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Vol. 14, Issue 7, 3027-3040, July 2003
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Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada, M5S 1A8
Submitted September 25, 2002;
Revised March 5, 2003;
Accepted March 6, 2003
Monitoring Editor: John Pringle
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Members of the amphiphysin family of
proteins play roles in endocytosis and actin cytoskeleton function in a
variety of organisms. For example, amphiphysin I and II are concentrated in
nerve terminals of mature neurons and are believed to act as adaptor proteins
in clathrin-mediated endocytosis.
The S. cerevisiae genes RVS167 and RVS161 were
first identified in a screen for mutants that exhibited reduced
viability upon starvation
(Bauer et al., 1993).
Deletion of RVS167 or RVS161 causes a phenotype consistent
with a role for the Rvs proteins in cortical actin cytoskeleton organization,
endocytosis, and membrane dynamics. The mutants exhibit loss of viability and
unusual cell morphology in poor growth medium or salt-containing medium,
delocalized actin distribution under suboptimal growth conditions, an abnormal
(random) budding pattern in diploids, and defects in endocytosis and
sporulation (Bauer et al.,
1993). Ultrastructural studies have revealed that rvs167
mutants accumulate late secretory vesicles at sites of membrane and cell wall
construction (Breton et al.,
2001). Consistent with a role for Rvs167p in the actin
cytoskeleton, Rvs167p colocalizes with cortical actin patches during
vegetative growth and is concentrated at the shmoo tip during mating
(Balguerie et al.,
1999).
Like its mammalian homologues, Rvs167p is a multidomain protein, capable of
interacting with a number of actin cytoskeleton proteins. Rvs167p forms a
heterodimer with Rvs161p through their respective BAR domains
(Navarro et al.,
1997; Sivadon et al.,
1997; Colwill et al.,
1999). In addition, Rvs167p, like the amphiphysins, has a Src
homology 3 (SH3) domain, a protein module well defined for binding prolinerich
sequences (Pawson and Scott,
1997). The middle portions of Rvs167p and the amphiphysins are not
conserved. In Rvs167p, the central portion of the molecule consists of a
region rich in glycine, proline, and alanine (the GPA region), most of which
is predicted to form no secondary structure elements and is notable because it
contains no charged amino acids over a stretch of 107 residues.
Consistent with the multidomain structure of Rvs167p, two-hybrid and phage
display screens have identified a large number of putative Rvs167p-interacting
proteins, many with previously characterized roles in the actin cytoskeleton.
These include Abp1p, Acf2p, Act1p, Las17p, Sla1p, Sla2p, and Srv2p
(Amberg et al., 1995;
Colwill et al., 1999;
Madania et al., 1999;
Bon et al., 2000;
Drees et al., 2001;
Tong et al., 2002).
For most of these proteins, however, there has been no demonstration that
their interaction with Rvs167p is direct, and the functional significance of
the interaction is not clear. In addition, a number of synthetic lethal or
negative growth synergistic interactions have been identified between
RVS167 and other actin cytoskeleton genes, including SLA1, SLA2,
SAC6, SRV2 (Lila and Drubin,
1997), YPT51/VPS21
(Singer-Krüger and Ferro-Novick,
1997), MYO1, MYO2, and certain alleles of ACT1
(Breton and Aigle, 1998), and
SLT2 and KRE6 (Breton
et al., 2001). Taken together, these data make clear that
Rvs167p plays an important but apparently redundant role in organizing the
actin cytoskeleton, possibly by acting as an adaptor to bring other proteins
together at specific places or times in the yeast life cycle. However, the
precise molecular role of Rvs167p remains to be defined.
A fundamental question in cytoskeleton biology is how the components of the
actin cytoskeleton are regulated to coordinate rapid remodeling of the
cytoskeleton. One likely mechanism is protein phosphorylation, which has been
implicated in regulation of the actin patch-associated proteins Pan1p, Sla1p,
Ent1p, and Clc1p in S. cerevisiae
(Chu et al., 1999;
Zeng and Cai, 1999;
Watson et al., 2001;
Zeng et al., 2001).
Rvs167p may also be regulated by phosphorylation. Rvs167p is a phosphoprotein
in log-phase cells and appears as an increased number of phosphoforms in cells
arrested in G1 phase by treatment with mating pheromone
(Lee et al., 1998).
In addition, Rvs167p could be phosphorylated in vitro by the cyclin-dependent
kinase (Cdk), Pcl2p-Pho85p, which had been immunoprecipitated from yeast
(Lee et al., 1998).
Pho85p is a multifunctional Cdk with roles in phosphate and glycogen
metabolism, targeted proteolysis, cell cycle progression, and cell polarity
(for reviews, see Carroll and O'Shea,
2002; Moffat et al.,
2000). Consistent with the multifunctional nature of Pho85p, 10
genes encoding Pho85p cyclins, Pcls, have
been identified and placed into subfamilies based on sequence similarity
within the cyclin-box region (Measday
et al., 1997). The Pcl1,2 subfamily includes three Pcls
that are specifically expressed in the G1 phase of the cell cycle (Pcl1p,
Pcl2p, and Pcl9p), as well as two that are not known to be regulated in the
cell cycle (Pcl5p and Clg1p). Lee et al.
(1998) showed that the
phenotype of rvs167
strains has some similarities with the
phenotypes of strains deleted for genes encoding Pho85p or members of the
Pcl1,2 group of cyclins. Interestingly, the mammalian homologue of Pho85p,
Cdk5, has been shown to phosphorylate the mammalian homologue of Rvs167p,
amphiphysin I, in vitro (Floyd et
al., 2001), suggesting that this regulation may be
conserved.
