubulin antibodies. (B) Graph showing Tec1 degradation upon exposure to mating pheromone in yeast cells expressing only wild-type Ste7. Tec1 levels in uninduced wild-type Ste7 are represented by an open triangle (U), uninduced Ste7-L194S are represented by an open
circle (○), induced wild-type Ste7are represented by a closed circle (●), and induced Ste7-L194S are represented by a closed triangle (▲). (C) Duplicate blots of Tec1 in uninduced yeast expressing wild-type Ste7 and Ste7-L194S. The relative amounts of Tec1 to Tubulin are shown below each lane.
0 30 60 90 120 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Time (min) Ste7 0 30 60 120 Ste7-L194S 0 30 60 120 Tub1 Tec1 Tub1 Tec1 min 1 2 3 4 5 6 7 8 A. B. Tub1 Tec1 Tec1 Tub1 2.0 1.5 1.5 1.7 C.
Figure 6.7: Models of Mpk1 activation. See text for explanation. (A) Wild-type Ste7. (B) Direct mechanism of phosphorylation of Mpk1 by Ste7-L194S. (C) Indirect
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CHAPTER 4
GENERAL DISCUSSION
There are five MAPK signaling modules in yeast. These MAPK modules are involved in signaling processes for mating, filamentous growth, high osmolarity adaptation, cell wall synthesis, and sporulation. It is becoming clear that signaling via MAPK modules in yeast is not a linear event but rather due to a network of proteins. It was originally proposed by Madhani et al. that there were distinct functions for the MAPKs Kss1 and Fus3 in mating and filamentous growth (11). Here they propose that Fus3 is the MAPK used in the mating pathway while Kss1 is the MAPK used for filamentous growth (11). This interpretation of their data was ultimately erroneous. Recent data from our lab and others has demonstrated that a linear signaling pathway is not a likely scenario. It has been demonstrated by various labs that Kss1 and Fus3 are dually phosphorylated by Ste7 to an equal extent in response to mating pheromone (1, 3, 12, 15). Additionally the transcriptional output in response to mating pheromone is identical when activated by Kss1 or Fus3 (14). One notable difference between the mechanism by which Ste7 phosphorylates Kss1 and Fus3 is that Fus3 phosphorylation requires the scaffold protein Ste5 while phosphorylation of Kss1 does not (1, 3, 9, 12).
Recent evidence has also demonstrated that MAPKs can play both positive and negative roles in various signaling pathways. One such example is the MAPK Kss1.
promotes filamentous growth (7, 11). In a similar fashion we demonstrated that unphosphorylated Fus3 was inhibitory for signaling in the mating pathway even when Kss1 was constitutively phosphorylated (12). This inhibition was removed only when Fus3 was deleted along with the scaffold protein Ste5 (12). These results suggest a synergistic effect both proteins have on inhibiting the transcriptional output of the mating pathway when Fus3 remains unphosphorylated.
Further evidence that MAPKs in yeast work in concert with each other is the inhibitory functions observed across pathways. One such example is the MAPK Fus3 inhibiting filamentous growth when activated by mating pheromone. It was first observed that deleting Fus3 had a stimulatory effect on invasive growth (6, 11). This effect was due to the fact that activated Fus3 leads to a phosphorylation and ubiquitin dependent degradation of the filamentous growth transcription factor Tec1 (2, 5). It was also demonstrated that deleting the MAPK Hog1 also had a stimulatory effect on
filamentous growth (10, 13). In addition to stimulating filamentous growth, deleting Hog1 also allowed for cross-talk between the high osmolarity signaling pathway and the mating pathway. The results of this cross-talk were transcriptional output and
morphogenic changes typically observed in haploid yeast in response to mating pheromone now occurred in response to high osmolarity (13).
The MAPK involved in cell wall synthesis, Mpk1, is activated in response to stimuli that alter the shape of the plasma membrane (reviewed in (8)). The polarized growth morphogenesis observed when haploid yeast encounter mating pheromone serves as a stimulus to activate Mpk1 (4, 8). This activation of Mpk1 in response to mating pheromone is dependent on the transcriptional output and translation of genes regulated
by Ste12 (4). Work done by Yashar et al. demonstrated that the MAPKK Ste7, which normally functions in the mating and filamentous growth MAPK signaling pathways, could compensate for deletions of the redundant MAPKKs Mkk1 and Mkk2, which activate Mpk1, when overproduced or in strains deleted for Ste5 and rescue yeast from lysis when heat shocked (16). These results suggest that the scaffold protein Ste5 promotes signaling specificity by tethering Ste7 and sequestering it from activating MAPKs other than Kss1 and Fus3 (16). We found further evidence to support this Ste7 cross-talk into the cell wall synthesis pathway with a substitution mutant derivative (Ste7-
L194S) that has reduced binding to Ste5. When this mutant derivative of Ste7 is expressed
in yeast cells we observe a 2-fold higher phosphorylation of Mpk1 in vegetatively growing cells when compared to cells expressing wild-type Ste7 (Chapter 3 of this thesis). In conjunction with this increased phosphorylation of Mpk1 yeast expressing
Ste7-L194S have higher levels of transcription of the filamentous growth reporter gene
Ty1-lacZ when compared to cells expressing wild-type Ste7.
The work done by others and in this thesis supports the hypothesis that signaling in yeast via MAPK modules is not a linear event but rather involves a network of MAPKs. One can speculate on how such a network could function in a differentiation