EUKARYOTIC CELL, Nov. 2005, p. 1794–1800
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 4, No. 11
Cyclic AMP-Independent Regulation of Protein Kinase A Substrate
Phosphorylation by Kelch Repeat Proteins
Ailan Lu and Jeanne P. Hirsch*
Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029
Received 27 July 2005/Accepted 27 August 2005
Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae is regulated by the kelch repeat-
containing proteins Gpb1p and Gpb2p, which act downstream of the G protein ?-subunit Gpa2p. Here we show
that deletion of GPB1 and GPB2 causes increased haploid invasive growth in cells containing any one of the
three protein kinase A (PKA) catalytic subunits, suggesting that Gpb1p and Gpb2p are able to inhibit each of
these kinases. Cells containing gpb1? gpb2? mutations also display increased phosphorylation of the PKA
substrates Sfl1p and Msn2p, indicating that Gpb1p and Gpb2p are negative regulators of PKA substrate
phosphorylation. Stimulation of PKA-dependent signaling by gpb1? gpb2? mutations occurs in cells that lack
both adenylyl cyclase and the high-affinity cyclic AMP (cAMP) phosphodiesterase. This effect is also seen in
cells that lack the low-affinity cAMP phosphodiesterase. Given that these three enzymes control the synthesis
and degradation of cAMP, these results indicate that the effect of Gpb1p and Gpb2p on PKA substrate
phosphorylation does not occur by regulating the intracellular cAMP concentration. These findings suggest
that Gpb1p and Gpb2p mediate their effects on the cAMP/PKA signaling pathway either by inhibiting the
activity of PKA in a cAMP-independent manner or by activating phosphatases that act on PKA substrates.
Activation of protein kinase A (PKA) by cyclic AMP
(cAMP) is a conserved feature of eukaryotic signaling systems.
In the yeast Saccharomyces cerevisiae, PKA regulates cell
growth and morphology in response to nutrient and stress
signals (31). Yeast PKA functions downstream of the mono-
meric G proteins Ras1p and Ras2p, which are activated by an
unknown mechanism (3). Ras1p and Ras2p stimulate adenylyl
cyclase to produce cAMP (8, 9, 35), which activates PKA
through the well-established mechanism of binding to the reg-
ulatory subunit of PKA and releasing active catalytic subunits
(33). There are three isoforms of the catalytic subunit, called
Tpk1p, Tpk2p, and Tpk3p, which are encoded by different
genes (34). Deletion of the three genes encoding the catalytic
subunits is lethal, but any one of the three genes can provide
the essential function of PKA. There is one regulatory subunit,
called Bcy1p, which is thought to bind to each of the three
catalytic subunits (33). Activation of PKA results in phosphor-
ylation of substrates that are involved in intermediary metab-
olism, stress responses, and filamentous growth (10, 31).
Yeast PKA also appears to function downstream of the G
protein ?-subunit Gpa2p (5, 17, 21). Gpa2p is coupled to a cell
surface receptor, called Gpr1p, that contains seven membrane-
spanning domains, a structure that is found in other G protein-
coupled receptors (16, 36, 37). The effects conferred by a
deletion of GPR1 are suppressed by constitutive activation of
Gpa2p, as would be expected for a G protein that acts down-
stream of its coupled receptor (22, 30). In contrast to the
situation with Ras proteins, little is known about the way in
which the Gpa2p pathway regulates cAMP/PKA signaling. The
effect of Gpa2p on PKA responses has recently been shown to
involve the kelch repeat-containing proteins Gpb1p and
Gpb2p (also called Krh2p and Krh1p, respectively) (1, 14).
Gpb1p and Gpb2p physically interact with Gpa2p, suggesting
that they function in the signaling pathway. Deletion of GPB1
and GPB2 results in phenotypes that are characteristic of in-
creased PKA signaling, indicating that Gpb1p and Gpb2p in-
hibit a component of the cAMP/PKA pathway. Gpb1p and
Gpb2p appear to transmit the signal generated by Gpa2p to
downstream components, because gpb1? gpb2? mutations sub-
stantially suppress the defects in pseudohyphal and invasive
growth conferred by a gpa2? mutation. Gpb1p and Gpb2p
appear to act upstream of PKA, because the increase in sig-
naling conferred by gpb1? gpb2? mutations is substantially
blocked by deletion of TPK2, which encodes a PKA catalytic
isoform. These results are consistent with a model in which
activation of Gpa2p relieves the inhibition of PKA that is
either directly or indirectly mediated by Gpb1p and Gpb2p,
resulting in increased PKA activity.