In this study we have examined the possible regulation of Rvs167p by phosphorylation both by Pcl-Pho85p and by the MAP kinase Fus3p, which is involved in the response to mating pheromone.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Other strains were made using standard yeast genetic
techniques (Sherman, 1991).
Strain BY1344 is a Ura- derivative of strain BY714
(Lee et al., 1998),
isolated by plating BY714 on SD medium containing 1 mg/ml 5-fluoroorotic acid
(Boeke et al., 1984).
Complete deletions of the indicated open reading frames (ORFs) were
constructed by integrative transformation
(Longtine et al.,
1998). Standard yeast media were used
(Sherman, 1991). To make
salt-containing minimal medium, NaCl was added to synthetic dextrose medium
(SD) to the final concentration (wt/vol) indicated. Phosphate-depleted medium
(YPGal-Pi) was prepared as described
(Warner, 1991). Where
indicated, 
-factor (from Louisiana State University Health Sciences
Center Core Laboratory, New Orleans, LA) was added to log-phase cultures in
YPD to a final concentration of 5 µM, and cells were grown for 1.5 h (2 h
for the slow-growing strains BY391 and BY1076) before harvesting.
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Insect Cell Culture and Protein Purification
Pho85p tagged N-terminally with GST + 6x His and Pcl2p tagged
N-terminally with 6x His were made by cloning the ORFs into the
Baculovirus Transfer Vectors pAcGHLT and pAcHLT-c, respectively (PharMingen,
San Diego, CA; Huang et al.,
1999a). Rvs167p was tagged N-terminally with GST + 6x His in
pAcGHLT. To produce active Fus3p, Fus3pK42R, or Kss1p kinase from
insect cells, cells were coinfected with viruses expressing the MAP kinase
module of STE5, STE11-4, STE7, and FUS3-myc,
FUS3K42R-myc, or KSS1-myc
(Breitkreutz et al.,
2001). Sf9 insect cells were coinfected with the recombinant
viruses and were harvested after 48 h. Insect cells were grown in Grace's
insect medium at 27°C, infected, and lysed using standard procedures
(Ausubel et al., 1994;
PharMingen). To prepare Pcl2p-Pho85p, cells were coinfected with viruses
expressing His-Pcl2p and His-GST-Pho85p, and then Cdk complexes were purified
over glutathione Sepharose (Amersham, Piscataway, NJ) as previously described
(Colwill et al.,
1999). The reconstituted MAP kinase modules were purified as
described by coimmunoprecipitation with anti-myc antibodies
(Breitkreutz et al.,
2001). Other GST-tagged proteins were purified over glutathione
Sepharose using standard procedures. His-tagged proteins were purified using
Ni-NTA agarose (Qiagen, Valencia, CA) using a native purification procedure
recommended by the manufacturer.
Plasmids
Plasmids are described in Table
2. Site-directed mutagenesis was done using either standard
two-step PCR mutagenesis techniques (Ho
et al., 1989; Warner,
1991) or the QuikChange XL Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA). Primers were designed to amplify the region
encoding each putative Pho85 phosphorylation site and surrounding area. The
sequences of primers used in this article are available upon request. The
integrity of all PCR products was confirmed by sequencing.
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In Vitro Kinase Assays and Quantitative Phosphorylation
Approximately 10 pmol GST-GPA-SH3 was phosphorylated by 3 pmol Pho85p and 1
pmol Pcl2p in a 30 µl reaction with 100 mM Tris.Cl, pH 7.5, 10 mM
MgCl2, 1 mM DTT, 50 µM ATP, and 50 µCi
[
-32P]ATP (Mandel, Guelph, Ontario, Canada). The reaction
was incubated at 30°C for 30 min. Proteins were separated on 7.5%
polyacrylamide gels, the gels were stained with Coomassie Blue and dried, the
radioactive proteins were detected by autoradiography, and the gel slice
containing the protein of interest was cut out and processed. To achieve
quantitative phosphorylation of Rvs167p, reactions were carried out as
described above with the following exceptions: ATP was added to a final
concentration of 5 mM and reactions were incubated at 25°C for 16 h, and
then an additional one half volume of kinase was added and the reaction was
incubated at 25°C for an additional 6 h. Reactions were done with
unlabeled ATP and, in parallel, "spiked" with
[-32P]ATP so that incorporation of PO4 could be
monitored. Quantification by phosphorimager showed that approximately 3 mol of
phosphate were incorporated per mole of Rvs167p. "Mock"
phosphorylation reactions were identical to quantitative phosphorylation
reactions except that kinase was left out.
The reconstituted MAP kinase modules were used to phosphorylate 
1 pmol
GST-Rvs167p or 20 pmol GPA-SH3-His in a 30-µl reaction using
[-32P]ATP as described by Breitkreutz et al.
(2001). Labeled proteins were
visualized by phosphorimager.
In Vivo Labeling of Rvs167p and Phosphopeptide Mapping
The galactose-inducible expression vector pGAL-RVS167
(Colwill et al.,
1999) was transformed into strains BY263 and BY1344, and
overexpression of RVS167 was confirmed by Western blot analysis
(Lee et al., 1998).
Transformants were grown to midlog phase in SD-Ura and then in YPD for about
two generations and then were transferred to YPGal-Pi and allowed to double
once. Because overexpression of RVS167 is toxic
(Colwill et al.,
1999), this doubling took 10–15 h. The cells were
pelleted and concentrated 20-fold to give a final volume of 2 ml in YPGal-Pi,
32P-orthophosphate was added to a final concentration of 1 mCi/ml,
and cells were incubated with shaking for 1 h. Cells were pelleted, washed
with 50 mM NaF, and flash frozen. 32P-labeled Rvs167p was
immunoprecipitated from cells (Lee et
al., 1998), run on a 7.5% polyacrylamide gel, and detected by
autoradiography.
