From the Department of Molecular Genetics, University of Texas
M. D. Anderson Cancer Center, Houston, Texas 77030
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INTRODUCTION |
The cdc42 gene has been highly conserved through
evolution and encodes a small GTPase belonging to the Rho family of
Ras-related guanine nucleotide-binding proteins (1-3). Homologs of
cdc42 have been cloned from the evolutionarily distant
yeasts Saccharomyces cerevisiae (4) and
Schizosaccharomyces pombe (5), the nematode Caenorhabditis elegans (6), insects (7), and mammals (8). Until recently, the cellular functions of Cdc42 proteins were unknown.
However, recent studies from a variety of model systems have provided
substantial insights into Cdc42 function. In mammalian cells, Cdc42 and
a related GTPase, Rac, have been shown to participate in regulation of
the actin cytoskeleton, cell cycle control, and mitogen-activated
protein kinase (MAPK)1
cascades (1-3). At least two types of proteins have been implicated as
effectors for Cdc42 in mammalian cells. The first are members of a
recently elucidated family of protein kinases referred to as
p21cdc42/rac-activated kinases, or PAKs (9). PAKs, like Cdc42,
are conserved from yeasts to mammals and are activated by Cdc42 and Rac
GTPases but not by other small GTPases, such as Ras and Rho. In a
recent study, evidence was provided that p65PAK
(
-Pak/Pak1) is required for Cdc42-induced activation of the c-Jun
N-terminal kinase/stress-activated protein kinase cascade, but not for
Cdc42-induced cytoskeletal remodeling or DNA synthesis (10). PAKs
induce c-Jun N-terminal kinase/stress-activated protein kinase
activation in vitro (11, 12), so it would appear that they
are likely mediators of Cdc42-induced c-Jun N-terminal
kinase/stress-activated protein kinase activation in vivo.
In another recent study, it was shown that dominant-activated mutants
of
-Pak/Pak1 induce dissolution of actin stress fibers and
reorganization of focal complexes (13). Thus, a role for PAKs in
cytoskeletal regulation is likely, although the exact nature of this
function is, at present, unclear. A second putative Cdc42 effector in
mammalian cells is the Wiskott-Aldrich syndrome protein, or WASP (14,
15). WASP binds to Cdc42, but not to Rac or Rho GTPases (14). WASP is highly enriched in polymerized actin (14), and T lymphocytes from
patients with Wiskott-Aldrich syndrome, an immunodeficiency disease,
exhibit highly aberrant cytoskeletal organization (15). Thus, WASP is
likely to mediate at least part of the cytoskeletal regulatory
functions of Cdc42.
Substantial insights into the function and regulation of Cdc42 GTPases
have come from studies using yeast model systems. In the budding yeast
S. cerevisiae, Cdc42 is required for activation of a mating
pheromone-induced MAPK cascade and for proper bud site selection, a
process involving reorganization of the actin cytoskeleton (1, 16).
Cdc42 is also required for induction of the filamentous growth phase of
S. cerevisiae, a process that involves some, but not all, of
the components of the pheromone signaling pathway, as well as Ras
protein function (17, 18). Two PAK homologs, Ste20 (19-22) and Cla4
(23), and perhaps a third, Skm1 (24), are probable effectors for Cdc42
in S. cerevisiae. Ste20 and Cla4 are partially redundant in
function. S. cerevisiae mutants deleted of the
cla4 gene are morphologically aberrant (23) but
mating-competent, while ste20 null mutants are sterile and
defective in filamentous growth induction (19, 20). Mutants deleted of
both STE20 and CLA4 are inviable and cannot
undergo cytokinesis (23). Thus, Ste20 and Cla4 share at least one
essential cellular function. While skm1 null mutants are
viable and exhibit no obvious phenotypic defects, overexpression of
skm1 leads to aberrant cell morphology, suggesting a role
for the Skm1 protein in morphological regulation (24).
The fission yeast S. pombe possesses a single known
cdc42 gene, which is essential for cell viability (5). Wild
type fission yeast cells are rod-shaped, whereas cdc42 null
cells are spheroidal in morphology and exhibit mislocalization of actin
(5). This phenotype suggests a role for Cdc42 in cytoskeletal
regulation. Chang et al. (25) showed that Cdc42 participates
in a Ras-mediated morphological control pathway in S. pombe.
These investigators provided genetic and biochemical evidence that
Cdc42 and Ras1, the single known S. pombe Ras homolog, are
part of a complex of interacting proteins that includes the putative
Cdc42 guanine nucleotide exchange factor Scd1 and Scd2, an SH3
domain-containing protein of unknown function. Scd1 and Scd2 are
homologous to Cdc24 and Bem1, respectively, which have been shown to
regulate Cdc42 function in S. cerevisiae (1). Previously, we
provided evidence that a Ste20/PAK homolog, Shk1 (also known as Pak1
(26)) is a critical effector for Cdc42 in S. pombe (27).
Cdc42 and Shk1 interact physically, as determined by both two-hybrid
assays (26, 27) and coprecipitation experiments (26). shk1,
like cdc42, is an essential gene, and the terminal
phenotypes of cdc42 and shk1 null mutants are
similar (26, 27). Furthermore, overexpression of shk1
partially suppresses the mating defect of S. pombe mutants expressing a dominant negative mutant allele of cdc42 (27). Like Cdc42, Shk1 is also linked to Ras function in S. pombe.
Overexpression of dominant negative forms of shk1 results in
inhibition of Ras-dependent mating responses (26, 27). In
addition, cooverexpression of shk1 and skb1, a
gene we recently described that encodes a second putative Shk1
regulator, restores elongate morphology to S. pombe ras1
null mutants (28). These various data suggest that Shk1 is a key
mediator of the Ras1/Cdc42 signaling complex in S. pombe.
In this report, we describe the cloning and characterization of
shk2, a novel gene encoding a second Ste20/PAK-related
protein kinase in S. pombe. Shk2 is more closely related in
structure to the S. cerevisiae PAKs Cla4 and Skm1 than to
other known yeast and metazoan PAKs. We provide evidence for physical
and functional interaction between Shk2 and Cdc42 and for involvement
of Shk2 in Ras1/Cdc42-mediated morphological control and mating
response pathways. We show that Shk2 is not essential for viability,
normal morphology, or mating in S. pombe and provide
evidence that its functions substantially overlap with those of Shk1,
with Shk1 being the dominant protein in function. We also provide
genetic evidence corroborating a role for Shk1 in MAPK
cascade-dependent mating response in S. pombe.
