(Received for publication, October 31, 1994; and in revised form, November 29, 1994)
From the
Conservation of the structure and function of Ras proteins has
been observed in a variety of eukaryotic organisms. However, the nature
of their downstream effectors appears to be quite divergent; adenylyl
cyclase and a protein kinase Raf-1, which do not share any structural
homology with each other, are effectors of Ras in the budding yeast and
in higher organisms, respectively. We show here that a protein kinase
Byr2, which has been known to act downstream of Ras1 in a mating
pheromone signal transduction system of Schizosaccharomyces
pombe, binds directly to Ras proteins in a GTP-dependent manner.
The region of Byr2 responsible for the Ras binding was mapped by a gene
deletion analysis to its N-terminal segment of 206 amino acid residues,
which does not possess any significant homology with the other
effectors of Ras. The affinity of the Byr2 N terminus for Saccharomyces cerevisiae Ras2 was determined by measuring its
activity to competitively inhibit Ras-dependent adenylyl cyclase
activity and found to be comparable with those of yeast adenylyl
cyclase and human Raf-1, with a dissociation constant (K) of about 1 nM. Furthermore,
Byr2 inhibited a Ras GTPase-activating activity of Ira2, a S.
cerevisiae homologue of neurofibromin. These results indicate that
Byr2 is an immediate downstream target of Ras1 in S. pombe.
The ras genes are widely conserved in a variety of
eukaryotic organisms from yeasts to mammals. They encode small
GTP-binding proteins that participate in signal transduction from
plasma membrane receptors to nuclei (for reviews, see (1) and (2) ). In mammalian cells the majority of cellular Ras in
quiescent cells is in the inactive GDP-bound form. Stimulation of cells
with mitogens or differentiation factors activates guanine nucleotide
exchange proteins and increases the abundance of the active GTP-bound
form. The GTP-bound Ras interacts directly with a serine/threonine
kinase Raf-1, which results in activation of a phosphorylation cascade
including Raf-1, MAP ()kinase kinases, and MAP
kinases(3, 4, 5, 6, 7) .
Similar pathways involving Ras have been identified in other species
including Caenorhabditis elegans and Drosophila
melanogaster(1) . In the budding yeast Saccharomyces
cerevisiae, a pair of Ras proteins, Ras1 and Ras2, interacts
directly with adenylyl cyclase in a GTP-dependent manner and regulates
its activity (for reviews, see Refs. 8 and 9). A domain comprising the
leucine-rich repeats structure has been defined as a Ras
protein-binding site of yeast adenylyl cyclase (10) . There is
no apparent structural homology between the leucine-rich repeats of
adenylyl cyclase and the Ras protein-binding region, the N-terminal
regulatory domain, of Raf-1 even though they bind mutually
competitively to the effector region of Ras(10) .
In the fission yeast Schizosaccharomyces pombe, its single Ras homologue, Ras1, is required for sexual responses induced by mating pheromones, namely conjugation in haploid cells and sporulation in diploid cells(11) . The function of Ras1 is mediated by control of a protein kinase cascade that ultimately regulates a member of the MAP kinase family. Spk1, which is essential for conjugation and sporulation in S. pombe, is structurally homologous to MAP kinases of vertebrates(12) , and a vertebrate MAP kinase can complement the loss of Spk1 in S. pombe(13) . Two genes BYR1 and BYR2, encoding putative protein kinases of distinctive structures, were isolated as multicopy suppressors of the ras1 defect(14, 15) , and BYR1 was shown to be epistatic to BYR2. Byr1 was found to be structurally related to vertebrate MAP kinase kinases(16) . This led to an assumption that Ras1, Byr2, Byr1, and Spk1 constitute a signal-transducing pathway analogous to the vertebrate Ras-MAP kinase cascade although direct protein-protein interactions among its constituents have not been biochemically verified. Recently from a study utilizing a yeast two-hybrid system came evidence suggesting that Byr2 interacts closely, if not directly, with Ras(7) . This led us to examine the mode of interaction of this protein with Ras by biochemical means.
Figure 1:
Measurement of Ras2
binding to GST-Byr2 by adenylyl cyclase inhibition assay. A,
adenylyl cyclase activity was measured in the presence of 0.5 pmol of
Ras2
with the addition of various amounts of
GST-Byr2(1-236) (
), GST-Byr2(1-206) (
),
GST-Byr2(1-151) (
), and GST-Byr2(88-236) (
).
An essentially similar assay was carried out in the presence of 2.5
mM Mn
instead of Mg
and
Ras2
with the addition of various amounts of
GST-Byr2(1-236) (
). B, adenylyl cyclase activities
dependent on various concentrations of Ras2
were
measured in the presence of various amounts of GST-Byr2(1-236) as
follows: 0 (
), 5 (
), 10 (
), and 20 pmol (
).
One unit of activity is defined as 1 pmol of cAMP formed in 1 min of
incubation with 1 mg of protein at 30 °C under standard assay
conditions. C, double-reciprocal plot analysis of the binding
reaction between GST-Byr2(1-236) and Ras2
. The
amounts of free and Byr2-bound Ras
were calculated as
described in the text.