The band corresponding to 32P-Rvs167p was cut out of the dried
gel. The gel slice was rehydrated in water for 1 h, and peptides were isolated
using an in-gel digestion procedure as described
(Figeys et al.,
2001), except that chymotrypsin (Roche Diagnostics, Indianapolis,
IN) was used instead of trypsin because the GPA region contains a stretch of
107 amino acids with no charged residues. Phosphopeptide mapping was done
using standard procedures (Boyle et
al., 1991; van der Geer
and Hunter, 1994). Chymotryptic peptides (200–2000 cpm
total) were spotted onto 20 cm x 20 cm thin-layer glassbacked cellulose
(TLC) plates (Merck, Darmstadt, Germany) at a spot 3 cm from the bottom and 7
cm from the cathode side. The plates were run in pH 4.72 electrophoresis
buffer on an HTLE-7000 electrophoresis apparatus at 1000 V for 3 h and
air-dried. Chromatography in the perpendicular direction (in
phosphochromatography buffer) was done for 12–14 h, and the plate was
exposed to a phosphorimager screen.
Western and Far Western Assays
Preparation of cell extracts and Western blot hybridization were done as
described (Lee et al.,
1998). Far Western hybridization was done as described
(Guichet et al.,
1997) using 35S-labeled Las17p or Ymr192p that had been
synthesized using a coupled T7 polymerasereticulocyte lysate system (Promega,
Madison, WI) primed with plasmid pBST-BEE1 or pGEM+YMR192w. To ensure that the
in vitro translation reaction products hybridized with the Western blot
contained no phosphatase activity, which could have removed phosphates from
the phosphorylated Rvs167p, 32P-labeled Rvs167p was run on a gel
and transferred to duplicate nitrocellulose membranes. One filter was
hybridized with mock in vitro translated protein (primed with no DNA), and the
other was stored at -20°C. After an overnight hybridization and washing,
the 32P-Rvs167p on the two filters was quantified using
phosphorimager analysis. This control experiment showed that no counts were
lost from the Rvs167p protein on the filter during incubation with the in
vitro translation mixture.
SGA Analysis and Complementation of Synthetic Lethality
Synthetic genetic array (SGA) analysis was done as described
(Tong et al., 2001)
with the modifications outlined below. A strain in which the RVS167
ORF had been replaced with the gene encoding nourseothricin resistance
Nat (Goldstein and McCusker,
1999) and that contained plasmid p416MET-RVS167-3A, which encodes
Rvs167p lacking the three phosphorylated serines
(Table 1), was crossed to an
ordered array of 4000 viable gene-deletion mutants (xxx).
The levels of Rvs167p in cells containing this plasmid under semirepressing
conditions (0.17 mM methionine) are similar to endogenous levels of Rvs167p
(see RESULTS). Diploids were sporulated, and double-mutant haploids
(rvs167 xxx) carrying p416MET-RVS167-3A were
selected and then tested for growth on SD + 4% NaCl at 34°C, conditions
under which some rvs167 double mutants have been shown to be inviable
(Lila and Drubin, 1997).
Strains that had apparent synthetic defects with RVS167-3A were
retested by mating in parallel to an rvs167 strain containing
vector, p416MET-RVS167-3A, or plasmid p416MET-RVS167, which encodes wild-type
Rvs167p. This screen for genetic backgrounds that require phosphorylation of
Rvs167p was not exhaustive because 1) an early (and thus incomplete) set of
the gene-deletion mutants was used and 2) deletion strains that themselves
grow slowly are beyond the sensitivity of this assay
(Tong et al.,
2001).
Complementation of the sla1 rvs167 strain
was assayed on SD-His containing 4.1% NaCl at 34°C. Complementation of the
end3 sla1 rvs167 strain was
assayed on SD-Ura containing 3.2% NaCl at 30°C. The degree of
complementation by RVS167-4A was transformant-dependent and highly variable in
penetrance (our unpublished results), possibly due to variability in levels of
expression of RVS167 from the plasmid.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). To establish Rvs167p as a bona fide in vivo substrate of
Pho85p, we sought to identify the Pcl2p-Pho85p phosphorylation sites. To this
end, we first analyzed the phosphopeptide map of bacterially expressed
GST-GPA-SH3 that had been phosphorylated in vitro using Pcl2p-Pho85p purified
from insect cells (Figure 1A). This identified three major phosphopeptides (numbered 1, 2, and 3 in
Figure 1A), suggesting that the
GPA-SH3 region is phosphorylated at three sites. We next asked whether the
same sites were phosphorylated in the context of the full-length protein by
comparing this map with that of GST-Rvs167p purified from insect cells and
phosphorylated in vitro with Pcl2p-Pho85p. We saw the same three major spots,
as well as a similar pattern of minor spots
(Figure 1B). This result
suggests that the amino-terminal BAR domain is not needed for normal
Pcl2p-Pho85p-dependent phosphorylation and is consistent with previous
findings that Pcl2p does not interact with the BAR domain in a two-hybrid
assay (Colwill et al.,
1999). Thus, we did our subsequent phosphopeptide mapping on
GST-GPA-SH3 mutant proteins purified from Escherichia coli.