Finally, we show that, despite the structural relatedness of the two
proteins, Shk2 cannot substitute for Cla4 in budding yeast, suggesting
that Shk2 and Cla4 are not functional homologs.
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EXPERIMENTAL PROCEDURES |
Yeast Strains, Manipulation, and Genetic Analysis--
S.
pombe strains used in this study were SP870
(h90 ade6-210 leu1-32 ura4-D18)
(from D. Beach), SP870D (h90 ade6-210
leu1-32 ura4-D18/h90 ade6-210 leu1-32
ura4-D18) (from V. Jung), CHP428 (h+
ade6-210 his7-366 leu1-32 ura4-D18) (from E. Chang), SP66
(leu2-32 ade6-216) (from D. Beach), SP42N17
(h90 ade6-216 leu1-32
ura4::adh1-cdc42N17) (27), SPGLD
(h90 ade6-210 leu1-32 ura4-D18
gpa1::LEU2/h90 ade6-210 leu1-32
ura4-D18 gpa1::LEU2) (29), SPRN1
(h90 ade6-210 leu1-32 ura4-D18
ras1
) (30), SPRN1D (h90 ade6-210
leu1-32 ura4-D18 ras1
/h90 ade6-210 leu1-32
ura4-D18 ras1
) (28), SP206U (h90
ade6-210 leu1-32 ura4-D18/h90 ade6-210
leu1-32 ura4-D18 shk1::ura4) (27), and SPSHK2U
(h90 ade6-210 leu1-32 ura4-D18
shk2::ura4) (see below). S. cerevisiae strains used were L40 (MATa ade2 his3 leu2 trp1
LYS2::lexA-HIS3 URA3::lexA-lacZ) (31), HF7c
(MATa ade2-101 his3-200 leu2-3, 112 lys2-801
trp1-901 ura3-52 gal4-542 gal80-538
LYS2::GAL1UAS-GAL1TATA-HIS3 URA3::GAL417mers(x3)-CYC1TATA-lacZ)
(32), SFY526 (MATa ade2-201 his3-200 leu2-3, 112 lys2-801 trp1-901 ura3-52 canR gla4-542
gal80-538,
URA3::GAL1UAS-GAL1TATA-lacZ)
(CLONTECH), FY40 (MATa, HMLa, HMRa, ho-Bgal,
ura3, HIS4, ade2-1, canI-100, met, his3, leu2-3, 112, trp1-1,
bar1::HisG, ste20::del, cla4::LEU2, Ycp
TRP1 cla4-75(c2816)), and MJY8 (MATa, cla4::LEU2
his3-100 ura3-1 leu2-3, 112 can1-100) (from F. Cvrckova,
Institute of Molecular Pathology, Wien, Austria). Standard yeast
culture media and genetic methods were used (33, 34). S. pombe cultures were grown on either 0.5% yeast extract, 3%
dextrose, and 75 mg/liter adenine (YEA) or Edinburgh minimal medium
(EMM) with appropriate auxotrophic supplements (34). S. cerevisiae cultures were grown on either 1% yeast extract, 2%
peptone, and 2% dextrose (YPD) or drop-out medium with appropriate
auxotrophic supplements (33). Yeasts were transformed by the lithium
acetate procedure (34). The shk2::ura4 strain,
SPSHK2U, was constructed by transforming SP870D with a 3.2-kb
HpaI-Ecl136II shk2::ura4 fragment from
the plasmid pBSIIshk2::ura4. Diploid transformants carrying a
single disrupted and a single wide-type copy of shk2 were
identified by Southern blot analysis, and
shk2::ura4 transformants were isolated by tetrad dissection.
Plasmids--
The two-hybrid plasmids pGADGH (for expression of
GAD fusions), pHP5, and pGBT9 (for expression of GBD fusions) and
pBTM116 and pVJL11 (for expression of LBD fusions) have been described previously (25, 31, 35, 36). The plasmids pGADCdc42, pGADCdc42(T17N), pGADCdc42(G12V), pGADShk1, pGADSTE20, pGADSkb1, pGADScd1, pGADScd2, pGADByr2, pGADByr1, pGADRas1, pGADSpk1, pGBDSkb1, pGBDScd1, pGBDScd2, pGBDByr1, pLBDCdc42, pLBDShk1, pLBDRaf, pLBDRas1,
pLBDRas1G17V, pLBDGpa1, pLBDByr2, pLBDScd1,
pLBDlamin, pSP206, pREP1Shk1, pAAUCMSkb1, pAAAU, and pAAAUByr2 have
also been described (25, 27, 28, 36). pLBDRac and pLBDRhoG were
provided by J. Camonis (Faculte de Medecine Lariboisiere, Paris).
pRSETCdc42-Hs(G12V) and pRSETHa-Ras(G12V) were provided by J. Frost and
M. Cobb (University of Texas Southwestern Medical Center, Dallas). The
S. pombe-Escherichia coli shuttle vector pAAUCM was used for
high level expression of coding sequences from the S. pombe
adh1 promoter (27). pREP1 (37) was used for expressing coding
sequences from the S. pombe nmt1 promoter. pREP1Shk2(K343R)
was provided by J. Chernoff (Fox Chase Cancer Center,
Philadelphia). FD44, a TRP1-based plasmid carrying the CLA4 gene, was provided by F. Cvrckova. pREP1Byr2 was made
by cloning a SalI-SacI fragment of the
byr2 coding sequence obtained from pAIS1 (38) into the
corresponding sites of pREP1 and allows for overexpression of
byr2 from the nmt1 promoter. A 2.1-kb fragment of
the shk2 gene was amplified by polymerase chain reaction
(PCR) using a plasmid harboring the shk2 gene (pSP204) as
template and the primer pair 5'-TGCATCGTGTCGACAATGCTTTTAAGTGTAAGT and
5'-AGGCAGGTCGACAGTTAACTAACG. The PCR-amplified shk2 fragment
was then digested by SalI and BclI and cloned
into SalI and BamHI sites of pREP1, generating pREP1Shk2. pBSIIShk2 was constructed by cloning a
SalI-Ecl136II fragment of shk2 from
pREP1Shk2 into the SalI -EcoRV sites of pBluescript II SK (pBSII). pHP5Shk2 was obtained by cloning a SalI-Ecl136II fragment of shk2 from
pBSIIShk2 into the SalI-NaeI sites of pHP5. A
SalI-SacI fragment of shk2 was
released from pREP1Shk2 and cloned into pAAUCM, pAUD6, and pAD5,
generating pAAUCMShk2, pAUD6Shk2, and pAD5Shk2, respectively.
