Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, 615 N Wolfe St, MD 21205, USA
Correspondence
David E. Levin
levin{at}jhmi.edu
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ABSTRACT |
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Present address: Wyeth Pharmaceuticals, Cambridge, MA 02140, USA.
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INTRODUCTION |
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A second essential function of Rho1 is to bind and activate protein kinase C (Kamada et al., 1996; Nonaka et al., 1995
), which is encoded by PKC1 (Levin et al., 1990
). Loss of PKC1 function, or of any of the components of the MAP kinase cascade under its control, results in a cell lysis defect that is attributable to a deficiency in cell wall construction (Levin & Bartlett-Heubusch, 1992
; Levin et al., 1994
; Paravicini et al., 1992
). The MAP kinase cascade is a linear pathway comprising a MEKK (Bck1; Costigan et al., 1992
; Lee & Levin, 1992
), a pair of redundant MEKs (Mkk1/2; Irie et al., 1993
) and a MAPK (Mpk1/Slt2; Lee et al., 1993
; Torres et al., 1991
). One of the consequences of signalling through the MAP kinase cascade is the activation of the SRF-like transcription factor, Rlm1 (Watanabe et al., 1997
; Jung et al., 2002
). Signalling through Rlm1 regulates the expression of at least 25 genes, most of which have been implicated in cell wall biogenesis (Jung & Levin, 1999
).
Cell wall integrity signalling is induced in response to several environmental stimuli. First, signalling is activated persistently in response to growth at elevated temperatures (e.g. 3739 °C; Kamada et al., 1995), consistent with the finding that null mutants in many of the pathway components display cell lysis defects only when cultivated at high temperature. Second, hypo-osmotic shock induces a rapid, but transient activation of signalling (Davenport et al., 1995
; Kamada et al., 1995
). Third, treatment with mating pheromone stimulates signalling at a time that is coincident with the onset of morphogenesis (Buehrer & Errede, 1997
; Errede et al., 1995
). Indeed, mutants defective in cell integrity signalling undergo cell lysis during pheromone-induced morphogenesis. Finally, agents that cause cell wall stress, such as caffeine and the chitin antagonist Calcofluor White, also activate signalling (Ketela et al., 1999
; Martin et al., 2000
).
The mechanisms by which cell wall stress is transmitted to Rho1 is an area of active investigation. Several regulators of Rho1 activity have been identified. Rom1 and Rom2 comprise a redundant pair of guanine nucleotide exchange factors (GEFs) for Rho1 (Ozaki et al., 1996). Bem2 and Sac7 are GTPase-activating proteins (GAPs) for Rho1 (Kim et al., 1994
; Peterson et al., 1994
; Schmidt et al., 1997
). Finally, a family of cell surface sensors for the activation of cell integrity signalling has been described. These include Wsc1, Wsc2, Wsc3, Mid2 and Mtl1 (Gray et al., 1997
; Verna et al., 1997
; Jacoby et al., 1998
; Ketela et al., 1999
; Rajavel et al., 1999
). Among these, Wsc1 and Mid2 are the major sensors dedicated to signalling wall stress during vegetative growth and pheromone-induced morphogenesis (Rajavel et al., 1999
; Ketela et al., 1999
). Indeed, a wsc1
mid2
double mutant displays a severe cell lysis defect, indicating that these genes have an overlapping function during vegetative growth. The cytoplasmic domains of both Wsc1 and Mid2 interact with the N-terminal domain of the Rom2 (and presumably Rom1) guanine nucleotide exchange factor to stimulate GTP loading of Rho1 (Philip & Levin, 2001
).
All members of the Wsc1/Mid2 family are transmembrane proteins that reside in the plasma membrane (Ketela et al., 1999; Lodder et al., 1999
; Rajavel et al., 1999
; Verna et al., 1997
). Their overall structures are similar in that they possess small cytoplasmic domains, each has a single transmembrane region and their extracellular domains are rich in Ser/Thr residues. These Ser/Thr-rich regions are highly O-mannosylated, probably resulting in extension and stiffening of the polypeptide. Therefore, the extracellular domains have been proposed to act as rigid probes of the extracellular matrix (Rajavel et al., 1999
). Despite the broad similarity among the proteins, there is very limited sequence identity among their cytoplasmic domains. Here we present a mutational analysis of the cytoplasmic domain of Wsc1.