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Like all Cdks, Pho85p is a proline-directed kinase: its target site for
phosphorylation is a serine or threonine followed by a proline
(O'Neill et al.,
1996). To identify phosphorylation sites on Rvs167p, we
constructed a series of mutants encoding single serine or threonine to alanine
substitutions at each of the eight S/TP sites in the GPA and SH3 regions of
Rvs167p and expressed these genes in E. coli. We then purified the
mutant proteins, phosphorylated the proteins in vitro using Pcl2p-Pho85p, and
repeated the phosphopeptide mapping. Comparison of wild-type GST-GPA-SH3 with
mutants T407A, T412A, and T454A showed that none of the three major
phosphopeptides was missing in the mutants (our unpublished results). In the
phosphopeptide map of mutant S390A, some of the faint phosphopeptides were
missing, suggesting that S390 may be a minor site for phosphorylation (our
unpublished results). In contrast, the phosphopeptide map of mutant S299A
lacked spot 3 (Figure 2A),
indicating that S299 is phosphorylated by Pcl2p-Pho85p in vitro. In addition,
mutant S379A gave a phosphopeptide map in which spot 1 was barely detectable
(Figure 2A). This suggests that
S379 is phosphorylated by Pcl2p-Pho85p but that the peptide containing S379
may comigrate with another (unidentified) minor peptide.
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By predicted mobility (Boyle et
al., 1991), spot 2 was predicted to contain a phosphopeptide
(DPATATSPTPTGY) with both a serine and a threonine that were followed
by a proline: S321 and T323 (italics). Mutant T323A gave the same
phosphopeptide map as did wild-type protein
(Figure 2B). In contrast, the
phosphopeptide map of mutant S321A gave three spots, but spot 2 was less
intense and had increased mobility in the chromatography dimension compared
with the same spot on the wild-type map
(Figure 2B). This suggested
that in the mutant protein, an alternative residue was being phosphorylated.
This would be consistent with phosphorylation of T323 in the absence of S321,
based on predicted mobility calculations
(Boyle et al., 1991).
Indeed, in the phosphopeptide map of the S321A and T323A double mutant, spot 2
disappeared (Figure 2, A and
B).
Because we used chymotrypsin, which is less specific than the more commonly
used trypsin (Antal et al.
2001), we sometimes saw spots that were products of cleavage at
secondary sites. The phosphopeptide containing S379, in particular, gave two
spots in some of our maps (Figure
2A: spots 1 and 1-A and Figure
2C). The peptide containing S379 has two leucine residues, which
are minor chymotrypsin digestion sites, and therefore a significant amount of
partially digested peptide might be expected. We found that the intensity of
peptide 1-A seemed to vary depending on the batch of chymotrypsin (compare
phosphopeptide maps of wild-type Rvs167p in
Figure 2, A and C).
In summary, we conclude that Pcl2p-Pho85p phosphorylates Rvs167p in vitro at three major sites: S299, S379, and S321, and at T323 if S321 is not present.
Phosphorylation of Rvs167p by Pcl-Pho85p In Vivo
To determine whether Rvs167p is phosphorylated at the same sites in vivo as
it is in vitro, we labeled wild-type cells with 32P-orthophosphate
and did phosphopeptide mapping on immunoprecipitated 32P-Rvs167p.
Even though overexpression of RVS167 is toxic and thus leads to a
slowed doubling time (Colwill et
al., 1999), we could only recover sufficient phosphorylated
Rvs167p to use in phosphopeptide mapping experiments if we overexpressed
RVS167 under the control of the GAL promoter on a high-copy
plasmid.
Rvs167p isolated from wild-type cells gave the same phosphopeptide map as did Rvs167p GPA-SH3 phosphorylated in vitro with Pcl2p-Pho85p (Figure 3, A and B). When in vitro–and in vivo–labeled proteins were analyzed after mixing, all the peptides colocalized, indicating that Rvs167p is phosphorylated on S299, S321, and S379 in vivo (Figure 3B). In repeated in vivo–labeling experiments we have noticed that phosphorylation of S299 appears variable (our unpublished results).
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To determine whether phosphorylation of Rvs167p was dependent on
Pcl-Pho85p, we performed phosphopeptide mapping on Rvs167p from a yeast strain
deleted for the genes encoding the five cyclins of the Pcl1,2 subfamily:
PCL1, PCL2, PCL5, PCL9, and CLG1
(Measday et al.,
1997). We chose to use this strain rather than one deleted for
PHO85 itself because pho85 strains are slow growing
and difficult to label in vivo, particularly because overexpression of
RVS167 is toxic (Colwill et
al., 1999). In protein from the mutant strain, spots 1 and
1-A, representing phosphorylation at S379, and spot 3, representing
phosphorylation at S299, were reduced in intensity
(Figure 3B). Spot 2 (S321) was
not reduced in intensity. We conclude that Pcl-Pho85p is required in log-phase
cells for phosphorylation of S379 and S299. However, because Pcl2p-Pho85p can
phosphorylate Rvs167p at S321 in vitro, it is also possible that both
Pcl-Pho85p and another kinase can phosphorylate Rvs167p at this site in vivo
in a redundant manner.
FUS3 Is Needed for Phosphorylation of Rvs167p
The results with the quintuple PCL delete strain showed that
another kinase must be able to phosphorylate Rvs167p in vivo. We were
therefore interested in identifying that kinase. Previous work showed that
phosphoforms of Rvs167p could be detected by Western blot in extracts from
log-phase cells, but multiple phosphoforms of Rvs167p were more clearly
visible in cells treated with -factor
(Lee et al., 1998).
We therefore used Western blots of both log-phase and
-factor–treated cells to detect Rvs167p phosphoforms in various
strains in which a single gene encoding a kinase had been deleted.
As observed previously (Lee et
al., 1998), the presence of phosphoforms of Rvs167p was
reduced in both log-phase and -factor–treated cells in a strain
deleted for PHO85 in comparison to wild-type
(Figure 4A). In contrast,
Rvs167p from strains deleted for SLT2, HOG1, ARK1, or PRK1
had levels of phosphorylation similar to those seen in wild-type cells under
both conditions (Figure 4A). However, the strain deleted for FUS3 also had reduced phosphorylation
of Rvs167p. The reduction in Rvs167p phosphoforms was barely detectable in
log-phase cells but was conspicuous in cells treated with -factor
(Figure 4A).