pTrcHisBShk2 was constructed by cloning a
SalI-PstI fragment from pBSIIshk2 into the
XhoI-PstI sites of pTrcHisB (Invitrogen).
pGADGHShk2 was constructed by two steps. First, an EcoRI
fragment of Shk2 was released form pTrcHisBShk2 and cloned into the
EcoRI sites of pGADGH producing pGADGHShk2-3'. A
shk2 5'-fragment was released by BamHI from
pHP5Shk2 and cloned into the 5'-end of pGADGHShk2-3' to generated
pGADGHShk2. pHP5Cdc42 was made by cloning a
BamHI-SalI fragment of cdc42 from
pGADHGHCdc42 into the corresponding sites of pHP5. Shk2P1/P2 was
obtained by PCR using pSP204 as template and the primer pair
5'-CCTAAAGAGCTCTCAGATATATAA and 5'-AGGCAGGTCGACAGTTAACTAACG. The
resulting fragment was cut by SacI and SalI and
cloned into the SacI-XhoI sites of pBSII to
produce pBSIIshk2P1/P2. To construct pBSIIshk2::ura4, a
0.9-kb HindIII fragment of pBSIIShk2P1/P2 (see Fig.
2A) was replaced by a 1.8-kb HindIII fragment of
the ura4 gene released from pBSIIura4.
-Galactosidase Assays--
The filter assay for testing
two-hybrid interactions was performed as described previously (35).
LexA two-hybrid experiments were conducted using LexA DNA binding
domain (LBD) and Gal4 activating domain (GAD) pairs of fusion proteins.
The liquid assay for
-galactosidase activity was performed as
described (33).
-galactosidase activity was calculated using the
following formula: (A420 × 1.7)/(0.0045 × protein concentration × extract volume × time). Protein
concentration is expressed as mg/ml, extract volume in ml, and time as
min.
Quantitative S. pombe Mating Assays--
Mating assays
were performed as described previously (39). Briefly, transformants
were grown on EMM agar for 4 days to induce sexual activity.
Zygotes, asci, and unmated cells within individual clones were then
quantitated by microscopy.
Preparation of Yeast Cell Lysates, Immunoprecipitations,
Immunoblotting, and Myelin Basic Protein (MBP) Kinase
Assays--
Yeast cultures were grown to about 107
cells/ml in either drop-out medium (for S. cerevisiae
strains) or EMM (for S. pombe strains), harvested by
centrifugation, resuspended with yeast lysis buffer (20 mM
HEPES (pH 7.6), 200 mM KCl, 2 mM EGTA, 2 mM EDTA, 10 mM sodium molybdate, 50 mM sodium fluoride, 2 mM sodium pyrophosphate,
1 mM sodium vanadate, 0.1% Nonidet P-40, 10% glycerol, 10 µM E64, 100 µM leupeptin, 1 µM pepstatin, 1 mM phenylmethanesulfonyl fluoride, 2 µg/ml aprotinin), and ground with glass beads. Crude lysates were centrifuged at 16,000 × g for 15 min, and
the supernatant and particulate fractions were aliquoted and quick
frozen in liquid nitrogen prior to storing at
80 °C.
Immunoprecipitations of c-Myc epitope-tagged proteins were performed by
incubating yeast lysate (1 mg of protein) with 5 µl of anti-c-Myc
monoclonal antibody 9E10 (40) ascites on an orbital rotator for 2 h at 4 °C. Immune complexes were washed three times with yeast lysis
buffer and then resuspended in SDS-PAGE sample buffer and boiled for 3 min prior to SDS-PAGE. After electrophoresis, proteins were transferred
to nitrocellulose membranes, and c-Myc-tagged proteins were detected by
immunoblotting using 9E10 ascites (1:2500 dilution).
For detection of Cdc42 proteins, particulate fractions (50 µg of
protein) of SP42N17 cell lysates were boiled in SDS-PAGE sample buffer
prior to SDS-PAGE. After electrophoresis, proteins were transferred to
nitrocellulose membranes, and Cdc42 proteins were detected using
anti-Cdc42-Hs polyclonal antibody sc-87 (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA).
MBP kinase assays were performed by resuspending immune complexes in 25 µl of MBP kinase buffer (50 mM Tris, pH 7.4, 0.1 M NaCl, 10 mM MgCl2, 1 mM MnCl2, 0.1 mg/ml MBP, 10 mM ATP,
0.4 µCi/µl [
-32P]ATP (6000 Ci/mmol)) and
incubating for 20 min at 30 °C. Reactions were stopped by adding
SDS-PAGE sample buffer and boiling for 3 min prior to SDS-PAGE. After
electrophoresis, gels were fixed by exchanging six times with 5%
trichloroacetic acid, 3% sodium pyrophosphate, dried, and exposed to
film.
Filter Binding Assay for Cdc42--
The filter binding assay for
detection of Cdc42-Shk interactions was performed as described (41).