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METHODS |
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WSC1 plasmids
All plasmids used in this study are shown in Table 1.
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For construction of point mutants, pRS314-WSC1HA (p1672) was used as template for site-directed mutagenesis by the PCR overlap extension method (Ho et al., 1989). The T7 and T3 primers were used in primary PCR reactions with forward and reverse mutagenic primers, respectively. The products of these reactions were mixed in secondary PCR reactions using only the T7 and T3 primers. The full-length products derived from these reactions were subcloned either as KpnISacI fragments into pRS314 (K301A, Y303A and Q304A) or as ApaIPstI fragments into pRS314-WSC1HA (L369A, V371A, V372A, N373A, P374A, D375A and D378A). YEp352-wsc1-S328/329/331A-T337/341AHA (p1847) was constructed stepwise by first mutating the two Thr residues in pRS314-WSC1HA, followed by mutagenesis of the three Ser residues and finally subcloning the pentuple mutant as an MfeISalI fragment into YEp352-WSC1HA. YEp352-wsc1-S319/320AHA (p1868), YEp352-wsc1-S322/323AHA (p1869) and YEp352-wsc1-S319/320/322/323AHA (p1850) were constructed by simultaneous mutation of two or all four Ser residues, followed by subcloning the double and quadruple mutants as a MfeISalI fragment into YEp352-WSC1HA. pRS314-wsc1-(1-346)-S319/320/322/323AHA (p1866) was constructed by subcloning the truncated C terminus of wsc1-(1-346)HA (from p1513) into pRS314-wsc1-S319/320/322/323AHA by an ApaIPstI fragment (the ApaI site spans residues 326/327). The complete DNA sequences of all inserts were determined. Sequence analysis was performed by the JHU Biosynthesis and Sequencing Facility. PCR was performed using Pfu polymerase (Stratagene). Primers are available upon request.
Two-hybrid plasmids and assays.
Sequences encoding the C-terminal tails (residues 291378) of wsc1 point mutants were amplified by PCR using primers that placed a BamHI site before the N-terminal residue and a PstI site after the C-terminal residue. These fragments were cloned into the BamHI and PstI sites of pGBT9 (Clontech Laboratories) so as to fuse the wsc1 sequences in-frame with the Gal4-DNA-binding domain. All fusions were confirmed by DNA sequence analysis. pGBT9 clones of mutant wsc1 tails were cotransformed with pGAD424-rom2-(1-660) (p1667) into yeast strain SFY526 (Clontech Laboratories) and transformants were tested as described in Philip & Levin (2001) for two-hybrid interactions.
Immunodetection and phosphatase treatment of Wsc1HA.
Extracts of yeast strain EG123 expressing forms of Wsc1HA from YEp352 were made and tested by immunoblotting with mouse mAb 12CA5, as described by Philip & Levin (2001) with the following modifications to the lysis buffer: phosphatase inhibitors (30 mM sodium pyrophosphate and 0·2 mM sodium vanadate) and 0·5 mM EGTA were added and 0·5 % NP40 was substituted for Triton X-100. The phosphorylation state of Wsc1HA forms was determined by treatment with
protein phosphatase (New England Biolabs). Extracts (1525 µg protein) were treated with 400 U
protein phosphatase for 2 h at 30 °C in
phosphatase buffer (50 mM Tris/HCl, pH 7·5, 0·1 mM EDTA, 5 mM DTT, 0·01 % Brij35 and 2 mM MnCl2) with or without phosphatase inhibitors (45 mM KF, 23 mM sodium pyrophosphate and 1·5 mM sodium vanadate). Treated extracts were subjected to SDS-PAGE on 415 % polyacrylamide gradient Ready Gels (Bio-Rad).