To prove
the competitive nature of the inhibition, we extended the analysis by
examining the patterns of inhibition in the presence of various
concentrations of GST-Byr2(1-236) or Ras2 (Fig. 1B). We assumed that GST-Byr2 and adenylyl
cyclase bound mutually exclusively to Ras2. At each point of Ras2
concentration in the curves obtained for various amounts of GST-Byr2,
we obtained free Ras2 concentration available for adenylyl cyclase
activation as that required for giving the same adenylyl cyclase
activity in the absence of the competitor. A difference between the
original and the free concentrations of Ras2 was regarded as that bound
to GST-Byr2(1-236), and a reciprocal of this value was plotted
against a reciprocal of the free Ras2 concentration (Fig. 1C). This gave a series of straight lines for
each value of GST-Byr2(1-236), which converged on the horizontal
axis. The data confirmed our assumption that GST-Byr2(1-236)
polypeptide bound directly to Ras2
protein and
competitively sequestered it from adenylyl cyclase. The K
value of GST-Byr2(1-236) for
Ras2
was calculated from the point of intersection
with the horizontal axis and determined to be about 1 nM,
which is comparable with the value of human Raf-1 for H-Ras, 3.5
nM, or of yeast adenylyl cyclase for Ras2, 7 nM (see (10) for the data and a detailed description on the kinetic
analysis).
Figure 2:
Inhibition of Ira2-stimulated GAP activity
of Ras2 by GST-Byr2(1-236). An openbar stands
for the amount of [-
P]GTP bound to Ras2 (column 1) or Ras2
(column 7) put
into each reaction at the start of incubation. Blackbars stand for the residual amounts of
[
-
P]GTP bound to Ras2 (columns
2-6) or Ras2
(columns 8 and 9) after 5 min of incubation. The reactions contained: none (column 2); 48 pmol of GST-Byr2(1-236) (column
3); the lysate from yeast cells harboring pKT10 vector (columns 4 and 8); the lysate from yeast cells
harboring pKT10-IRA2 (columns 5 and 9); 48 pmol of
GST-Byr2(1-236) and the lysate from yeast cells harboring
pKT10-IRA2 (column 6). The experiment was carried out as
described under ``Experimental
Procedures.''
In S. cerevisiae two proteins, Ira1 and Ira2, were identified as GAPs for Ras1 and Ras2(19) . Next we examined the effect of GST-Byr2 on the stimulatory activity of Ira2 protein on Ras2 GTPase. As shown in Fig. 2, the addition of GST-Byr2(1-236) efficiently inhibited the GAP activity of Ira2 in vitro. This inhibition may be caused by competitive sequestration of Ira2 from Ras2 by association of the Byr2 N-terminal polypeptide with Ras2.
Figure 3:
Binding of Ras to GST-Byr2(1-236). A, Ras2 and H-Ras, either in a GDP-bound or GTPS-bound
form, were visualized by Western immunoblotting with an anti-Ras2
polyclonal antibody for Ras2 (17) and with a monoclonal
antibody Y13-259 for H-Ras. B, Ras2 and its
mutants in a GTP
S-bound form were visualized by Western
immunoblotting with the anti-Ras2 polyclonal antibody used in A. In each experiment, a aliquot of Ras protein used for the
binding assay (shown by an arrowhead in the lower
panel) and a aliquot of Ras protein bound to
GST-Byr2(1-236)-bound resin (shown by an arrow in the upper panel) were applied onto each lane. Measurement
of Ras binding was carried out as described under ``Experimental
Procedures.''
We have demonstrated for the first time that S. pombe Byr2 binds directly to the GTP-bound conformation of Ras but not
to its GDP-bound form. The binding was competitive with that of yeast
adenylyl cyclase and abolished by an effector mutation of Ras,
suggesting that the region of Ras recognized by Byr2 includes the
effector domain. Measurement of a K of the Byr2 N
terminus for Ras2 yielded a value of as low as 1 nM, which was
comparable with those of the other effectors, human Raf-1 and S.
cerevisiae adenylyl cyclase, for their homologous Ras proteins (10) . Furthermore, Byr2 inhibited the GTPase stimulatory
action of Ira2 on Ras2. This may provide other evidence that the
interaction between Ras and Byr2 is direct considering that Ira2
interacts directly with Ras. Similar inhibition of Ras GAP activity was
observed for human Raf-1 (5, 6) and presumably was
ascribable to overlapping of the binding sites between GAP and the
effector molecules. Although the physiological significance of the
inhibition of GAP activity remains to be clarified, it may have a
facilitating role in the signal transduction. Considering the observed
genetic epistasis of the BYR2 over the RAS1(15) , the data indicate that Byr2 is an immediate
downstream target of Ras1 in S. pombe.
S. pombe cells bearing the ras1 defect fail to conjugate and sporulate and are round in shape, unlike an elongated appearance of the wild-type cells(11) . In contrast, cells defective in Byr2 retain the normal elongated shape even though they possess deficiency in both conjugation and sporulation(15) . This may suggest that Ras1 has another effector molecule than Byr2 that affects the cell shape in S. pombe. Recently a candidate for this effector has been identified(21) . In mammalian cells, phosphatidylinositol 3-OH-kinase was proposed as a target of Ras(22) . Ras protein was shown to interact directly with the catalytic subunit of the enzyme in a GTP-dependent manner. In addition, a putative MAP kinase kinase kinase distinct from Raf-1 was shown to be regulated by Ras in mammalian cells although their mode of interaction remains to be clarified(23) .
We have shown that the N-terminal domains of both human Raf-1 and S. pombe Byr2 can effectively compete for Ras proteins with yeast adenylyl cyclase ( (10) and this paper). Strangely, the three targets of Ras, Byr2, Raf-1, and adenylyl cyclase, do not share any detectable homology among amino acid sequences of their Ras-binding domains even though all of them can bind mutually competitively to Ras proteins and discriminate between the GTP-bound and GDP-bound forms of Ras. In contrast, primary structures of both the effector region and its immediately flanking region of Ras proteins are unusually well conserved from yeasts to mammals. This raises the following two possibilities: 1) the three target molecules share a common tertiary structure for Ras binding that is presently unpredictable from their primary amino acid sequences and 2) they possess considerable differences in their binding recognition sequences of Ras. A further binding study using a set of Ras mutants and determination of the three-dimensional structure of the Ras-target complexes will be needed to solve these fundamental problems.