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. 2010 Sep;30(17):4293-307.
doi: 10.1128/MCB.00361-10. Epub 2010 Jun 28.

Dynamic localization of Fus3 mitogen-activated protein kinase is necessary to evoke appropriate responses and avoid cytotoxic effects

Raymond E Chen  1 , Jesse C Patterson, Louise S Goupil, Jeremy Thorner
Affiliations

Dynamic localization of Fus3 mitogen-activated protein kinase is necessary to evoke appropriate responses and avoid cytotoxic effects

Raymond E Chen et al. Mol Cell Biol. 2010 Sep.

Abstract

Cellular responses to many external stimuli are mediated by mitogen-activated protein kinases (MAPKs). We investigated whether dynamic intracellular movement contributes to the spatial and temporal characteristics of the responses elicited by a prototypic MAPK, Fus3, in the mating pheromone response pathway in budding yeast (Saccharomyces cerevisiae). Confining Fus3 in the nucleus, via fusion to a histone H2B, reduced MAPK activation and diminished all responses (pheromone-induced gene expression, cell cycle arrest, projection formation, and mating). Elimination of MAPK phosphatases restored more robust outputs for all responses, indicating that nuclear sequestration impedes full MAPK activation but does not abrogate its functional competence. Restricting Fus3 to the plasma membrane, via fusion to a lipid-modified CCaaX motif, led to MAPK hyperactivation yet severely impaired all response outputs. Fus3-CCaaX also caused aberrant cell morphology and a proliferation defect. Unlike similar phenotypes induced by pathway hyperactivation via upstream components, these deleterious effects were independent of the downstream transcription factor Ste12. Thus, appropriate cellular responses require free subcellular MAPK transit to disseminate MAPK activity optimally because preventing dynamic MAPK movement either markedly impaired signal-dependent activation and/or resulted in improper biological outputs.