Immunodetection of Mpk1HA and activated Mpk1 after heat shock.
Yeast cells grown to an OD600 of 0·51·0 at 23 °C in YEPD were exposed to a mild heat shock (39 °C) by 1 : 1 dilution with fresh medium prewarmed to 55 °C and maintained at 39 °C for the indicated times. The cell response was terminated by further dilution (1 : 1) with ice-cold stop mix (Kamada et al., 1995). Extracts were made and Mpk1HA was detected by immunoblot after samples (5 µg protein) were subjected to SDS-PAGE using mouse mAb 12CA5 (BaBCo) and horseradish peroxidase-linked secondary antibody (Amersham) as described by Kamada et al. (1995)
. Activated Mpk1 was detected with rabbit polyclonal anti-phospho-p44/p42 MAPK (Thr202/Tyr204) antibody (New England Biolabs) essentially as described by Martin et al. (2000)
except that 20 µg protein was fractionated and primary antibody was used at a 1 : 1000 dilution.
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RESULTS AND DISCUSSION |
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To determine the contribution of the phosphorylated seryl residues to Wsc1 function, we introduced the S4A mutation into the wsc1(1-346) truncation allele. Fig. 5(d) shows that eliminating these phosphorylation sites partially suppressed the growth defect associated with wsc1(1-346), supporting the conclusion that phosphorylation of Wsc1 serves a negative regulatory role. Phosphorylation site mutants of Wsc1 did not display enhanced two-hybrid interaction with Rom2 (data not shown). However, because only the cytoplasmic domain of Wsc1 was expressed in the two-hybrid clone to allow nuclear localization, it may not be phosphorylated in this setting. Finally, we tested the effect of the S4A mutation on Mpk1 activity by following dual phosphorylation of the MAP kinase (Martin et al., 2000
). We did not detect constitutive activity of Mpk1 in cells expressing the WSC1-S4A allele under non-inducing conditions (Fig. 5e
). However, a shift from 23 to 39 °C reproducibly induced a more rapid activation of Mpk1 than observed in wild-type cells (Fig. 5e
), suggesting that dephosphorylation of Wsc1 potentiates signal transduction. Full activation of Mpk1 in response to mild heat shock normally requires 30 min (Kamada et al., 1995
; Fig. 5e
). By contrast, Mpk1 was fully activated within 10 min after temperature upshift in the WSC1-S4A mutant.
These results, taken together, suggest a model for the regulated interaction of Wsc1 with Rom2. We propose that two regions of the Wsc1 cytoplasmic tail interact with Rom2 (Fig. 6). One of these includes tyrosine 303, which is proximal to the plasma membrane. Because a truncation that removes most of the cytoplasmic tail [wsc1(1-316)] was partially functional, we conclude that this region is sufficient for Rom2 stimulation in the absence of other cytoplasmic domain sequences. The other Rom2-interacting region is at the extreme C terminus of Wsc1, defined by residues 369375. Disruption of either of these interactions in the context of the full-length protein prevents Rom2 interaction. The presence of a negative regulatory region between residues 316 and 345 also influences function. We identified a cluster of seryl residues within this region of Wsc1 (S319, 320, 322 and 323) that appear to be phosphorylated. Seryl-to-alanyl mutation of this cluster of residues partially suppressed the growth defect of a Wsc1 mutant missing the C-terminal interaction region. Moreover, a WSC1 mutant missing these phosphorylation sites potentiated activation of Mpk1 by mild heat shock. Therefore, we propose that phosphorylation of the Wsc1 cytoplasmic tail interferes with Rom2 interaction (Fig. 6
) and that dephosphorylation provides a means of regulating this interaction in response to cell wall stress. However, we have not been able to detect stress-induced dephosphorylation of Wsc1. Perhaps only a small fraction of the Wsc1 is dephosphorylated in response to wall stress. It will be interesting to identify the protein kinase and phosphatase that are responsible for the regulation of Wsc1.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 April 2004;
revised 12 July 2004;
accepted 15 July 2004.
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