(Received for publication, February 7, 1996; and in revised form, February 29, 1996)
From the
We have investigated the role of the essential Rho1 GTPase in cell integrity signaling in budding yeast. Conditional rho1 mutants display a cell lysis defect that is similar to that of mutants in the cell integrity signaling pathway mediated by protein kinase C (Pkc1), which is suppressed by overexpression of Pkc1. rho1 mutants are also impaired in pathway activation in response to growth at elevated temperature. Pkc1 co-immunoprecipitates with Rho1 in yeast extracts, and recombinant Rho1 associates with Pkc1 in vitro in a GTP-dependent manner. Recombinant Rho1 confers upon Pkc1 the ability to be stimulated by phosphatidylserine, indicating that Rho1 controls signal transmission through Pkc1.
The PKC1 gene of the budding yeast Saccharomyces
cerevisiae encodes a homolog of mammalian protein kinase C (PKC) ()(1) that regulates a MAP kinase (MAPK)-activation
cascade comprised of a MEKK (Bck1), a redundant pair of MEKs (Mkk1/2),
and a MAPK (Mpk1)(2, 3) . Mutants in this signaling
cascade, called the cell integrity pathway, undergo cell lysis
resulting from a deficiency in cell wall construction that is
exacerbated by growth at elevated temperatures. We have reported that
thermal stress activates the cell integrity pathway and proposed that
weakness in the cell wall that develops during growth at high
temperature induces the signal for pathway activation(4) .
Pkc1 most closely resembles the conventional isoforms of mammalian
PKC, which require phospholipids, Ca, and
diacylglycerol (DAG) as cofactors to stimulate their catalytic
activity(1) . However, in vitro studies of this yeast
protein kinase have failed to demonstrate stimulation by cofactors,
despite the finding that mutations in PKC1 predicted to
relieve cofactor dependence have an activating effect on the
enzyme(5, 6) . This suggested that one or more
components required for cofactor-dependent stimulation of Pkc1 was
missing from in vitro reconstitution experiments.
Members
of the Rho family of small GTPases (RhoA, Cdc42, and Rac) regulate
various aspects of actin cytoskeleton organization and activation of
the SRF transcription factor in mammalian
cells(7, 8, 9, 10) . Cdc42 and Rac,
but not RhoA, stimulate the signaling pathway that contains the
JNK/SAPK (Jun NH-terminal kinase or stress-activated
protein kinase) MAPK homolog in mammalian cells (11, 12, 13) . Downstream effectors of RhoA
have not been identified(14, 15) . The yeast RHO1 gene encodes a homolog of mammalian RhoA that resides at sites of
cell growth (16) and whose function is essential for
viability(17) . A rho1
mutant is partially
suppressed by expression of human RhoA, but a residual cell lysis
defect is apparent at high temperature(18) , suggesting that RHO1 may function within the cell integrity pathway.
Additionally, an activated allele of PKC1 was isolated
recently as a dominant mutational suppressor of this
defect(19) , further supporting the notion that these signaling
molecules act through a common pathway. In this communication, we
demonstrate that Rho1 associates with Pkc1 in a GTP-dependent manner
and confers upon this protein kinase the ability to respond to
phosphatidylserine as an activating cofactor.
Yeast extract preparation, immunoprecipitation,
immunodetection and protein kinase assays of Mpk1 were
conducted as described previously(4) . Preparation of cell
extracts and immunoprecipitations for experiments with
Rho1 were carried out as in (4) with some
modifications. Lysis buffer without p-nitrophenyl phosphate
and with 1% Nonidet P-40 was used. The extract (700 µg of protein)
was precleared by incubation with 20 µl of a 50% suspension of
protein A-Sepharose for 1 h prior to immunoprecipitation to eliminate
nonspecific binding of proteins to immune complexes. Beads were boiled
in SDS-PAGE sample buffer, and samples were applied to 7.5% (for Pkc1
blots) or 15% (for
Rho1 blots) SDS-PAGE gels. For Pkc1
kinase assays, all as described previously(5) , except for the
addition of recombinant GTPases (see below). A synthetic peptide
corresponding to the sequence surrounding Ser
of Bck1, a
phosphorylation site for Pkc1, was used as substrate in Pkc1 kinase
assays(5) .