Briefly, His6-tagged Shk1, Shk2, Cdc42-Hs(G12V),
Ha-Ras(G12V), and TrcHis peptide (THP) were purified from bacterial
cell lysates using nickel-agarose following the manufacturer's
instructions (Invitrogen). GST protein was purified from bacterial
lysates using glutathione-agarose and the manufacturer's instructions
(Amersham Pharmacia Biotech). GST and His6-tagged Shk1,
Shk2, and THP were immobilized on nitrocellulose membranes using a
vacuum dot blotter. The membranes were blocked for 2 h at room
temperature in 5% dried milk. 10 µg of Hiss-tagged Cdc42-Hs(G12V) and Ha-Ras(G12V) were each incubated with 10 µCi of
[
-32P]GTP (6000 Ci/mmol) for 10 min at 30 °C in 30 µl of 50 mM Tris, pH 7.5, 5 mM EDTA, and 0.5 mg/ml bovine serum albumin. Nucleotide exchange was stopped on ice by
adding MgCl2 to 10 mM. The nitrocellulose filter was washed twice with buffer A (50 mM Tris-HCl, pH
7.5, 100 mM NaCl, 5 mM MgCl2, 0.1 mM dithiothreitol) and incubated in 10 ml of buffer A
containing 5% skim milk and [
-32P]GTP-bound
Cdc42-Hs(G12V) or Ha-Ras(G12V). After incubation for 5 min at 4 °C,
the membrane was washed three times with cold buffer A containing 5%
dried milk and subjected to autoradiography to visualize bound
Cdc42-Hs(G12V) or Ha-Ras(G12V) protein.
 |
RESULTS |
Cloning and Sequence Analysis of the shk2 Gene--
In a previous
study, we reported on the cloning and characterization of
shk1, a S. pombe gene encoding a homolog of the
S. cerevisiae PAK Ste20 (27). shk1 was cloned
independently by Ottilie et al. (26), who named the gene
pak1. The original shk1 fragment was amplified
from S. pombe genomic DNA using the PCR and degenerate
oligonucleotide primers based on peptide sequences in the catalytic
domain of the S. cerevisiae Ste20 protein kinase (27). The
product resulting from this PCR contained the partial shk1
gene fragment as well as a partial fragment of a related sequence that
we named shk2, for Ste20 homologous
kinase 2. Analysis of several recombinant
plasmids generated from the PCR for STE20-related S. pombe sequences indicated that shk1 and shk2
were represented in roughly equal proportions, with no other sequences
being identified. The full-length shk2 gene was isolated by
using the PCR-derived shk2 fragment as a probe to screen a
S. pombe genomic DNA library. The nucleotide sequence of the
full-length shk2 gene (GenBankTM accession
number U45981) revealed an intronless open reading frame of 1770 base
pairs encoding a predicted protein 589 amino acids in length (Fig.
1A).

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Fig. 1.
Shk2 sequence analysis and alignment with
representative yeast and mammalian PAKs. A, deduced
amino acid sequence of the shk2 gene product. The
shk2 nucleic acid sequence was submitted to
GenBankTM (accession number U45981). B,
predicted structural organization of the Shk2 protein. A predicted
protein kinase catalytic domain composes residues ~295-589. The
protein kinase subdomain I signature sequence
GXGXXG comprises residues 316-321 (GQGASG). The
sequence VAIK, containing the invariant subdomain II lysine, lies at
positions 340-343. The predicted regulatory domain (amino residues 1 to ~294) can be subdivided into three subdomains. A highly conserved
CRIB motif is designated as R2 and occupies residues 129-184. The
sequence N-terminal to the R2/CRIB subdomain is designated as R1, and
the sequence between the R2/CRIB and catalytic domains is designated as
R3. C, amino acid sequence alignments of rat -Pak/Pak1
(Pak1), S. pombe Shk2 and Shk1, and S. cerevisiae
Cla4. Identical amino acid residues are indicated by black
boxes. Cdc42/Rac1-binding domains (R2/CRIB) are highlighted
by the outlined shaded box.
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The structural organization of the predicted Shk2 protein is similar to
that of previously described PAKs (Fig. 1B). The N-terminal half of Shk2 (amino acid residues 1 to ~294) comprises a presumptive regulatory domain containing a potential Cdc42/Rac interactive binding
(CRIB) (41) sequence (amino acid residues 129-184), while the
C-terminal half of Shk2 (residues ~295-589) contains a predicted
protein kinase catalytic domain. We have designated the CRIB sequence
as regulatory subdomain 2, or R2; the regulatory sequence N-terminal to
the CRIB domain (residues 1-128) as R1; and the domain between the
R2/CRIB and catalytic domains (residues 185-294) as R3 (Fig.
1B). Shk2 is most closely related in structure to the
S. cerevisiae PAKs Cla4 (45% identity) and Skm1 (44%
identity); it exhibits a lesser degree of homology to Shk1 (38%
identity), Ste20 (39% identity), and mammalian
-Pak/Pak1 (41%
identity). An alignment of Shk2 with S. pombe Shk1, S. cerevisiae Cla4, and mammalian
-Pak/Pak1 is shown in Fig.
1C. The catalytic domain of Shk2 is highly homologous to
other yeast and mammalian PAKs (52-60% identity). The R1 subdomain of
Shk2 exhibits greatest homology to the corresponding domains of Shk1
(28% identity), Skm1 (28% identity), and Cla4 (24% homology). The
subdomains of Cla4 and Skm1 corresponding to the Shk2 R1 domain were
shown by others to exhibit homology with the pleckstrin homology domain consensus sequence (24). Shk2 exhibits a degree of homology with the
pleckstrin homology domain consensus sequence similar to that of Skm1
and Cla4, although the extent of this homology is greatly dependent on
the parameters used to generate the alignments (data not shown). By
contrast, the Shk1 R1 subdomain lacks any discernible homology to
either the corresponding domains of Cla4 and Skm1 or to the pleckstrin
homology consensus sequence, despite the fact that it exhibits marked
homology with the Shk2 R1 domain. The R2/CRIB and catalytic domains of
Shk2 exhibit greatest similarity to the corresponding domains of Skm1
and Cla4 (56 and 52% identity, respectively). The R3 subdomain of Shk2
exhibits little homology to the corresponding domains of any other
previously described PAK.
shk2 Is a Nonessential Gene--
To examine the function of Shk2
in S. pombe, a disruption of the shk2 gene was
made by replacing the majority of the shk2 protein coding
sequence with the ura4 gene (see Fig.
