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. 1999 Oct;10(10):3301–3316. doi: 10.1091/mbc.10.10.3301

Figure 4.

Figure 4

The RAS/STE pathway and Cdc28 tyrosine phosphorylation independently modulate filamentous growth. (A) Filament formation of Cdc28 tyrosine phosphorylation mutants with altered RAS/STE function on synthetic nitrogen (SC) or nitrogen starvation (SLAD) agar. Mutants with decreased tyrosine phosphorylation lacking the inhibitory Swe1 kinase or its phosphorylation site on Cdc28 (swe1/swe1 and CDC28-T18A, Y19F [AF]) show impaired filamentous development on SC and SLAD media compared with the wild-type strain. On the other hand, lack of the reactivating phosphatase Mih1 (mih1/mih1) confers constitutively increased Cdc28 tyrosine phosphorylation and a moderately enhanced filamentous phenotype. Plasmids expressing the STE11-4 or RAS2-Val19 allele suppress the impaired filamentation of both the swe1/swe1 and CDC28-T18A, Y19F strains and further enhance the filamentous phenotype of mih1/mih1 strains. (B) STE11-4/STE11-4 CDC28-AF/CDC28-AF double mutants on YPD or SLAD agar. The nonphosphorylatable CDK mutant CDC28-T18A, Y19F does not block an ectopic RAS/STE signal. (C) Agar invasion assay. Cells were treated as described in Figure 1B. Like the STE11-4 mutant, the double mutant is hyperinvasive in YPD agar. (D) Cell morphology in liquid YPD media. Highly elongated cells that grow as adherent cell aggregates reminiscent of the STE11-4 florets are apparent in STE11-4/STE11-4 CDC28-AF/CDC28-AF double mutant cultures. Bars, 50 μm. (E) Flow cytometry. Cells were prepared as described in MATERIALS AND METHODS. The STE11--4/STE11-4 CDC28-AF/CDC28-AF double mutant reveals a marked shift to 4N DNA content relative to the CDC28-AF/CDC28-AF control and comparable to that of the STE11-4/STE11-4 mutant alone.