Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae.

Abstract

Haploid cells of budding yeast Saccharomyces cerevisiae respond to mating pheromones by inducing genes required for conjugation, arresting cell cycle progression, and undergoing morphological changes. The same cells respond to nutrient deprivation by altering budding pattern and inducing genes required for invasive growth. Both developmental alternatives to vegetative proliferation require the MAP kinase Kssl and the transcriptional transactivator Stel2. Using a two-hybrid screen for gene products that interact with Kssl, two homologous and previously uncharacterized loci (DIG1 and DIG2, for down-regulator of invasive growth) were identified. DIG2 is pheromone-inducible, whereas DIG1 is constitutively expressed. Digl~ colocalizes with Kssl in the nucleus, coimmunoprecipitates with Kssl from cell extracts in a pheromone-independent manner, and is phosphorylated by Kssl in immune complexes in a pheromone-stimulated manner. Kssl binds specifically to a GST-Digl fusion in the absence of any other yeast protein. Using the two-hybrid method, both Digl and Dig2 also interact with the other MAP kinase of the pheromone response pathway, Fus3.However, neither digl or dig2 single mutants, nor a digl dig2 double mutant, have a discernible effect on mating. In contrast, digl dig2 cells constitutively invade agar medium, whereas a digl dig2 stel2 triple mutant does not, indicating that Digl and Dig2 share a role in negatively regulating the invasive growth pathway. High-level expression of Digl suppresses invasive growth and also causes cells to appear more resistant to pheromone-imposed cell cycle arrest. Stel2 also binds specifically to GST-Digl in the absence of any other yeast protein. Collectively, these findings indicate that Digl, and most likely Dig2, are physiological substrates of Kssl and suggest that they regulate Stel2 function by direct protein-protein interaction.

Comparative genomics of MAP kinase and calcium–calcineurin signalling components in plant and human pathogenic fungi

“…In S. cerevisiae, two nuclear protein substrates of Kss1, Dig1 and Dig2, negatively regulate the invasive growth pathway by repressing Ste12 action (Cook et al, 1996). We failed to detect Dig1 and Dig2 orthologues in any of the species analyzed except for the close relative A. gossypii, suggesting that a regulatory mechanism other than that mediated by Dig1 and Dig2 must be operating in filamentous fungi.…”

Transcriptional regulation of protein complexes within and across species

“…For instance, the predicted targets of the cell cycle regulator Fkh2 are enriched for the cell cycle and DNA processing functions (P ϭ 7.93 ϫ 10 Ϫ6 ). Another example is Dig1 whose set of predicted targets is enriched for genes involved in cell differentiation (P ϭ 6.47 ϫ 10 Ϫ5 ), in agreement with its functional role (30,31). Overall, for all 10 TFs having more than five predicted targets, their target sets were significantly enriched for functional categories consistent with the function(s) of the regulating TF (SI Table 6).…”

A walk-through of the yeast mating pheromone response pathway

“…The Dig1 and Dig2 proteins bind to and repress Ste12 [29,135]. In strains lacking Dig1 and Dig2, pheromone-induced genes are constitutively upregulated [10,122,135].…”

“…Fus3 and/or Kss1 must be catalytically active in order for pheromone-induced changes in gene expression to occur [53]. Furthermore, Ste12 [17,65], as well as Dig1 and Dig2 [29,135], are substrates of Fus3 and Kss1. Finally, Dig1 and Dig2 appear to bind Ste12 less tightly following pheromone stimulation [29,135].…”

“…Furthermore, Ste12 [17,65], as well as Dig1 and Dig2 [29,135], are substrates of Fus3 and Kss1. Finally, Dig1 and Dig2 appear to bind Ste12 less tightly following pheromone stimulation [29,135]. These data collectively suggest that MAPK-dependent phosphorylation of Ste12 and/or Dig1/2 alters the ability of Dig1/2 to bind to and repress Ste12.…”