2A and "Experimental Procedures"). The shk2::ura4 DNA fragment was
used to transform the wild type S. pombe diploid strain
SP870D. Two independent shk2+/shk2::ura4 diploids
were sporulated, and asci containing four spores were dissected for
tetrad analysis. Most asci produced two viable Ura+ spores
and two viable Ura
spores. Southern blotting was used to
confirm that cells derived from Ura+ spores contained a
disrupted copy of shk2 and lacked the wild type
shk2 gene (data not shown). These results demonstrated that shk2 is not an essential gene. shk2-deleted cells
exhibited no obvious growth defects at either 30 or 36 °C (Fig.
2B), and microscopic analysis revealed that they are
indistinguishable from wild type S. pombe cells in
morphology (Fig. 2, C and D). By contrast,
S. pombe shk1 null mutants are inviable and spheroidal in
shape (26, 27). We also determined that shk2 null cells
mated with about the same efficiency as wild type cells (data not
shown), indicating that Shk2 is dispensable for mating in S. pombe.

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Fig. 2.
Analysis of the shk2 null
mutant. A, map of the shk2 gene showing the
fragment deleted by ura4 in construction of the
shk2::ura4 mutant strain, SPSHK2U (see
"Experimental Procedures"). The bottom bar
corresponds to the sequence encoding the Shk2 protein (Shk2p),
subdivided into regulatory subdomains R1, R2, and R3, and the catalytic
domain. B, the shk2 null mutant grows normally at
30 and 36 °C. S. pombe wild type (SP66) cells
(left side of panel) and
shk2::ura4 (SPSHK2U) cells (right
side of panel) were streaked onto YEA as
described under "Experimental Procedures" and grown at either
30 °C (top of panel) or 36 °C
(bottom of panel) for 3 days. C and
D, photomicrographs of wild type (C) and
shk2::ura4 (D) S. pombe
cells. shk2::ura4 cells are indistinguishable from
wild type cells in morphology.
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Shk2 Overexpression Restores Viability and Normal Morphology to the
shk1 Deletion Mutant--
Having observed that shk2-deleted
cells exhibit no obvious phenotypic defects, we asked whether Shk1 and
Shk2 might be partially overlapping in function by determining whether
overexpression of shk2 could suppress the shk1
null mutation. To do this, we constructed a LEU2-based
plasmid, pREP1Shk2, for overexpressing shk2 from the
thiamine-repressible nmt1 promoter (see "Experimental Procedures"). A
shk1+/shk1::ura4 diploid
strain was transformed with pREP1Shk2, the control plasmid pREP1,
pWH5Shk1, a LEU2-based plasmid carrying the genomic
shk1 sequence, and pREP1Shk2(K343R), which expresses a
mutant Shk2 protein in which the invariant protein kinase subdomain II
lysine is substituted by arginine. Transformants were induced to
sporulate, and then spores were scored for viability. Viable shk1::ura4 spores were recovered from pWH5Shk1-
and pREP1Shk2-transformed cells, but not from pREP1 or
pREP1Shk2(K343R)-transformed cells. This result suggested that
overexpression of functional shk2 can restore viability to
shk1-deleted cells. Microscopic analysis of
pREP1Shk2-transformed shk1::ura4 cells indicated
that they are similar to wild type cells in morphology (Fig.
3, top panels). When grown in thiamine-containing medium, pREP1Shk2-transformed shk1::ura4 cells became spheroidal in morphology
and growth-inhibited, whereas pWH5Shk1-transformed
shk1::ura4 cells remained elongated (Fig. 3,
bottom panels). These results demonstrate that
shk2 can act as a high dosage suppressor of the viability
and morphology defects of the shk1 null mutant.

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Fig. 3.
Overexpression of shk2 restores
viability and wild type morphology to the shk1 null
mutant. The
shk1+/shk1::ura4 diploid
strain SP206U was transformed with either a plasmid harboring a genomic
copy of the shk1 gene (pWH5Shk1), a plasmid for
thiamine-repressible overexpression of shk2 (pREP1Shk2), or
the control plasmid pREP1. Transformants were sporulated, and then
random spore analysis was performed by micromanipulation. Viable
Ura+ spores were recovered only from pWH5Shk1- and
pREP1Shk2-transformed cells. pWH5Shk1- (left side
of panel) and pREP1Shk2- (right side
of panel) transformed shk1::ura4 cells
were grown in the absence (top of panel) or
presence (bottom of panel) of thiamine in EMM
medium. pREP1Shk2 restored elongate morphology to the
shk1::ura4 mutant (top,
right). Incubation of the pREP1Shk2-transformed
shk1::ura4 mutant in thiamine (15 µM) resulted in reversion to the spheroidal morphology
characteristic of the shk1 null phenotype
(bottom, right).
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Genetic and Biochemical Evidence for Interaction between Shk2 and
Cdc42--
Previously characterized PAKs have been shown to bind Cdc42
and Rac GTPases but not other small GTPases, such as Ras and Rho. The
two-hybrid assay was used to determine whether Shk2, like other known
PAKs, interacts with Cdc42 or Rac or with proteins besides Cdc42 that
are involved in Cdc42-dependent signaling in S. pombe. A summary of the two-hybrid interactions tested by the
-galactosidase filter assay are shown in Table
I, and results of a smaller subset of
quantitative liquid
-galactosidase assays are shown in Table
II. Shk2 was found to form detectable
two-hybrid complexes both with Cdc42 and, to a much lesser degree, with
Rac1, but not with Ras1 or RhoG (Tables I and II). Shk2 interacted in
the two-hybrid assay with both wild type and activated (G12V) forms of
Cdc42 but not with a dominant negative mutant of Cdc42, Cdc42(T17N)
(Tables I and II). The T17N mutation is analogous to mutations
identified at the corresponding positions of yeast and mammalian Ras
proteins that result in defective guanine nucleotide exchange (42-44).
The inability of Cdc42(T17N) and Shk2 to interact in the two-hybrid
assay suggests that the Cdc42-Shk2 interaction is
GTP-dependent.