Here we show that Gpb1p and Gpb2p affect signaling by
altering the level of phosphorylation of PKA substrates. How-
ever, the function of Gpb1p and Gpb2p does not require
changes in the intracellular concentration of cAMP. These
results imply that the signaling process initiated by the Gpa2p
?-subunit does not constitute a linear pathway that acts solely
by stimulating the production of cAMP but rather has at least
one component that acts downstream of adenylyl cyclase.
MATERIALS AND METHODS
Plasmid construction. To construct a HIS3 disruption of TPK1, a 1.7-kb XbaI
fragment from pHIS3-Bs.1 was cloned into the XbaI sites of plasmid pTPK1-
50.1, which consists of vector YCp50 with a 1.7-kb insert containing the TPK1
gene, to produce plasmid ptpk1-1::HIS3. To construct a HIS3 disruption of
TPK2, a 1.7-kb EcoRI-NotI fragment from pHIS3-Bs.2 was cloned into the
EcoRI-NotI sites of plasmid pTPK2N-Bs.1 to produce plasmid ptpk2-2::HIS3.
Plasmid pTPK2N-Bs.1 consists of a 2.0-kb BglII fragment containing the TPK2
gene cloned into the BamHI site of pBluescript, in which a NotI site was inserted
* Corresponding author. Mailing address: Department of Pharma-
cology and Biological Chemistry, Mount Sinai School of Medicine, Box
1603, 1 Gustave L. Levy Place, New York, NY 10029. Phone: (212)
241-0224. Fax: (212) 996-7214. E-mail: firstname.lastname@example.org.
before the stop codon by site-directed mutagenesis. To construct a HIS3 disrup-
tion of TPK3, a 1.7-kb EcoRI-XhoI fragment from pHIS3-Bs.1 was cloned into
the EcoRI-XhoI sites of a plasmid with a 2.7-kb HindIII fragment containing the
TPK3 gene to produce plasmid ptpk3-3::HIS3. Plasmid pCYR1.Bs contains a
4.5-kb KpnI-XbaI fragment from pCYR1 cloned into the KpnI-XbaI sites of
pBluescript. To construct a URA3 disruption of CYR1, a 1.0-kb HindIII-NsiI
fragment from pURA3.Bs (1) was cloned into the HindIII-NsiI sites of plasmid
pCYR1.Bs to produce pcyr1::URA3.Bs.
Strain construction and media. Strains used in the present study are listed
in Table 1. Construction of strains containing the pde2::HIS3 allele (36) and
the gpb1::URA3 and gpb2::HIS3 alleles (1) was described previously. The
tpk1-1::HIS3 allele was made by transformation of cells with the 2.4-kb EcoRV-
AatII fragment from plasmid ptpk1-1::HIS3. The tpk2-2::HIS3 allele was made
by transformation of cells with the 2.6-kb SacI-SalI fragment from plasmid
ptpk2-2::HIS3. The tpk3-3::HIS3 allele was made by transformation of cells with
the 2.7-kb NcoI-EcoRV fragment from plasmid ptpk3-3::HIS3. The gpb2::TRP1
allele was made by transformation of a gpb2::HIS3 strain with a 3.8-kb SmaI/
XhoI fragment from marker swap plasmid pHT6 (7). The cyr1::URA3 allele was
made by transformation of cells with the 2.9-kb KpnI-XbaI fragment from plas-
mid pcyr1::URA3.Bs. The cyr1::TRP1 allele was made by transformation of cells
with the 3.7-kb SmaI fragment from marker swap plasmid pUT11 (7). The
pde1::kanMX allele was made by transformation of cells with a pde1::kanMX
cassette amplified from genomic DNA of a strain containing this allele (Open
Biosystems). Strain constructions involving transformations were confirmed by
Southern blot or PCR.
Strains were grown on YEPD (yeast extract-peptone-dextrose) with 2% glu-
cose, and strains under selection were grown on synthetic dropout media, as
described previously (13).