To examine the role of RHO1 in the cell integrity signaling pathway, we isolated a set of 11 temperature-sensitive rho1 alleles by in vitro random mutagenesis. Some of these mutants displayed cell lysis defects at the restrictive temperature (e.g. rho1-5), but others did not (e.g. rho1-3; Fig. 1A). Additionally, overexpression of PKC1 suppressed exclusively rho1-5 (Fig. 1B). Because of this allele-specific behavior, we chose rho1-3 and rho1-5 for further study.
Figure 1:
Overexpression of PKC1 suppresses the cell lysis defect of a rho1 mutant. A, the rho1-5 allele lyses at
restrictive temperature. Yeast strains patched on a YPD plate were
incubated at 23 °C for 3 days, then shifted overnight to 37 °C.
The patches were assayed in situ for release of alkaline
phosphatase as an indication of cell lysis. 1, wild type; 2, rho1-3; 3, rho1-5; 4, pkc1
(stt1-1;
SYT11-12A). B, an episomal plasmid (YEp352) with or without PKC1 was transformed into the rho1
mutants (rho1-3 and rho1-5). Transformants were
streaked onto a YPD plate and incubated at 37 °C for 3
days.
The Mpk1 MAPK is activated via Pkc1 in response to
brief heat shock treatment(4) . To determine if RHO1 is required for cell integrity pathway signaling, we tested the
ability of rho1 mutants to activate Mpk1 upon
heat shock. Mpk1, tagged at its COOH terminus with the HA epitope
(Mpk1
), was immunoprecipitated from extracts of heat
shock-treated cells and assayed for protein kinase activity in
vitro using myelin basic protein (MBP) as substrate. Heat
shock-induced activation of Mpk1 was completely blocked in the rho1-3 mutant (Fig. 2), indicating that RHO1 function is essential for Mpk1 activation. The rho1-5 mutant allowed some Mpk1 activation, suggesting that this allele
retains some function at restrictive temperature. Residual function of
the rho1-5 allele at high temperature might also explain
the allele-specific suppression of this mutant by PKC1 overexpression if Rho1 function is required for Pkc1 activation.
Figure 2:
RHO1 is required for Mpk1
activation in response to heat shock. Top panel,
phosphorylation of MBP by Mpk1 immunoprecipitated from
extracts of cells shifted from growth at 23-39 °C for 30 min.
This treatment did not affect the viability of the mutant strains (data
not shown). Mpk1 activity in rho1-5 (lanes 4 and 5) and rho1-3 (lanes 6 and 7) relative to wild type (RHO1; lanes
1-3) maintained at 23 °C (lane 1) is indicated. Bottom panel, immunoblot of immunoprecipitated
Mpk1
.
The yeast Cdc42 GTPase interacts with and stimulates the Ste20
protein kinase, which regulates the MAPK activation cascade of the
yeast pheromone response pathway(24, 25) .
Additionally, both recombinant human Cdc42 and Rac stimulate a
mammalian protein kinase that is closely related to Ste20
(PAK65)(26, 27) . Because Ste20 and Pkc1 function at
analogous positions in their respective MAPK signaling
pathways(2, 3) , we examined the possibility that Rho1
interacts directly with Pkc1 in vivo. Rho1, tagged at its
NH terminus with the HA epitope (
Rho1), was
immunoprecipitated from yeast extracts, and the resultant
immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with
anti-Pkc1 antibody. Pkc1 was co-immunoprecipitated with
Rho1 (Fig. 3A, lanes 4 and 6),
suggesting that Pkc1 associates with Rho1 in vivo. This
interaction was observed both in cells growing at 23 °C and after
heat shock.
Figure 3:
Pkc1 associates with Rho1 in vivo and in vitro. A, Rho1 was
immunoprecipitated from extracts of cells growing at 23 °C (lane 4) or shifted from 23 to 39 °C for 30 min (lane
6).
Rho1 immunoprecipitates (left) and
whole-cell extracts (100 µg of protein; right) were
analyzed by immunoblot with anti-Pkc1 antibodies (top panels)
or with anti-HA (to detect
Rho1; bottom panels).
