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Vol. 11, Issue 11, 4033-4049, November 2000
Department of Molecular and Cell Biology, Division of Biochemistry
and Molecular Biology, University of California, Berkeley, California
94720-3202
Ste5 is essential for pheromone response and binds components
of a mitogen-activated protein kinase (MAPK) cascade: Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK). Pheromone
stimulation releases G
(Ste4-Ste18), which recruits Ste5 and
Ste20 (p21-activated kinase) to the plasma membrane, activating
the MAPK cascade. A RING-H2 domain in Ste5 (residues 177-229)
negatively regulates Ste5 function and mediates its interaction with
G. Ste5(C177A C180A), carrying a mutated RING-H2 domain, cannot
complement a ste5
mutation, yet supports mating even
in ste4 ste5 cells when
artificially dimerized by fusion to glutathione
S-transferase (GST). In contrast, wild-type Ste5 fused
to GST permits mating of ste5 cells, but does not
allow mating of ste4 ste5 cells. This differential behavior provided the basis of a genetic selection for STE5 gain-of-function mutations. MATa
ste4 ste5 cells expressing Ste5-GST
were mutagenized chemically and plasmids conferring the capacity to
mate were selected. Three independent single-substitution mutations
were isolated. These constitutive STE5 alleles induce cell cycle arrest, transcriptional activation, and morphological changes normally triggered by pheromone, even when G is absent. The first, Ste5(C226Y), alters the seventh conserved position in the
RING-H2 motif, confirming that perturbation of this domain constitutively activates Ste5 function. The second, Ste5(P44L), lies
upstream of a basic segment, whereas the third, Ste5(S770K), is
situated within an acidic segment in a region that contacts Ste7. None
of the mutations increased the affinity of Ste5 for Ste11, Ste7, or
Fus3. However, the positions of these novel-activating mutations
suggested that, in normal Ste5, the N terminus may interact with the C
terminus. Indeed, in vitro, GST-Ste5(1-518) was able to associate
specifically with radiolabeled Ste5(520-917). Furthermore, both the
P44L and S770K mutations enhanced binding of full-length Ste5 to
GST-Ste5(1-518), whereas they did not affect Ste5 dimerization. Thus,
binding of G to the RING-H2 domain may induce a conformational change that promotes association of the N- and C-terminal ends of Ste5,
stimulating activation of the MAPK cascade by optimizing orientation of
the bound kinases and/or by increasing their accessibility to
Ste20-dependent phosphorylation (or both). In accord with this model,
the novel Ste5 mutants copurified with Ste7 and Fus3 in their activated
state and their activation required Ste20.
c/o Luo Laboratory, Life Sciences Division,
Lawrence Berkeley National Laboratory, Donner Laboratory, Berkeley,
California 94720-3206.
Corresponding author. E-mail address:
jeremy{at}socrates.berkeley.edu.
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