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Fus3p is a MAP kinase involved in the pheromone response and is partially
functionally redundant with the homologous MAP kinase Kss1p (for review see
Breitkreutz and Tyers, 2002;
Gustin et al., 1998).
We examined the dependence of Rvs167p phosphorylation on FUS3 and
KSS1 in the EG123 strain background
(Peter et al., 1993).
In wild-type EG123 cells, we saw multiple Rvs167p phosphoforms in cells
treated with -factor (Figure
4B). In EG123 cells deleted for FUS3, there was a
reduction in the number of phosphoforms of Rvs167p
(Figure 4B), similar to that
seen in the S288C strain background (Figure
4A). Rvs167p phosphoforms looked similar to those seen in
wild-type cells in a strain deleted for KSS1, whereas in cells
deleted for both FUS3 and KSS1, Rvs167p phosphoforms
resembled those in fus3 cells. Thus FUS3, but not
KSS1, is needed for efficient phosphorylation of Rvs167p in vivo.
We next asked whether Fus3p was able to phosphorylate Rvs167p directly in
vitro, using a purified MAP kinase module reconstituted in insect cells from
recombinant components (Breitkreutz et
al., 2001). Insect cells were coinfected with various
combinations of baculoviruses that expressed STE11-4 (a
constitutively active form of STE11), STE5, STE7, and
FUS3-myc, KSS1-myc, or FUS3K42R-myc. MAP kinase
complexes were then immunopurified from the cell lysates. Both the
Fus3p-containing complex and the Kss1p-containing complex have been shown to
phosphorylate Ste12p, a known substrate for both kinases
(Breitkreutz et al.,
2001). In our assay, Fus3p, but neither a catalytically inactive
Fus3p (Fus3K42R) nor Kss1p, was able to phosphorylate full-length
GST-Rvs167p (Figure 5A, top
panel). We also tested whether Fus3p could phosphorylate Rvs167p GPA-SH3-His,
which lacks the entire BAR domain. The GPA-SH3 fragment was phosphorylated
efficiently by Fus3p, but not by Fus3-K42R or Kss1p
(Figure 5A, bottom panel),
indicating that, in vitro, the BAR domain of Rvs167p is not needed for
phosphorylation by Fus3p. Phosphopeptide mapping of Rvs167p GPA-SH3
phosphorylated by Fus3p in vitro revealed that the same sites were
phosphorylated as with Pcl2p-Pho85p, although not all with the same intensity
(Figure 5B).
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In summary, the MAP kinase Fus3p is able to phosphorylate Rvs167p in vitro
and is required for full phosphorylation of Rvs167p in response to
-factor in vivo.
Role of Phosphorylation In Vivo
We next sought to discover the biological significance of Rvs167p
phosphorylation by searching for conditions in which phosphorylation of
Rvs167p was essential in vivo. We constructed plasmids encoding versions of
Rvs167p that had serine or threonine to alanine substitutions in the residues
that we had shown to be phosphorylated. pMET-RVS167-3A encodes Rvs167p with
S299A, S321A, and S379A substitutions, and pMET-RVS167-4A encodes Rvs167p with
these three changes plus a T323A substitution. T323 is a minor phosphorylation
site (Figure 2B), and we have
seen no differences in complementation between pMET-RVS167-3A and
pMET-RVS167-4A (our unpublished results). Western blots revealed that, as
expected, Rvs167-4A was underphosphorylated relative to wild-type Rvs167p in
log-phase cells, as seen by the absence of slower-migrating phosphoforms
(Figure 6A).
|
To assess genetically the consequences of a failure to phosphorylate
Rvs167p, we needed to confront the genetic redundancy of the actin
cytoskeleton. RVS167 is not an essential gene, although it is
required for viability under certain growth conditions
(Bauer et al., 1993;
Colwill et al., 1999)
and in strains deleted for a number of other actin cytoskeleton genes
(Lila and Drubin, 1997;
Singer-Krüger and Ferro-Novick,
1997; Breton and Aigle,
1998; Breton et al.,
2001). Furthermore, expression of the RVS167 BAR domain
alone is able to largely complement rvs167 defects, even
though the Rvs167p SH3 domain is known to bind biologically important ligands
and presumably plays a significant role in wild-type cells
(Lila and Drubin, 1997;
Bon et al., 2000;
Tong et al., 2002).
Because the Rvs167p phosphorylation sites that we have identified all lie in
the GPA region, outside the BAR domain, we reasoned that a requirement for
phosphorylation would probably only be apparent in genetic backgrounds that
are compromised for actin cytoskeletal function and thus would be sensitized
to defects in Rvs167p function. Consistent with this idea, we found that in
rvs167 strains, expression of wild-type RVS167 or
RVS167-4A complemented defects in growth on carbon and nitrogen
starvation media and on medium containing salt, as well as defects in
endocytosis and sporulation, with equal efficiency (our unpublished results).
In addition, overexpression of RVS167-4A resulted in inhibition of
growth to the same extent as did overexpression of wild-type RVS167
(our unpublished results).