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Table I
Analysis of Shk2 two-hybrid interactions
Values represent the presence of transformed colonies that expressed
detectable -galactosidase activity (+) or did not ( ). Activating
domain fusions were to the activating domain of S. cerevisiae Gal4 (GAD). GBD-GAD combinations were expressed in the
Gal4 two-hybrid tester strains HF7c and/or SF7526. LBD-GAD combinations
were tested in the LexA tester strain, L40. At least eight independent
transformants were tested for each determination.
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Table II
Quantitative -galactosidase assays for representative Shk2
two-hybrid tests
Representative yeast transformants tested by the -galactosidase
filter assay (Table I) were also tested by quantitative liquid
-galactosidase assays. Lamin, Cdc42, Cdc42(T17N), Rac1, Ras1, and
RhoG were tested as LBD fusion proteins in the two-hybrid tester strain
L40. Snf4 and Skb1 were tested as GBD fusion proteins in SFY526. The
GBD-Skb1 fusion protein tested lacks the first 23 amino acids of the
full-length Skb1 protein and was used because the full-length Skb1 GBD
fusion protein weakly autoactivates in the two-hybrid assay.
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The Shk2-Cdc42 interaction was confirmed biochemically by a filter
binding assay (41) using bacterially expressed recombinant proteins.
His6-tagged Shk1 and Shk2 proteins, as well as
His6-tagged pTrcHis peptide (His6-THP) and GST
protein, were immobilized on nitrocellulose membranes using a dot
blot apparatus. After blocking, the membranes were incubated with
His6-Cdc42-Hs(G12V)·[
-32P]GTP or
His6-Ha-Ras(G12V)·[
-32P]GTP and then
washed and exposed to film. As shown in Fig.
4, His6-Cdc42-Hs(G12V)·[
-32P]GTP bound to
both His6-Shk1 and His6-Shk2, but not to
His6-THP or GST. This result demonstrates that both Shk1
and Shk2 bind directly to Cdc42. Binding was not detected between
His6-Ha-Ras(G12V)·[
-32P]GTP and either
His6-Shk1 or His6-Shk2.

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Fig. 4.
Direct binding of Cdc42 to Shk1 and Shk2
proteins. Approximately 100 ng of His6-tagged Shk1,
Shk2, and THP and approximately 10 µg of GST were each immobilized
onto nitrocellulose filters using a vacuum dot blotter and incubated
with [ -32P]GTP·Cdc42-Hs(G12V) (top) or
[ -32P]GTP·Ha-Ras(G12V) (bottom) as
described under "Experimental Procedures." After washing, bound
GTPase was visualized by autoradiography.
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Shk2 also formed a two-hybrid complex with Skb1 (Tables I and II), a
protein that we previously identified by a two-hybrid screen for
Shk1-interacting proteins and that we showed by genetic analyses to
positively modulate Shk1 function (28). However, we were unable to
recapitulate the Shk2-Skb1 interaction in vitro by
coprecipitation experiments using recombinant bacterially expressed proteins.
Having determined that Cdc42 and Shk2 interact physically, we examined
whether the two proteins interact functionally in S. pombe.
We first examined whether cooverexpression of shk2 and cdc42 affects cell growth differently than overexpression of
each gene separately. cdc42 was overexpressed from a plasmid
containing the thiamine-repressible nmt1 promoter
(pREP1Cdc42), while shk2 was overexpressed from a plasmid
containing the constitutive adh1 promoter (pAAUCMShk2). The
wild type S. pombe strain CHP428 was cotransformed with
pREP1Cdc42, pAAUCMShk2, and/or control plasmids, and transformants were
assayed for growth in the presence and absence of thiamine. As shown in
Fig. 5A, cells transformed
with pREP1Cdc42 or pAAUCMShk2 grew equally well on medium with or
without thiamine. However, cells cotransformed with pREP1Cdc42 and
pAAUCMShk2 produced normal size colonies only when grown on thiamine.
When grown in the absence of thiamine, cells cotransformed with
pREP1Cdc42 and pAAUCMShk2 produced only microcolonies, indicating that
cooverexpression of cdc42 and shk2 is toxic. We
also examined cells overexpressing cdc42 and/or
shk2 microscopically. Cells overexpressing cdc42 (Fig. 5C) were virtually indistinguishable from wild type
cells (Fig. 5B) in morphology, while a substantial
percentage of cells overexpressing shk2 alone were somewhat
distorted in shape (Fig. 5D). However, cells
cooverexpressing both cdc42 and shk2 were drastically distorted in morphology and much larger than wild type
cells or cells overexpressing cdc42 or shk2 alone
(Fig. 5E). These results provide evidence for functional
interaction between Cdc42 and Shk2 in regulating cell morphology in
S. pombe.

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Fig. 5.
Evidence for functional interaction between
Cdc42 and Shk2 in S. pombe. A, wild type
(CHP428) S. pombe cells were transformed with pREP1Cdc42
(for overexpression of cdc42), pAAUCMShk2 (for
overexpression of shk2), and/or the control plasmid pREP1 or
pAAUCM and plated onto EMM medium containing 15 µM
thiamine for repression of cdc42 expression. Transformants
were then streaked onto EMM with (left) or without
(right) thiamine (15 µM) and grown at 30 °C
for 4 days. Overexpression of cdc42 or shk2 alone
did not affect the growth rate of cells. However, cooverexpression of
both genes was toxic, suggesting that the Cdc42 and Shk2 proteins
functionally interact in vivo. B-E, the same transformants
shown in A were grown in liquid EMM with thiamine, washed,
and grown overnight in EMM without thiamine and observed
microscopically. B, cells cotransformed with pREP1 and
pAAUCM; C, cells cotransformed with pREP1Cdc42 and pAAUCM;
D, cells cotransformed with pREP1 and pAAUCMShk2 (note
aberrant, bulbous shape of cells indicated by arrows);
E, cells cotransformed with pREP1Cdc42 and pAAUCMShk2 (note
that most cells are highly aberrant in shape).
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Overexpression of shk2 Restores Elongate Morphology to the ras1
Deletion Mutant--
Given the physical and functional interactions
observed previously among Ras1, Cdc42, and Shk1 (25, 27, 28), we asked whether shk2 overexpression might suppress the morphological
defect resulting from deletion of the ras1 gene.
ras1 null mutants are spheroidal in shape (Fig.
