Dimerization of Ste5, a mitogen-activated protein kinase cascade scaffold protein, is required for signal transduction - PubMed (original) (raw)

Dimerization of Ste5, a mitogen-activated protein kinase cascade scaffold protein, is required for signal transduction

D Yablonski et al. Proc Natl Acad Sci U S A. 1996.

Abstract

The mitogen-activated protein kinase cascade of the Saccharomyces cerevisiae pheromone response pathway is organized on the Ste5 protein, which binds each of the kinases of the cascade prior to signaling. In this study, a structure-function analysis of Ste5 deletion mutants uncovered new functional domains of the Ste5 protein and revealed that Ste5 dimerizes during the course of normal signal transduction. Dimerization, mediated by two regions in the N-terminal half of Ste5, was first suggested by intragenic complementation between pairs of nonfunctional Ste5 mutants and was confirmed by using the two-hybrid system. Coimmunoprecipitation of differently tagged forms of Ste5 from cells in which the pathway has been activated by Ste5 overexpression further confirmed dimerization. A precise correlation between the biological activity of various Ste5 fragments and dimerization suggests that dimerization is essential for Ste5 function.

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Figures

Figure 1

Figure 1

Complementation of ste5_Δ and_ste5 ts by STE5 deletion mutants. (A) Mating assay of strains SMβ-2 and SMβ2-5Δ carrying representative STE5 deletion mutants on a 2 μ plasmid. Mating was assayed by a standard plate mating assay. Products of the mating reaction were replica plated to minimal media to select for diploids, shown here. (B) Results of mating assays of all of the Ste5 deletion mutants. Assays were performed as in_A_. (C) Mating assay of the_ste5_Δ strain β5Δ carrying pairs of_ste5_ mutant alleles. Group 1 plasmids are a subset of the plasmids assayed in A and B. Group 2 plasmids are based on the centromeric vector pRS414 (24) into which the indicated STE5 alleles were inserted.

Figure 2

Figure 2

Summary of Ste5 domains. (A) The region of Ste5 required for mating and the Ste5 dimerization domain as defined in this work. For comparison, location of the Lim-like domain, the putative leucine zipper, and the Far1 homology domain are indicated, as well as the binding domains of Ste5, determined by others (6, 31). The Ste7 binding domain is poorly defined. The C-terminal region (thick line) is necessary for Ste7 binding but may not be sufficient, since the entire region shown (thick and thin lines) is the smallest fragment that bound Ste7 (6). (B) Sequence of the Ste5 LIM-like domain. The LIM consensus sequence is shown (32) as well as a LIM-like consensus derived from a comparison of Ste5, Far1, Pep5, and PS43 (12). (C) Sequence of the Ste5 putative leucine zipper. Characteristic hydrophobic residues at positions a and d of each heptad are indicated (33).

Figure 3

Figure 3

Coimmunoprecipitation of GST-Ste5 with Ste5M and with Ste11M. GST-Ste5, Ste5M, and Ste11M were expressed under control of the GAL1/10 promoter in a _ste5_Δ strain. Where indicated, 10 μg/ml α-factor was added for the last 5 or 20 min of growth. Cells were lysed as described in the text. (A) A 17-μg sample of each lysate was separated by SDS/PAGE (7% acrylamide) and blotted with affinity-purified anti-Ste5 antibodies. (B) A 300-μg sample of each lysate was immunoprecipitated with anti-Myc monoclonal antibody or with anti-IGF-1R monoclonal antibody in a final volume of 220 μl. Immune complexes were separated by electrophoresis on a 4–10% gradient gel and blotted. Ste5M and GST-Ste5 were detected with anti-Ste5 antibodies (Upper). The blot was erased and reprobed with anti-Myc antibodies to detect Ste5M and Ste11M (Lower). (C) Cells were lysed in NLB buffer without (left lane) or with (six right-hand lanes) 5 μM zinc acetate. A 300-μg sample of each lysate was immunoprecipitated as in B, except that prior to the addition of anti-Myc-bound protein G beads, lysates were preincubated at 4°C for 1 hr in the presence of chelators at the concentrations indicated. Immunoprecipitation was in a final volume of 132 μl. Ste5M and GST-Ste5 were detected with anti-Ste5 antibodies (Upper). A shorter exposure allows comparison of the amount of Ste5M in each lane (Lower).

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