To overcome the problem of functional redundancy, we tested whether the
RVS167 phosphorylation site mutant was able to complement the
synthetic lethality of various strains, both under standard conditions and in
the presence of NaCl at 34°C, conditions that have been shown to
exacerbate the phenotype of some rvs167 double mutants
(Lila and Drubin, 1997) No
difference in complementation by wild-type RVS167 and
RVS167-4A was seen in rvs167 sac6,
rvs167 srv2, or rvs167
sla2 strains (our unpublished results). In contrast, the
growth defect of the rvs167 sla1 strain was
complemented more efficiently by wild-type RVS167 than by the
RVS167 phosphorylation site mutant on medium containing 4.1% NaCl at
34°C (Figure 6B). Thus, in
at least one strain background, under certain growth conditions,
phosphorylation of Rvs167p is important for viability. To screen for other
genetic backgrounds in which phosphorylation of Rvs167p is essential, we used
a modified version of the SGA technique
(Tong et al., 2001;
see MATERIALS AND METHODS). This screen yielded two genetic backgrounds that
were not efficiently complemented for growth on 4% NaCl at 34°C by
RVS167-3A but were complemented by wild-type RVS167:
sla1 rvs167 (as already found by the candidate
approach; see above) and end3 rvs167.
The finding that both SLA1 and END3 had synthetic growth
defects in combination with the RVS167 phosphorylation site mutant
was of particular interest because End3p and Sla1p have recently been shown to
form a complex with a third protein, Pan1p
(Tang et al., 2000).
PAN1 is an essential gene (Tang
and Cai, 1996), whereas a sla1 end3 double mutant strain
has a reduced growth rate (Tang et
al., 2000). Tang et al.
(2000) have suggested that the
Pan1p-Sla1p-End3p complex may be essential but that the contributions of Sla1p
and End3p are partially redundant. We constructed an end3
sla1 rvs167 strain (which had a more severe
growth defect than the end3 sla1 strain) and
found that this strain was complemented for growth on salt-containing medium
by wild-type RVS167 but not by RVS167-4A
(Figure 6C). Western blot
analysis showed that Rvs167p was present in the strain expressing
RVS167-4A (Figure 6C).
These data show that under certain physiological circumstances,
phosphorylation of Rvs167p is important for cell viability.
Role of Phosphorylation In Vitro
We next explored the biochemical consequences of Rvs167p phosphorylation in
vitro. Our experiment was based on several genetic observations. Pan1p has
recently been shown to be an activator of the Arp2/3 complex, which stimulates
actin nucleation, in an in vitro system
(Duncan et al.,
2001). Because Pan1p, End3p, and Sla1p form a trimeric complex
(Tang et al., 2000),
the Arp2/3-activation activity may be due to this complex. Another activator
of the Arp2/3 complex in yeast is Las17p
(Madania et al.,
1999; Winter et al.,
1999), which binds to the SH3 domain of Rvs167p
(Bon et al., 2000;
Tong et al., 2002).
When combined with a las17-6 allele (which encodes a protein with a
truncation of the Arp2/3 activation domain), a truncation of the Pan1p carboxy
terminus (which is thought to be involved in Arp2/3 activation) leads to
temperature sensitivity, suggesting that the Arp2/3 activation function
carried out by Las17p may be redundant with that of Pan1p
(Duncan et al.,
2001). These findings, coupled with our observation that a version
of Rvs167p that cannot be phosphorylated is lethal under some growth
conditions in the absence of Sla1p and End3p, suggest that phosphorylation of
Rvs167p may be important in some function that involves Las17p.
To determine whether phosphorylation of Rvs167p had any effect on its interactions with Las17p, we tested Rvs167 GPA-SH3 that had been quantitatively phosphorylated or mock phosphorylated in vitro (see MATERIALS AND METHODS) for its ability to bind to Las17p, using a Far Western assay. Phosphorylated and unphosphorylated Rvs167p were run on a gel and blotted to nitrocellulose, and the blot was hybridized with in vitro–translated Las17p that had been labeled with [35S]methionine (Figure 7, middle panel). Controls established that there were equal amounts of phosphorylated and unphosphorylated GPA-SH3 on the filter (Figure 7, left panel) and that the in vitro translation reaction hybridized with the Western blot contained no phosphatase activity, which could have removed phosphates from the phosphorylated Rvs167p (see MATERIALS AND METHODS). Phosphorimager analysis revealed that 6.0 times more Las17p bound to mock-phosphorylated Rvs167p GPA-SH3 than to phosphorylated GPA-SH3. Thus, phosphorylation of Rvs167p inhibited its interaction with Las17p.
|
We next asked if the effect of phosphorylation of the GPA region of Rvs167p
on binding to the SH3 domain was specific for Las17p. Many proteins have been
reported to interact with Rvs167p in two-hybrid
(Bon et al., 2000;
Drees et al., 2001;
Tong et al., 2002)
and phage display (Tong et al.,
2002) assays, but few of these interactions have been confirmed in
vitro. Among these interactions, the SH3 domain of Rvs167p has been reported
to bind to an uncharacterized gene product, Ymr192p. Using affinity
chromatography, we have shown that Ymr192p from yeast extracts binds to the
SH3 domain of Rvs167p (Friesen, Colwill, and Andrews, manuscript in
preparation). We tested Rvs167p GPA-SH3 that had been phosphorylated or
mock-phosphorylated in vitro for its ability to bind to Ymr192p using a Far
Western assay. As with Las17p, binding of Ymr192p to the SH3 domain of Rvs167p
was inhibited by phosphorylation (Figure
7, right panel). Quantification by phosphorimager revealed a
5.8-fold difference. Thus, phosphorylation of the GPA region by Pcl2p-Pho85p
inhibits binding of at least two proteins to the SH3 region of Rvs167p.
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The actin-regulating kinase, Prk1p, phosphorylates Pan1p
and Sla1p, leading to disruption of the End3p/Pan1p/Sla1p complex
(Zeng and Cai, 1999;
Zeng et al., 2001).