6A). When transformed with a
plasmid harboring the ras1 gene, elongate morphology is
restored to the ras1 mutant (Fig. 6B). As shown
in Fig. 6, C and D, overexpression of
shk2 restores elongate morphology to the ras1
deletion mutant, providing further evidence that Shk2, like Shk1 and
Cdc42, participates in the Ras1-dependent morphological
control pathway in S. pombe.

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Fig. 6.
Overexpression of shk2 restores
elongate morphology to the ras1 null mutant. A
S. pombe ras1 deletion mutant (SPRN1) was transformed with
the control plasmids pAAUCM and pREP1 (A); pAUR, which
carries the wild type ras1 gene (B); or pREP1Shk2
for overexpression of Shk2 (C and D).
Overexpression of shk2 restored elongate morphology to
ras1-deleted cells. Transformants were patched onto EMM and
grown overnight at 30 °C prior to photomicroscopy.
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Overexpression of Byr2 Suppresses the Mating Defect of an S. pombe
Mutant Partially Defective for Shk1 Function--
A role for Shk1 in
mediating sexual responses in S. pombe was suggested from
previous studies in which it was shown that overexpression of dominant
negative mutants of shk1 impaired sexual responses (26, 27).
To further explore this aspect of Shk1 function, we tested whether
pREP1Shk2-transformed shk1::ura4 cells exhibit a
defect in mating. As shown in Table III,
the mating efficiency of pREP1Shk2-transformed
shk1::ura4 cells was nearly 30-fold lower than
that of shk1+ cells. Two conclusions can be
drawn from this experiment: (i) the shk1 gene is required
for normal mating in S. pombe, and (ii) overexpression of
shk2, while capable of restoring both viability and normal
morphology to the shk1 deletion mutant, does not fully restore mating functions to the shk1 deletion mutant.
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Table III
shk1-deleted cells overexpressing shk2 exhibit a mating defect that
is partially suppressed by overexpression of byr2
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Other investigators have demonstrated by genetic analyses that the
S. cerevisiae PAK Ste20 functions upstream of the protein kinase Ste11 (19), a MAPK kinase kinase homolog required for pheromone-induced signal transduction (46). Furthermore, it has been
shown that Ste20 immune complexes can phosphorylate Ste11 protein
in vitro, implicating Ste11 as a potential Ste20 substrate (47). The fission yeast protein kinase Byr2 is a structural and
functional homolog of S. cerevisiae Ste11 (29). Because shk1 is an essential gene, it had been difficult to test for
a genetic interaction between shk1 and byr2.
However, the above described finding that pREP1Shk2-transformed
shk1::ura4 cells exhibit a significant mating
defect made this test possible. Indeed, we found that
pREP1Shk2-transformed shk1::ura4 cells that
overexpressed byr2 mated with about 16-fold higher
efficiency than shk1
pREP1Shk2 cells transformed with a
control plasmid and with only about 2-fold lower efficiency than
shk1+ cells (Table III). Thus, byr2
is a high dosage suppressor of the mating defect resulting from partial
loss of shk1. These results provide the first direct genetic
evidence for interaction between PAKs and the MAPK module/cascade
required for mating response in S. pombe.
Next, we examined the genetic interaction between cdc42 and
shk2 with respect to mating. We showed previously that
S. pombe cells overexpressing the dominant negative
cdc42(T17N) mutant gene exhibit a marked mating defect,
which can be partially suppressed by overexpression of Shk1 (27). As
shown in Table IV, cells harboring an
integrated copy of an adh1-cdc42(T17N) fusion gene and
transformed with a high copy shk2 plasmid mated with about 4-fold greater efficiency than cdc42(TN17)-expressing cells
transformed with a control plasmid. This result suggests that Shk2,
like Shk1, participates in the Ras and Cdc42-dependent
mating response pathway of S. pombe. To rule out the trivial
explanation that differences in the level of expression of the
cdc42(T17N) gene are responsible for the differences in
mating efficiency, lysates were prepared from the various
cdc42(T17N) strains tested and subjected to immunoblot analysis. As shown in Fig. 7, comparable
levels of Cdc42 protein were detected in all strains, except the one
transformed with a high copy plasmid for overexpression of wild type
Cdc42, from which, as expected, a substantially greater amount of Cdc42
protein was detected.
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Table IV
Overexpression of shk2 results in partial suppression of the mating
defect caused by expression of the cdc42T17N mutant gene
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Fig. 7.
Level of Cdc42(T17N) in S. pombe
transformants shown in Table IV. Cell lysates were prepared
from S. pombe strains listed in Table IV as described under
"Experimental Procedures." Particulate fractions of the cell
lysates (50 µg of protein) were resolved by SDS-PAGE and transferred
to nitrocellulose, and immunoblots were performed using -Cdc42-Hs
monoclonal antibody for detection of Cdc42. The wild type S. pombe strain SP66, which carries only the endogenous
cdc42 gene, was used as a control to show that the Cdc42
protein detected in SP42N17 cultures was predominantly Cdc42(T17N),
except for the pREP1Cdc42-transformed culture, which overexpresses both
wild type and T17N forms of Cdc42.
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Shk2 Cannot Substitute for Cla4 in S. cerevisiae--
As already
noted, Shk2 is most similar in structure to the S. cerevisiae PAK Cla4. This prompted us to examine whether Shk2 and
Cla4 are functionally related. S. cerevisiae cla4 deletion mutants are morphologically aberrant but competent for mating (23).
S. cerevisiae ste20 mutants are sterile but normal in morphology (19, 20). Deletion of both ste20 and
cla4 is lethal (23). To determine whether shk2
can substitute for cla4 or ste20, we constructed
the plasmid pAUD6Shk2 for overexpressing shk2 from the
strong S. cerevisiae ADH1 promoter. Two different
cla4 mutants, one a deletion mutant and the other a
temperature-sensitive mutant, were transformed with either pAUD6Shk2, a
plasmid containing the cla4 gene, or a control plasmid.
pAUD6Shk2 failed to restore normal morphology to either the deletion
(Fig. 8C) or
temperature-sensitive (data not shown) mutant of cla4, nor
did it restore viability to a cla4 ste20 double mutant (data
not shown). pAUD6Shk2 also failed to restore mating ability to a
ste20 deletion mutant (data not shown). pAUD6Shk2 expresses
a c-Myc epitope-tagged Shk2 protein (CMShk2). To confirm that CMShk2
was expressed in S. cerevisiae, lysates were prepared from
the cla4
mutant transformed with either pAUD6 or
pAUD6Shk2 and c-Myc-tagged proteins precipitated using monoclonal
antibody 9E10. Immunoprecipitates were then subjected to Western
blotting or assayed for MBP kinase activity. As shown in Fig.