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Figures

FIG. 1.
FIG. 1.
Subcellular localization of plasma membrane- and nucleus-restricted alleles of Fus3. Exponentially growing cultures of strain RCY9320 (fus3 kss1) carrying the plasmids indicated below were examined by bright-field and/or fluorescence microscopy. (A) PFUS3-FUS3-GFP (pRC202) (left), PFUS3-FUS3-GFP-CCaaX (pRC205) (middle), or PFUS3-FUS3-GFP-SSaaX (pRC206) (right). (B) PTPI1-FUS3-GFP (pRC225) (top left), PTPI1-FUS3-GFP-CCaaX (pRC226) (bottom), or PTPI1-FUS3-GFP-SSaaX (pRC227) (top right). (C) PFUS3-FUS3-GFP (pRC202) (top left), PFUS3-FUS3-GFP-HTB2 (pRC251) (top middle), PTPI1-FUS3-GFP (pRC225) (bottom left), PTPI1-FUS3-GFP-HTB2 (pRC252) (bottom right), or empty vector (pRS315) (top right).
FIG. 2.
FIG. 2.
Anisotropic membrane distribution of Fus3-GFP-CCaaX is independent of core pheromone response pathway components. Strains of the indicated genotypes (BY4741, RCY9320, RCY9335, RCY9336, RCY9337, RCY9338, RCY9339, RCY9340, RCY9341, RCY9342, RCY9343, and RCY9344) were transformed with a plasmid encoding Fus3-GFP-CCaaX (pRC226). Exponentially growing cultures were examined by fluorescence microscopy.
FIG. 3.
FIG. 3.
Membrane distribution of Fus3-GFP-CCaaX is conferred by the Fus3 sequence. Wild-type (WT) (BY4741) and fus3 kss1 (RCY9320) cells were transformed with galactose-inducible expression vectors encoding PGAL1-FUS3-GFP-CCaaX (pRC249), PGAL1-GFP-CCaaX (pRC285), PGAL1-FUS3-GFP-SSaaX (pRC250), or PGAL1-GFP-SSaaX (pRC286). Exponentially growing 26°C cultures were examined by fluorescence microscopy 2.5 h after addition of galactose.
FIG. 4.
FIG. 4.
Restriction of Fus3 localization affects its phosphorylation of substrates. (A) rvs167 (RCY9339) and fus3 kss1 cells (RCY9320) were transformed with an empty vector (V; pRS315) or plasmids encoding Fus3-GFP (WT; pRC225), Fus3-GFP-Htb2 (H; pRC252), or Fus3-GFP-CCaaX (C; pRC226). Exponentially growing cultures were incubated in the absence (−) or presence (+) of 3 μM α-factor (pheromone) for 1.5 h, after which whole-cell extracts were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting (IB) with anti-Rvs167 antibody and with anti-Cdc10 antibody to confirm equivalent sample loading. Arrowheads indicate phosphorylated forms of Rvs167. (B) fus3 kss1 cells (RCY9352) were transformed with plasmids encoding Dig1-(HA)3 (YCpDIG1-3HA), Fus3-GFP (WT; pRC225), Fus3-GFP-Htb2 (H; pRC252), Fus3-GFP-CCaaX (C; pRC226), Fus3(D137A)-GFP (kd; pRC288), and/or empty vectors (V; pRS315 or pRS316) as indicated. Exponentially growing cultures were incubated in the absence (−) or presence (+) of 3 μM α-factor (pheromone) for 1.5 h, after which whole-cell extracts were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting with anti-HA antibody (two SDS-PAGE-immunoblots of the same samples are shown). Lower arrowhead, Dig1; upper arrowhead, phosphorylated Dig1. The anti-Cdc10 immunoblot serves as a loading control.
FIG. 5.
FIG. 5.
Basal and pheromone-induced Fus3 activation depend on its subcellular localization. Strains RCY9320 (fus3 kss1 STE5+) and RCY9346 (fus3 kss1 ste5) were transformed with an empty vector (pRS315) or plasmids encoding PFUS3-FUS3-GFP-CCaaX (pRC205), PFUS3-FUS3-GFP-SSaaX (pRC206), PTPI1-FUS3-GFP-CCaaX (pRC226), PTPI1-FUS3-GFP-SSaaX (pRC227), PFUS3-FUS3-GFP (pRC202), PFUS3-FUS3-GFP-HTB2 (pRC251), PTPI1-FUS3-GFP (pRC225), or PTPI1-FUS3-GFP-HTB2 (pRC252). Exponentially growing cultures were incubated in the absence (−) or presence (+) of 2 μM α-factor (pheromone) for 15 min, after which whole-cell extracts were prepared and resolved by SDS-PAGE. Total and activated levels of Fus3 were assessed by immunoblotting with anti-GFP and anti-phospho-MAPK antibodies, respectively. Arrowheads indicate corresponding bands in the anti-GFP and anti-phospho-MAPK immunoblots. The asterisk denotes a band present in all lanes, which likely corresponds to the phosphorylated form of the MAPK Slt2/Mpk1.
FIG. 6.
FIG. 6.