Phosphorylation of the yeast epsin homologue, Ent1p, by Ark1p or Prk1p
negatively regulates its interaction with Pan1p
(Watson et al., 2001)
In this work, we have shown that Pcl-Pho85p phosphorylates the actin
cytoskeleton protein, Rvs167p.
Interestingly, the mammalian homologue of Rvs167p, amphiphysin I, is also a
phosphorylated protein with its phosphorylation sites clustered in the center
of the protein (Floyd et al.,
2001). Amphiphysin is phosphorylated in vitro by p35-Cdk5, the
mammalian functional homologue of Pcl-Pho85p
(Huang et al.,
1999a). Analogous to the situation with Rvs167p, for which we have
shown that phosphorylation inhibits binding of two proteins to the SH3 domain,
phosphorylation of amphiphysin inhibits its interaction with AP-2 and clathrin
(Slepnev et al.,
1998). This remarkable conservation of both a kinase and its
substrate demonstrates a conserved pathway involving the regulation of an
endocytosis protein.
Which Pho85 Cyclin Phosphorylates Rvs167p?
Several substrates for Pho85p kinase in conjunction with different cyclins
have been identified: Pho80p-Pho85p phosphorylates Pho4p with the consensus
SPXL/I (O'Neill et al.,
1996); Pcl10p-Pho85p phosphorylates Gsy2p at S/TPXDL
(Huang et al., 1996),
Pcl5p-Pho85p phosphorylates Gcn4p at TPVL
(Meimoun et al.,
2000; Shemer et al.,
2002), and Pho85p complexed with an unidentified cyclin
phosphorylates Sic1p at TPPR
(Nishizawa et al.,
1998). We have shown that in a strain deleted for the five genes
encoding the Pcl1,2 subfamily of cyclins, phosphorylation of Rvs167p at S379
and S299 is substantially reduced (Figure
3). One Pcl-Pho85p-dependent site on Rvs167p, S379, has the
sequence SPPL, consistent with the requirement for a hydrophobic
residue at position +3 seen with Pho4p and Gcn4p, whereas the other site,
S299, has the sequence SPVS.
Because of functional redundancy among the Pcls (Measday et al.,
1994,
1997;
Lee et al., 1998), we
cannot say which Pcl or Pcls are responsible for phosphorylation of Rvs167p.
Preliminary Western blot analysis of Rvs167p from samples taken at various
times in the cell cycle has shown that Rvs167p is specifically phosphorylated
in the G1 phase of the cell cycle (J. Moffat, personal
communication). This suggests that Rvs167p is likely to be phosphorylated in
vivo by one or more of the Pcls that is expressed specifically in G1: Pcl1p,
Pcl2p, or Pcl9p (Measday et al.,
1997). Pho85p, in association with its G1 cyclins, has previously
been demonstrated to have a role in cell integrity and polarity
(Lee et al., 1998;
Lenburg and O'Shea, 2001;
Huang et al., 2002).
Rvs167p is the first substrate that has been identified for Pho85p in
association with a G1 cyclin.
Phosphorylation of Rvs167p by the MAP Kinase Fus3p
Our data indicate that one or more kinases, in addition to Pcl-Pho85p, are
required for complete phosphorylation of Rvs167p during vegetative growth.
Because Pcl2p-Pho85p can phosphorylate Rvs167p in vitro at all of the three
sites that are known to be phosphorylated in vivo, we cannot say whether in a
wild-type strain phosphorylation at these sites is redundant or whether the
S321 site is phosphorylated only by another kinase in vivo. We have found that
the MAP kinase Fus3p is required for full phosphorylation of Rvs167p in cells
treated with factor.
Fus3p and Kss1p are MAP kinases that associate tightly with the MAPK kinase
(MEK) Ste7p. A putative binding site for Fus3p/Kss1p has been mapped to the
N-terminal 22 amino acids of Ste7p (Bardwell et al.,
1996,
2001;
Bardwell and Thorner, 1996). A
region with a high degree of sequence similarity to the N terminus of Ste7p is
found within the BAR domain of Rvs167p, extending from amino acid
148–156 (Aaron Neiman, personal communication). This suggests the
possibility that Fus3p could bind to and phosphorylate Rvs167p. We have found
that Fus3p, but not the related kinase Kss1p, phosphorylates Rvs167p in vitro.
The putative Fus3p/Kss1p docking site on Rvs167p is not required for this
phosphorylation (Figure 5A);
however, this may be due to our in vitro phosphorylation conditions. Our
experiments do not reveal whether Fus3p is the kinase that is redundant with
Pcl-Pho85 for phosphorylation of Rvs167p during vegetative growth. It is
possible that Fus3p phosphorylates Rvs167p in the presence of factor
and that another (as yet unidentified) kinase phosphorylates Rvs167p during
log phase in a manner redundant with Pcl-Pho85p.
In phosphopeptide mapping experiments with in vivo-labeled Rvs167p, we do
not detect any Fus3p-dependent phosphopeptides of Rvs167p in log-phase cells
(our unpublished results). Since Fus3p activity is stimulated upon treatment
with mating pheromone (Peter et
al., 1993), it is possible that Fus3p phosphorylates Rvs167p
specifically upon activation by mating pheromone; however, we have been unable
to test this because under the conditions we require for in vivo labeling of
Rvs167p (low phosphate medium containing galactose compounded by the fact that
RVS167 overexpression is toxic), cells do not mount a response to
pheromone (our unpublished results).