8D, an approximately 68-kDa c-Myc-tagged protein (the predicted size for CMShk2) was detected in c-Myc immune complexes from
lysates of cells expressing pAUD6Shk2 but not pAUD6, indicating that
CMShk2 protein is expressed from the pAUD6Shk2 plasmid. Furthermore, MBP kinase activity was detected in pAUD6Shk2, but not pAUD6, immunoprecipitates (Fig. 8D), indicating that CMShk2 is
catalytically active. We conclude that the Shk2 and Cla4, despite their
similarity in structure, are not functionally interchangeable.

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Fig. 8.
Shk2 cannot substitute for Cla4 in S. cerevisiae. A cla4 mutant, MJY8, was
transformed with FD44 (carries the cla4 gene)
(A), pAUD6 (B), or pAUD6Shk2, for expression of
c-Myc epitope-tagged Shk2 from the strong ADH1 promoter
(C). Note that Shk2 expression could not restore normal
morphology to the cla4 mutant. D, pAUD6- and
pAUD6Shk2-transformed cla4 cells were lysed, and
CM-tagged proteins were immunoprecipitated using -c-Myc monoclonal
antibody 9E10. Immune complexes were either subjected to immunoblot
analysis using 9E10 antibody for detection of c-Myc-tagged proteins
(left side of panel) or assayed for
MBP kinase activity (right side of
panel). MBP kinase activity was detected only in c-Myc
immune complexes isolated from pAUD6Shk2-transformed cells.
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DISCUSSION |
In this report, we have described the cloning and characterization
of shk2, a gene encoding a novel PAK in the fission yeast, S. pombe. Our results suggest that Shk2, like the previously
described fission yeast PAK Shk1 (26-28), participates in Ras- and
Cdc42-dependent morphological control and mating response
pathways. While shk2 deletion mutants exhibit no obvious
defects in growth, morphology, or mating, overexpression of
shk2 restores viability and elongate morphology to the
S. pombe shk1 null mutant. Overexpression of shk2
does not restore full mating competence to the shk1 null mutant. These results suggest that Shk1 and Shk2 may be partially redundant, with Shk1 being the dominant protein in function. We cannot
rule out the possibility that an additional Shk2-related PAK exists in
S. pombe. However, from our PCR-based cloning approach, which utilized degenerate oligonucleotide primers based on the S. cerevisiae Ste20 protein sequence, we identified only the
shk1 and shk2 genes.
Results of previous studies by us and others provided evidence for
involvement of Shk1 in the S. pombe mating response pathway. First, overexpression of a catalytically defective mutant of Shk1 inhibited mating of S. pombe cells (26). Second,
overexpression of the N-terminal regulatory domain of Shk1 attenuated
the hypersexual response of S. pombe cells expressing the
dominant activated ras1(G17V) mutant (27). Finally,
overexpression of shk1 partially bypassed the mating defect
of S. pombe cells expressing the dominant inhibitory cdc42(T17N) allele (27). In this report, we have shown that S. pombe cells deleted of shk1 but overexpressing
shk2 exhibit a significant mating defect. Furthermore, we
have shown that this defect can be largely suppressed by overexpression
of the MAPK kinase kinase Byr2 and, additionally, that overexpression
of shk2 partially bypasses the mating defect of S. pombe cells expressing the dominant inhibitory
cdc42(T17N) allele. Our results corroborate a role for Shk1
in the S. pombe mating response pathway, which was suggested
in previous studies (26, 27). In addition, our results suggest that,
with regard to mating responses, Byr2 acts downstream from the Shk
kinases. Our results provide the first direct genetic evidence linking
PAKs to regulation of a MAPK module S. pombe. A homolog of
Byr2, Ste11, has been similarly implicated as a downstream target for
the Shk1 homolog Ste20 in S. cerevisiae (19, 47).
Although our results suggest that Shk1 function is dominant over that
of Shk2, it is possible that Shk2 is required for cellular functions
for which we have not assayed. Interestingly, the R3 subdomains of Shk1
and Shk2 lack any discernible structural homology. It is possible
that these domains might specify unique molecular functions for each
kinase. Further insights into Shk2 function and perhaps PAK functions
in general may be gained by conducting genetic screens for S. pombe mutants that are synthetically lethal with the
shk2 null mutation.
In both S. pombe and S. cerevisiae, PAKs are
required not only for mating responses but also for essential cellular
functions unrelated to mating. The specific nature of these essential
functions has yet to be defined in either yeast. S. cerevisiae possesses three PAK-encoding genes. Two of these,
STE20 and CLA4, are partially overlapping in
function (23). The third, Skm1, is completely dispensable (24).
Deletion of STE20 results in sterility (19, 20), while
deletion of CLA4 results in aberrant morphology (23). However, deletion of both CLA4 and STE20 genes is
lethal (23). PAK wiring is clearly different in S. pombe, in
which a single PAK, Shk1, has essential functions not shared by other
PAKs (26, 27). These differences are not surprising, given that fact
that S. cerevisiae and S. pombe are a half
billion years diverged in evolution (45). It remains to be determined
whether the essential functions of PAKs in S. pombe and
S. cerevisiae are conserved in higher organisms or, for that
matter, between the two distantly related yeasts.
We thank F. Cvrckova for plasmids and
strains; Jonathan Chernoff for plasmids and for communicating
unpublished results; Anjana Kundu and Erin Mooney for technical
assistance; and Jenny Henkel and Anthony Polverino for comments on the
manuscript. We especially thank Michael Wigler (Cold Spring Harbor
Laboratory), in whose laboratory this project was initiated.