Restriction of Fus3 localization impairs mating. (A) fus3 kss1 (RCY9320) and fus3 kss1 msg5 ptp2 ptp3 (RCY9401) cells were transformed with an empty vector (V; pRS315) or plasmids encoding Fus3-GFP (WT; pRC225), Fus3-GFP-Htb2 (H; pRC252), or Fus3-GFP-CCaaX (C; pRC226). Mating proficiency was determined relative to fus3 kss1 cells carrying Fus3-GFP. Averages are shown; bars depict standard errors of the means (n = 2). (B) fus3 kss1 (RCY9352) cells were transformed with two plasmids as indicated: V, vector (pRS315 or pRS316); WT, Fus3-GFP (pRC225); H, Fus3-GFP-Htb2 (pRC283); and C, Fus3-GFP-CCaaX (pRC226). Mating proficiency was determined relative to cells carrying Fus3-GFP. Averages are shown; bars depict standard errors of the means (n ≥ 4). (C) fus3 kss1 (RCY9320) and fus3 kss1 msg5 ptp2 ptp3 (RCY9401) cells were transformed with an empty vector (V; pRS315) or plasmids encoding Fus3-GFP (WT; pRC225), Fus3-GFP-Htb2 (H; pRC252), or Fus3-GFP-CCaaX (C; pRC226). Exponentially growing cultures were incubated in the absence (−) or presence (+) of 2 μM α-factor (pheromone) for 15 min, after which whole-cell extracts were prepared and resolved by SDS-PAGE. Total and activated levels of Fus3 were assessed by immunoblotting with anti-GFP and anti-phospho-MAPK antibodies, respectively. Arrowheads indicate corresponding bands in the anti-GFP and anti-phospho-MAPK immunoblots.
FIG. 7.
FIG. 7.
Pheromone-inducible gene expression and growth arrest depend on the localization of Fus3. (A) Strains RCY9380 (FUS3+ KSS1+ FUS1-GFP), RCY9382 (fus3 kss1 FUS1-GFP), RCY9384 (FUS1+ KSS1+ PRM1-GFP), and RCY9386 (fus3 kss1 PRM1-GFP) were transformed with plasmids encoding Fus3 (WT; pRC224), Fus3-Htb2 (H; pRC287), Fus3-CCaaX (C; pRC273), Fus3-SSaaX (s; pRC274), or an empty vector (V; pRS315). Exponentially growing cultures were incubated in the absence (−) or presence (+) of 2 μM α-factor (pheromone) for 1.5 h, after which whole-cell extracts were prepared and resolved by SDS-PAGE. Levels of Fus1-GFP, Prm1-GFP, and Cdc10 (loading control) were assessed by immunoblotting with anti-GFP and anti-Cdc10 antibodies. (B) The strains described above for panel A were assessed for pheromone-induced growth arrest by an agar diffusion (halo) assay. (C) fus3 kss1 (RCY9320) and fus3 kss1 sst2 (RCY9358) cells carrying plasmids encoding PFUS3-FUS3-GFP (pRC202), PFUS3-FUS3-GFP-HTB2 (pRC251), PFUS3-FUS3-GFP-CCaaX (pRC205), PFUS3-FUS3-GFP-SSaaX (pRC206), PTPI1-FUS3-GFP (pRC225), PTPI1-FUS3-GFP-HTB2 (pRC252), PTPI1-FUS3-GFP-CCaaX (pRC226), PTPI1-FUS3-GFP-SSaaX (pRC227), or an empty vector (pRS315) were assessed for pheromone-induced growth arrest by an agar diffusion (halo) assay.
FIG. 8.
FIG. 8.
Pheromone-inducible polarized growth depends on the localization of Fus3. (A) Strain RCY9320 (fus3 kss1) was transformed with plasmids encoding Fus3-GFP (WT; pRC202, pRC225), Fus3-GFP-Htb2 (H; pRC251, pRC252), Fus3-GFP-SSaaX (s; pRC206, pRC227), Fus3-GFP-CCaaX (C; pRC205, pRC226), or empty vector (V; pRS315). Exponentially growing cultures were incubated with 2 μM α-factor for 1.5 to 2.5 h and examined by microscopy. (B) Strains RCY9320 (fus3 kss1) and RCY9401 (fus3 kss1 msg5 ptp2 ptp3) were transformed with plasmids encoding Fus3-GFP (WT; pRC225), Fus3-GFP-Htb2 (H; pRC252), or an empty vector (V; pRS315). Exponentially growing cultures were treated with or without 2 μM α-factor for 2 h and examined by microscopy.
FIG. 9.
FIG. 9.
Restriction of Fus3 to the plasma membrane causes aberrant cell morphology. (A) Exponentially growing cultures of fus3 kss1 (RCY9320) cells carrying plasmids encoding Fus3-GFP-SSaaX (pRC227), Fus3-GFP-CCaaX (pRC226), Fus3(D137A)-GFP-SSaaX (pRC284), or Fus3(D137A)-GFP-CCaaX (pRC254) were examined by microscopy. (B) Wild-type (BY4741) and isogenic ste5 (RCY9337), far1 (RCY9341), and bni1 (RCY9344) mutant cells were transformed with plasmids encoding PGAL1-FUS3-GFP-CCaaX (pRC249) or PGAL1-FUS3-GFP-SSaaX (pRC250), cultivated at 26°C in media containing 2% raffinose, 0.2% sucrose, and 2% galactose, and examined by microscopy. (C) Wild-type (BY4741) and isogenic fus3 kss1 (RCY9320) cells were transformed with plasmids encoding PGAL1-FUS3-GFP-CCaaX (pRC249), PGAL1-FUS3-GFP-SSaaX (pRC250), or an empty vector (pRS315), cultivated at 26°C in media containing 2% raffinose, 0.2% sucrose, and 2% galactose, and examined by microscopy.
FIG. 10.
FIG. 10.
The aberrant morphology caused by Fus3-GFP-CCaaX is rescued at elevated temperature. (A) An experiment otherwise identical to that described in the legend to Fig. 9C was performed using cultures cultivated in galactose-containing media at 37°C. (B) Wild-type (BY4741) and fus3 kss1 (RCY9320) cells were transformed with an empty vector (pRS315) or plasmids encoding PGAL1-FUS3-GFP-CCaaX (pRC249) or PGAL1-FUS3-GFP-SSaaX (pRC250) and cultivated at 37°C in media containing dextrose or galactose. Whole-cell extracts were prepared from exponentially growing cultures, and levels of Fus3 and Cdc10 (loading control) were assessed by immunoblotting with anti-GFP and anti-Cdc10 antibodies, respectively.