Although Rvs167p is hyperphosphorylated in cells treated with
factor (Figure 4), no clear
role for Rvs167p in mating has been identified
(Brizzio et al.,
1998). One possible role for phosphorylation of Rvs167p during
mating is revealed by ultrastructural studies that showed that rvs167
mutants are slowed in the process of digesting the septum, suggesting a role
for Rvs167p in cell fusion during mating
(Breton et al.,
2001). Thus, one role for phosphorylation of Rvs167p by Pcl-Pho85p
and/or by Fus3p may be in regulating the actin cytoskeleton during mating. One
member of the G1 class of Pho85p cyclins, PCL2, is induced upon
treatment with mating pheromone. It is clear, however, that Rvs167p is also
phosphorylated during vegetative growth.
Phosphorylation Inhibits Interaction of Rvs167p with Las17p and
Ymr192p
Phosphorylation has been shown to disrupt protein-protein interactions
between the SH3 domain of amphiphysin I and dynamin and between the central
domain of amphiphysin and AP-2 (Slepnev
et al., 1998). We have shown, using Far Western analysis,
that phosphorylation of Rvs167p inhibits its interaction in vitro with two
proteins that bind to the SH3 domain of Rvs167p: Las17p and Ymr192p. In
contrast, we have mapped the phosphorylation sites of Rvs167p to amino acids
S299, S321, and S379 in the GPA region, amino-terminal to the SH3 domain
(Figure 2). The mechanism by
which phosphorylation at a distant site affects binding to the SH3 domain of
Rvs167p remains unclear. One possibility is that phosphorylation affects
Rvs167p binding activity by inducing a conformational change due to the
introduction of negative charges in the uncharged GPA region. Deletion of the
GPA region has no detectable phenotype, suggesting that the role of the GPA
may be simply to link the N- and C-terminal domains
(Sivadon et al.,
1997). A conformational change in the GPA could alter the way the
Rvs167p domains are linked and could affect their interactions with other
proteins. This type of phosphoregulation is believed to exist in the case of
the mammalian cytoskeleton protein moesin
(Huang et al.,
1999b). In another example, regulation of the Src kinase Hck by a
phosphorylation-induced conformational change has been demonstrated in
structural studies (Sicheri et
al., 1997).
A second possible explanation for our finding that phosphorylation in the
GPA region inhibits binding to the SH3 domain of Rvs167p is that Las17p and
Ymr192p may make interactions with both the GPA and the SH3 domains of
Rvs167p. Rvs167p could have two independent binding faces for these proteins
with the SH3 interaction providing the bulk of the binding energy but the
interaction with the GPA, which would be inhibited by phosphorylation, still
making a substantial contribution. Results from two-hybrid assays support this
latter possibility (Colwill et
al., 1999; Madania et
al., 1999; Bon et
al., 2000).
A Model to Suggest a Biological Role for Phosphorylation of
Rvs167p
Our biochemical and genetic studies provide evidence for conserved Cdk and
MAP kinase regulation of protein complex formation by the amphiphysin
homologue Rvs167p. To explain our data, we suggest a model for phosphorylation
of Rvs167p. We propose that the major role for phosphorylation of Rvs167p is
to prevent binding or to release its interaction with Las17p. This model is
based on the following observations: 1) We have found that under certain
growth conditions, the gene encoding a version of Rvs167p that cannot be
phosphorylated leads to synthetic growth defects in combination with deletions
of SLA1 and/or END3. 2) Sla1p and End3p form a complex with
the essential protein Pan1p (Tang et
al., 2000). 3) PAN1 has a synthetic growth defect
with LAS17 with respect to their roles as activators of the Arp2/3
complex (Duncan et al.,
2001). We suggest that Las17p must be free of Rvs167p in order to
carry out its function as an activator of the Arp2/3 complex. This function is
redundant with some important function carried out by Sla1p and/or End3p (as
indicated by our synthetic lethal data). We suggest that in the absence of
Las17p-mediated activation of the Arp2/3 complex, the Pan1p/Sla1p/End3p
complex becomes essential for activating Arp2/3. Thus in a cell containing a
version of Rvs167p that cannot be phosphorylated, Rvs167p inappropriately
binds to Las17p, which therefore cannot activate Arp2/3, making Sla1p and
End3p (in a complex with Pan1p) essential. In support of this model, we found
that phosphorylation of Rvs167p inhibits its interaction with Las17p
(Figure 7).
Rvs167p is believed to be an adaptor protein like its mammalian homologue
amphiphysin. Our model must represent only one example of how Rvs167p
functions in the cell, a role that is regulated by the activity of the Cdk
Pcl-Pho85p. We have observed that RVS167-4A is not able to complement
an rvs167 sla1 end3 strain for
growth on salt-containing medium, but is able to partially complement for
growth on salt-free medium (Figure
6C). This is not surprising, because biochemical and genetic data
indicate that Rvs167p has roles in multiple complexes in the cell, some of
which are predicted to be independent of Pcl-Pho85p phosphorylation. We note
that the model we have proposed does not take into account our finding that
phosphorylation of Rvs167p also inhibits binding to Ymr192p, whose function is
unknown. We can hypothesize that in its putative role as an adaptor protein, a
certain population of Rvs167p in the cell is localized in a complex that
allows it to sequester Las17p via its SH3 domain, whereas another
population of Rvs167p is localized in a complex that allows it to bind Ymr192p
in an analogous phosphorylation-dependent manner. Future studies will be
directed toward identifying which Rvs167p complexes are formed in vivo in
response to a variety of regulatory stimuli to control cell polarity and other
functions of the actin cytoskeleton.
|
ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
|---|
Present addresses: Department of Biological Sciences, University of
Alberta, Edmonton, Canada, T6G 2E9
Present addresses: Samuel Lunenfeld Research Institute, Mount Sinai
Hospital, Toronto, Canada M5G 1X5.
Corresponding author. E-mail address:
brenda.andrews{at}utoronto.ca.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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