Hsl7p, a negative regulator of Ste20p protein kinase in the Saccharomyces cerevisiae filamentous growth-signaling pathway

Abstract

In the budding yeast, Saccharomyces cerevisiae, protein kinases Ste20p (p21(Cdc42p/Rac)-activated kinase), Ste11p [mitogen-activated protein kinase (MAPK) kinase kinase], Ste7p (MAPK kinase), Fus3p, and Kss1p (MAPKs) are utilized for haploid mating, invasive growth, and diploid filamentous growth. Members of the highly conserved Ste20p/p65(PAK) protein kinase family regulate MAPK signal transduction pathways from yeast to man. We describe here a potent negative regulator of Ste20p in the yeast filamentous growth-signaling pathway. We identified a mutant, hsl7, that exhibits filamentous growth on rich medium. Hsl7p belongs to a highly conserved protein family in eukaryotes. Hsl7p associates with the noncatalytic region within the amino-terminal half of Ste20p as well as Cdc42p. Deletions of HSL7 in haploid and diploid strains led to cell elongation and enhancement of both haploid invasive growth and diploid pseudohyphal growth. However, deletions of STE20 in haploid and diploid greatly diminished these hsl7-associated phenotypes. In addition, overexpression of HSL7 inhibited pseudohyphal growth. Thus, Hsl7p may inhibit the activity of Ste20p in the S. cerevisiae filamentous growth-signaling pathway. Our genetic analyses suggest the possibility that Cdc42p and Hsl7p compete for binding to Ste20p for pseudohyphal development when starved for nitrogen.

Figure 1

(A) Restriction map of HSL7. Positions of HSL7 ORF (open arrow) and the transposon insertion sites (triangles) are indicated. Structures of the fragment used to disrupt HSL7 are noted. (B) Sequence alignment of S. cerevisiae Hsl7p with S. pombe Skb1p, C. elegans C34E10.5, Drosophila capsuleen, and human Skb1p proteins. Numbers indicate amino acid residue position. Identical and conserved residues are shown in white letters against black and shaded letters, respectively. GenBank accession nos.: Sc-Hsl7p, U65920; Sp-Skb1p, U59684; Ce-C34E10.5, U10402; Dm-capsuleen, AJ002740; Hs-Skb1p, AF015913.

Figure 2

Interaction of Hsl7p and Ste20p. Resin-binding assay was done to determine interactions between Hsl7p and Ste20p. GST fusion proteins with full-length Ste20p, Ste20p^1–495, Ste20p^258–495, and Ste20p^Δ258–842. GST were bound to glutathione-Sepharose beads and incubated with MBP-Hsl7p. After washing, proteins eluted with SDS-sample buffer were subjected to SDS/PAGE in 12.5% (upper left blot), 7.5% (upper right blot), and 5% (lower blots) gels and immunoblotted for the presence of GST, GST-Ste20p fusion proteins (upper blots), and MBP-Hsl7p (lower blots) with antibodies to GST (upper blots) and MBP (lower blots), respectively. Interactions between Ste20p fragments and Hsl7p are summarized at the bottom.

Figure 3

Disruption of HSL7 stimulates haploid invasive growth and diploid pseudohyphal development. (A) Elongated cell morphology of the haploid hsl7Δ and diploid hsl7Δ/hsl7Δ strains. Deletion of STE20 abolished their elongated cell morphology. Yeast strains were cultured at 30°C in yeast extract/peptone/dextrose (YPD) medium for 3 days. Cells were visualized under the microscope with Nomarski optics. (B) Disruption of HSL7-stimulated invasive growth in haploid cells. (Left) Genotypes of the strains examined. (Center) Total growth. (Right) Haploid invasive growth. Σ1278b-derived strains: YAF1108 (MATa HSL7⁺ STE20⁺ leu2 ura3) and its otherwise isogenic derivatives YAF1191 (MATa hsl7Δ∷LEU2), YAF1192 (MATa ste20Δ), and YAF1193 (MATa hsl7Δ∷LEU2 ste20Δ). Strains were streaked onto YPD plates and grown for 3 days at 30°C. The plate shown in Center was rinsed under a stream of running water to distinguish surface growth from invasive growth by the degree of agar penetration (8). (C) Disruption of HSL7 enhanced pseudohyphal growth in diploid cells. Diploid strain YAF1111 (MATa/MATα HSL7/HSL7 STE20/STE20 leu2/leu2 ura3/ura3) was constructed from YAF1108 and YAF1110 (MATα HSL7⁺ STE20⁺ leu2 ura3) in the Σ1278b background. Diploid strains YAF1194 (MATa/MATα hsl7Δ∷LEU2/hsl7Δ∷LEU2), YAF1195 (MATa/MATα ste20Δ/ste20Δ) and YAF1196 (MATa/MATα hsl7Δ∷LEU2/hsl7Δ∷LEU2 ste20Δ/ste20Δ) are YAF1111 derivatives. Every diploid strain contained vectors carrying URA3 and/or LEU2 to complement the ura3 and/or leu2 auxotrophies. These transformants were streaked on SLAD medium (15) and allowed to grow for 4 days. Resulting colonies were photographed. (D and E) Expression of the FRE-lacZ reporter gene. The eight strains described in B and C were transformed with a plasmid YEpU-FTyZ (6) (and a LEU2-containing vector to complement leu2), and β-galactosidase activity was measured. (D) Haploid strains on SD medium. (E) Diploid strains on nitrogen-starved medium. Activities were normalized to protein concentration and are shown as relative to the activities of wild-type strain. Values are the averages of measurements made in triplicate.

Figure 4

Pseudohyphal growth of the hsl7Δ/hsl7Δ diploid strain on nitrogen-rich medium. Wild-type diploid (YAF1111) and hsl7Δ/hsl7Δ (YAF1194) strains containing a URA3-containing vector and/or a LEU2-containing vector to complement ura3 and/or leu2 were streaked on SLAD medium containing 5 mM ammonium sulfate and were incubated for 2 weeks at 30°C.

Figure 5

(A) Overproduction of HSL7 inhibits diploid pseudohyphal growth. Wild-type diploid strain YAF1111 containing a LEU2-vector (pRS315) was transformed with plasmids described at the top of the images. Cells of those strains were streaked on SLAD medium and incubated for 4 days at 30°C. (Left) Wild-type diploid cells containing vector, YEUp3. (Right) Wild-type diploid cells containing the plasmid for HSL7 overproduction, YEUp-HSL7Lp. (B) Simultaneous expression of CDC42 or STE20 reversed the inhibitory effect to pseudohyphal development of HSL7-overexpressed cells. Strain YAF1111 was transformed with plasmids described at the top of the images. Cells of those strains were streaked on SLAD medium and incubated for 4 days at 30°C. YCLp-CDC42 is the LEU2 plasmid for slight overproduction of CDC42; YELp-STE20 is the LEU2 plasmid for overexpression of STE20.

Figure 6

Model for the role of Hsl7p during filamentous growth. Hsl7p binds to Ste20p to repress its activity. In response to nitrogen starvation, Ras2p activates Cdc42p and activated Cdc42p interferes with binding of Hsl7p to Ste20p. Two 14–3-3 proteins, Bmh1p and Bmh2p, which are essential for the pathway leading to pseudohyphal growth, also interact with the C terminus of Ste20p (37).