©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Yeast Protein Kinase C by Rho1 GTPase (*)

(Received for publication, February 7, 1996; and in revised form, February 29, 1996)

Yoshiaki Kamada (1) Hiroshi Qadota (2) Christophe P. Python (1) Yasuhiro Anraku (2) Yoshikazu Ohya (2) David E. Levin (1)(§)

From the  (1)Department of Biochemistry, Johns Hopkins University School of Public Health, Baltimore, Maryland 21205 and the (2)Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The PKC1 gene of the budding yeast Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C (PKC) (^1)(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(2)-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 rho1Delta 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.


EXPERIMENTAL PROCEDURES

Yeast Strains and Mutant Construction

All strains used in this study were derived from YPH500(20) . Error-prone PCR (21) was used to introduce random mutations into the RHO1 sequence. The PCR-amplified RHO1 fragment was inserted into the EcoRI/BglII gap of pY0701 and introduced into yeast strain YOC706, which harbors a rho1Delta and a plasmid expressing RHO1 under the control of the GAL1 promoter(18) . We examined 4000 transformants for growth on YPD (yeast extract/peptone/dextrose) plates at 23 and 37 °C and identified 41 rho1 mutations. Among these, 11 rho1 alleles (designated rho1-1 to rho1-11) contained single or double base changes. All of these alleles were reconstructed by site-directed mutagenesis and integrated at the ADE3 locus (22) of diploid strain YOC701 (RHO1/rho1Delta::HIS3). Haploid strains used in this study (YOC764 (RHO1), YOC729 (rho1-3), and YOC755 (rho1-5)) were derived from YOC701 integrants by standard genetic techniques. A single copy plasmid (pYO904) that carries HA-tagged RHO1 was constructed in vector pRS314, as described previously(16) , and introduced into yeast strain YOC701. A segregant bearing rho1Delta::HIS3 and pYO904, and a wild-type (RHO1) segregant lacking the plasmid were used for co-immunoprecipitation experiments.

Antibodies, Extracts, Immunoprecipitation, Protein Kinase Assays, and Immunodetection

Anti-HA antibodies (12CA5; BAbCo, Inc.) were used for immunoprecipitation and immunodetection of Rho1, Mpk1, and Pkc1. Polyvalent Pkc1 antibodies (used for immunodetection of Pkc1) were raised by Cocalico Biologicals (Reamstown, PA) in New Zealand White rabbits against a TrpE::Pkc1 fusion protein that contains amino acids 470-664 of Pkc1. This antiserum was used (at 1:3000 dilution) for immunodetection of Pkc1. Secondary antibodies used were horseradish peroxidase-conjugated donkey anti-rabbit (Amersham Corp.; at 1:10,000 dilution).

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) .

Recombinant Rho1 and Cdc42

Recombinant GST-Rho1 and GST-Cdc42 were expressed and purified from baculovirus-infected insect (Sf9) cells, as described(23) . For in vitro association with Pkc1, GST-Rho1 was not eluted from the glutathione-agarose beads used for purification. GST-Rho1-bound beads were incubated with cell extract in immunoprecipitation buffer (4) for 5 h at 4 °C, followed by three washes with this buffer. For use in Pkc1 protein kinase assays, GST-Rho1 and GST-Cdc42 were eluted from the beads with reduced glutathione. Purified GST-Rho1 displayed no protein kinase activity against the Bck1 peptide in the absence of Pkc1 (not shown).


RESULTS AND DISCUSSION

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(2) 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 GTPS-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 GTPS, 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(2)) 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 GTPS-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-beta-glucan synthase (GS) complex(31) , the enzyme responsible for constructing polymers of 1,3-beta-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.(^2)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.




FOOTNOTES

*
This work was supported by Grant GM48533 from the National Institutes of Health, American Cancer Society Grant FRA-446 (to D. E. L.), Ministry of Education, Science, Sports and Culture of Japan Grants 07740581 and 07254203 (to Y. O.), and by grants form the Japan Society for the Promotion of Science for Japanese Junior Scientists (to H. Q.) and the Swiss National Science Foundation (to C. P. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Johns Hopkins University School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-9825; Fax: 410-955-2926; levin{at}welchlink.welch.jhu.edu.

(^1)
The abbreviations used are: PKC, protein kinase C; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MEK, MAPK-activating kinase; MEKK, MEK-activating kinase; DAG, diacylglycerol; SRF, serum response factor; JNK, Jun NH(2)-terminal kinase (also known as SAPK, stress-activated protein kinase); PCR, polymerase chain reaction; HA, influenza hemagglutinin; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PS, phosphatidylserine; PMA, phorbol myristate acetate; GS, 1,3-beta-glucan synthase; MBP, myelin basic protein; GTPS, guanosine 5`-O-(thiotriphosphate).

(^2)
C. Zhao and D. E. Levin, unpublished results.


ACKNOWLEDGEMENTS

We thank Y. Zheng for baculovirus constructions, K. Lee for TrpE::Pkc1 fusion protein, M. Watanabe for Pkc1 expertise, B. J. Blacklock for Sf9 cells, and K. Fujimura-Kamada for helpful discussion.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.