Yeast methods and protein assays. Invasive growth assays were performed
according to the method of Kuchin et al. (18). Yeast transformations were
performed by the lithium acetate method according to standard procedures (13).
Cell lysates for immunoblots were prepared from cells growing in log phase
(optical density at 600 nm ? 0.5 to 0.85). Cells were washed once with cold water
and resuspended in lysis buffer, made essentially as described previously (27),
with the exception that 25 mM ?-glycerophosphate was substituted by 15 mM
p-nitrophenyl phosphate. Cells were disrupted by shaking in the presence of glass
beads for 5 min at 4°C and cleared by centrifugation for 3 min. For immunoblots,
proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) and transferred to nitrocellulose. Blots were probed with
the following antibodies: anti-myc 9E10 at a dilution of 1/1,000, anti-phospho-
CREB (Ser133) (Cell Signaling) at a dilution of 1/1,000, and anti-green fluores-
cent protein (GFP) serum (Molecular Probes) at a dilution of 1/1,000. Secondary
antibodies were donkey anti-rabbit immunoglobulin conjugated to horseradish
peroxidase (Amersham) at a dilution of 1/10,000 and goat anti-mouse immuno-
globulin conjugated to horseradish peroxidase (Amersham) at a dilution of
1/10,000. Immune complexes were detected with an ECL or an ECL Plus kit
Heat shock assays and yeast RNA extraction were performed as described
previously (36) except that cells were incubated for 15 min at 50°C. Northern
blots were performed as described previously (1).
Effect of gpb1? gpb2? mutations is not specific to Tpk2p.
Deletion of GPB1 and GPB2 causes an increase in diploid
pseudohyphal growth and haploid invasive growth, phenotypes
that are characteristic of increased activation of the cAMP/
PKA signaling pathway (1, 14). It was therefore of interest to
determine which pathway component is the target of inhibition
by Gpb1p and Gpb2p. In yeast, total PKA activity results from
the combined activities of the three catalytic subunits of
cAMP-dependent kinase, Tpk1p, Tpk2p, and Tpk3p (34). To
test the effects of gpb1? gpb2? mutations on the individual
PKA catalytic isoforms, phenotypes associated with filamen-
tous growth were investigated in strains in which two of the
three TPK genes were deleted. Previous results have shown
that TPK2 has a positive effect on filamentous growth and that
TPK1 and TPK3 have negative effects on filamentous growth
when TPK2 is present (26, 28). However, in cells containing
either TPK1 only or TPK3 only, a small but reproducible in-
crease in invasive growth was observed in gpb1? gpb2? cells
compared to wild-type cells (Fig. 1A). The most straightfor-
ward interpretation of these results is that TPK1 and TPK3
have a modest positive effect on invasive growth in the absence
of TPK2 and that GPB1 and GPB2 inhibit that effect. Expres-
sion of FLO11, which encodes a cell surface flocculin that is
required for both pseudohyphal and invasive growth (20), was
undetectable in strains containing either TPK1 only or TPK3
only (Fig. 1B, lanes 3, 4, 7, and 8), indicating that the signal
generated in cells lacking TPK2 is quite low. Cells containing
TPK2 only display very high levels of invasive growth and
FLO11 RNA that were not affected by gpb1? gpb2? mutations
(Fig. 1A and B, lanes 5 and 6). In this strain, the high level of
Tpk2p activity is due to lack of inhibition by Tpk1p and Tpk3p.
However, detectable FLO11 RNA abundance in wild-type cells
depends on TPK2, and gpb1? gpb2? mutations increase the
level of FLO11 RNA in wild-type cells (Fig. 1B, lanes 1 and 2).
These results imply that the presence of GPB1 and GPB2
causes a decrease in Tpk2p activity. In summary, these findings
are consistent with the idea that GPB1 and GPB2 are able to
inhibit each of the PKA catalytic isoforms.