Untagged Rho1 was used as a negative control (lanes 1, 2, and 7). The band indicated by * is derived from
immunoprecipitating antibodies. B, recombinant GST-Rho1 (1
µg), purified from Sf9 insect cells and bound to glutathione
agarose beads, was preloaded with the indicated guanine nucleotide (lanes 2-5). Soluble yeast cell extract (400 µg of
protein) containing Pkc1
was incubated with the beads (lanes 1, 3, and 5), and bound Pkc1
was
detected by immunoblot analysis. A control in which naked
glutathione-agarose beads were used (lane 1) demonstrates
dependence of Pkc1
binding on
GST-Rho1.
To determine if the association between Rho1 and Pkc1
depends on the activation state of Rho1, we examined the effect of
different guanine nucleotides on this interaction in vitro.
Recombinant GST-Rho1, immobilized on glutathione-agarose beads, was
preloaded with either GTPS or GDP prior to incubation with a yeast
extract containing soluble Pkc1 tagged at its COOH terminus with the HA
epitope (Pkc1
). After washing the beads, bound Pkc1
was detected by SDS-PAGE and immunoblotting with anti-HA
antibody. Fig. 3B shows that GTP
S-bound GST-Rho1
associated with Pkc1 (lane 5), but GDP-bound protein did not (lane 3).
We also tested the possibility that Pkc1 activity
is stimulated by Rho1. Pkc1 was immunoprecipitated from
yeast extracts, and its protein kinase activity was measured in the
presence or absence of GST-Rho1 using a synthetic Bck1 peptide as
substrate. Fig. 4A shows that GST-Rho1 did not
stimulate Pkc1 activity alone but, when bound to GTP
S, conferred
upon the protein kinase the ability to respond to activating cofactors
(PS, DAG, and Ca
). This stimulatory effect is
specific to Rho1, because GST-Cdc42 did not confer cofactor-dependent
stimulation on Pkc1. In the presence of GTP-bound GST-Rho1, Pkc1 was
strongly activated by phosphatidylserine (PS) as a lone cofactor (Fig. 4B). The conventional isoforms of mammalian PKC
are not stimulated by PS alone(28, 29) . In contrast,
this behavior is characteristic of the atypical
isoform of
PKC(28, 30) . No additional stimulation was observed
by addition of Ca
, DAG, or phorbol ester (PMA) as a
DAG substitute. This behavior is also exclusively characteristic of
PKC
(28, 30) . Interestingly, the Cys-rich region
of Pkc1, which is predicted to be a DAG-binding domain, has been
reported to interact with Rho1 in two-hybrid experiments(19) .
Therefore, Rho1 may replace DAG in the activation of Pkc1.
Figure 4:
Rho1 allows cofactors to activate Pkc1. A, phosphorylation of synthetic Bck1 peptide by Pkc1 immunoprecipitated from 50 µg of soluble yeast cell extract
protein. Recombinant GST-Rho1 or GST-Cdc42 (1 µg) was preloaded
with the indicated guanine nucleotide. Cofactors (80 µg/ml PS, 8
µg/ml DAG, and 100 µM CaCl
) were added to
the reaction where indicated. Lanes 1 and 2 are
control reactions with no GTPase. Mean and standard error for three
experiments is shown. B, PS alone is sufficient to stimulate
Pkc1 fully in the presence of Rho1. Phosphorylation of Bck1 peptide by
Pkc1
in the presence of GTP
S-bound GST-Rho1 and the
indicated cofactors. Conditions were as in A, except for PMA
(16 ng/ml). Concentrations of PS as low as 8 µg/ml fully activated
Pkc1 (data not shown).
This
study provides the first example of a PKC isoform whose stimulation by
cofactors is dependent on a GTPase. We have identified recently a
second role for Rho1 in the maintenance of cell integrity.
Specifically, Rho1 is an essential component of the 1,3--glucan
synthase (GS) complex(31) , the enzyme responsible for
constructing polymers of 1,3-
-glucan in the cell wall. We have
found that thermal induction of the FKS2 gene, which encodes
another component of the GS(32, 33) , is under the
control of PKC1 and MPK1.(
)Based on these
findings, we propose the following model. A signal induced by weakness
created in the cell wall during growth (and exacerbated at high
temperature) stimulates guanine nucleotide exchange of Rho1 at the
growth site. The GTP-bound Rho1 stimulates cell wall construction
directly by activating GS and indirectly by stimulating Pkc1-dependent
gene expression in support of this process (Fig. 5).
Figure 5: Model for the dual role of Rho1 in the maintenance of cell integrity.