FIG. 11.
FIG. 11.
Pheromone pathway activation is reduced at elevated temperature. (A) Wild-type (BY4741) cells were transformed with plasmids encoding Fus3-GFP-CCaaX (pRC226), Fus3-GFP-SSaaX (pRC227), or an empty vector (pRS315) and cultivated at the indicated temperatures. Whole-cell extracts were prepared from exponentially growing cultures, and levels of total Fus3-GFP-CCaaX/-SSaaX, activated Fus3-GFP-CCaaX/-SSaaX, and Cdc10 (loading control) were assessed by immunoblotting with anti-GFP, anti-phospho-MAPK, and anti-Cdc10 antibodies, respectively. (B) PFUS1-HA-eGFP (YJP73) cells were grown at 30°C or 37°C and incubated with the indicated concentration of α-factor (pheromone) for 1 h. Cells were examined by microscopy, and GFP expression was quantified. The averages of three independent trials are shown; bars depict standard errors of the means across the three trials. In total, between 72 and 185 cells were examined for each condition. (C) PFUS1-HA-eGFP (YJP73) and hog1 PFUS1-HA-eGFP (YJP131) cells were grown at 30°C or 37°C and incubated in the absence (−) or presence (+) of 1 M sorbitol for 2 h. Cells were examined by microscopy, and GFP expression was quantified. The averages and standard errors of the means are shown (n = 28 to 82 cells).
FIG. 12.
FIG. 12.
The cell proliferation defect caused by Fus3-GFP-CCaaX is dependent on expression strength and temperature. (A) fus3 kss1 (RCY9320) cells were transformed with an empty vector (pRS315) or plasmids encoding PFUS3-FUS3-GFP-CCaaX (C; pRC205), PFUS3-FUS3-GFP-SSaaX (s; pRC206), PTPI1-FUS3-GFP-CCaaX (C; pRC226), or PTPI1-FUS3-GFP-SSaaX (s; pRC227). Cultures were spotted in 10-fold dilution series on plates and assessed for growth at the indicated temperatures. (B) Wild-type (BY4741) and fus3 kss1 (RCY9320) cells were transformed with an empty vector (pRS315) or plasmids encoding PGAL1-FUS3-GFP-CCaaX (C; pRC249) or PGAL1-FUS3-GFP-SSaaX (s; pRC250). Cultures were spotted in 10-fold dilution series on plates containing dextrose (Dex) or galactose (Gal) as the carbon source and assessed for growth at the indicated temperatures.
FIG. 13.
FIG. 13.
Restriction of Fus3 to the plasma membrane impairs cell proliferation. (A) Strains BY4741, RCY9335, RCY9320, RCY9340, RCY9342, RCY9343, RCY9336, RCY9337, RCY9341, RCY9344, RCY9339, RCY9368, RCY9337, and RCY9366, with corresponding genotypes indicated, were transformed with plasmids encoding PGAL1-FUS3-GFP-CCaaX (C; pRC249) or PGAL1-FUS3-GFP-SSaaX (s; pRC250). Cultures were spotted in 10-fold dilution series on plates containing dextrose or galactose as the carbon source and assessed for growth at 26°C. (B to D) Strains BY4741, RCY9320, RCY9337, RCY9343, RCY9336, and RCY9341, with genotypes indicated, were transformed with plasmids encoding PGAL1-GFP-CCaaX (C, G; pRC285), PGAL1-FUS3-GFP-CCaaX (C, FG; pRC249), PGAL1-fus3(D137A)-GFP-CCaaX (C, f*G; pRC259), PGAL1-GFP-SSaaX (s, G; pRC286), PGAL1-FUS3-GFP-SSaaX (s, FG; pRC250), or PGAL1-fus3(D137A)-GFP-SSaaX (s, f*G; pRC261). Cultures were spotted in 10-fold dilution series on plates containing dextrose or galactose as the carbon source and assessed for growth at 26°C.
FIG. 14.
FIG. 14.
MAPK dynamics and the induction of pathway responses. (A) The signal transduction cascade of the pheromone response pathway is depicted as a curve emanating from membrane-associated upstream components and terminating in the active MAPK. Restricting the localization of an intermediate pathway component (e.g., Ste11) does not prevent activation of normal downstream components or normal outputs (left), whereas restricting the localization of the terminal effector MAPK confines its activity to only some of its targets and biases its action toward inappropriate ones (right). (B) The activity of the upstream signal transduction pathway is directed to cellular processes according to the subcellular localization of Fus3. Only the free MAPK can access all of its relevant substrates efficiently.
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