TABLE 1. Strains used in this study
MATa ura3-52 trp1::hisG leu2::hisG his3::hisG
MATa gpb1::URA3 gpb2::HIS3
MATa gpb1::URA3 gpb2::HIS3 pde1::kanMX
MATa gpb1::URA3 gpb2::HIS3 tpk2-2::HIS3
MAT? cyr1::URA3 pde2::HIS3
MAT? cyr1::TRP1 pde2::HIS3
MAT? gpb1::URA3 gpb2::HIS3 cyr1::URA3 pde2::HIS3
MATa gpb1::URA3 gpb2::HIS3 tpk1-1::TRP1 tpk3-3::HIS3
MATa tpk1-1::TRP1 tpk3-3::HIS3
MATa gpb1::URA3 gpb2::HIS3 tpk2-2::TRP1 tpk3-3::HIS3
MATa gpb1::URA3 gpb2::HIS3 tpk1-1::HIS3 tpk2-2::TRP1
MATa tpk2-2::TRP1 tpk3-3::HIS3
MATa tpk1-1::HIS3 tpk2-2::TRP1
S. Palecek and S. Kron
aStrains with a SKY762 background are indicated with an asterisk. SKY762 is derived from ?1278b (19).
VOL. 4, 2005Gpb1p AND Gpb2p CONTROL PKA ACTIVITY1795
PKA activity is increased in gpb1? gpb2? cells. The exper-
iments described above indicate that strains containing gpb1?
gpb2? mutations display phenotypes that are characteristic of
increased activation of PKA. However, these experiments do
not demonstrate whether gpb1? gpb2? mutations affect the
activity of PKA itself. The results are also consistent with the
possibility that gpb1? gpb2? mutations affect the activity of a
related kinase that regulates the same physiological processes
as PKA, such as Sch9p (32). To test directly whether Tpk2p
activity is affected by the presence of Gpb1p and Gpb2p, the
phosphorylation state of a Tpk2p substrate was examined in
different mutant backgrounds.
Expression of FLO11 is regulated by the transcriptional re-
pressor Sfl1p, which is thought to be an in vivo substrate of
Tpk2p based on the following observations. Sfl1p acts down-
stream of Tpk2p because deletion of SFL1 restores FLO11
expression in tpk2? mutant cells (27, 28). Sfl1p binds specifi-
cally to Tpk2p, and in vitro phosphorylation of Sfl1p by Tpk2p
inhibits its binding to the FLO11 promoter (6, 27, 28). Finally,
phosphorylation of Sfl1p in vivo requires the presence of
Tpk2p (27). Phosphorylation of Sfl1p by Tpk2p can be de-
tected as a change in mobility on a gel (6, 27, 28). In wild-type
cells, deletion of GPB1 and GPB2 results in a significant in-
crease in the phosphorylated form of Sfl1p (Fig. 2A, lanes 1
and 2). There was no detectable phosphorylated Sfl1p in
cells containing a tpk2? mutation, and deletion of GPB1 and
GPB2 had no effect in this mutant background (Fig. 2A,
lanes 3 and 4).
One phenotype that is affected by all three forms of PKA is
the response to stress, which is mediated, in part, by the PKA
substrate Msn2p. Msn2p is a stress-activated transcription fac-
tor that is negatively regulated by PKA phosphorylation (11,
29). Msn2p is phosphorylated on serine 620 by PKA, and the
phosphorylated epitope is recognized by an antibody specific
for the phosphorylated form of mammalian CREB (12).
Strains transformed with a construct expressing an Msn2p-
GFP fusion protein were used to assay the level of Msn2p
phosphorylation in different mutant backgrounds by compar-
ing the signal obtained with the anti-phospho-CREB antibody
to that obtained with an anti-GFP antibody. A substantial
increase in the level of Msn2p phosphorylation at serine 620
was observed in cells containing gpb1? gpb2? mutations com-
pared to wild-type cells (Fig. 2B, lanes 1 and 2). A pronounced
effect on Msn2p phosphorylation was also seen in cells
containing an activated allele of RAS2, which is known to
increase PKA activity by stimulation of adenylyl cyclase activity
(Fig. 2B, lanes 3 and 4). These results establish that gpb1?
FIG. 1. PKA activity is increased by gpb1? gpb2? mutations.
(A) The following strains were patched onto YEPD–2.5% agar me-
dium, incubated for 4 days at 25°C, and photographed before (Total
growth) and after (Invasive growth) rubbing the surface of the plate with
a glass rod under a stream of water: wild-type strain SKY762, gpb1?
gpb2? strain HS182-3B, tpk1? tpk2? strain HS234-1C, gpb1? gpb2?
tpk1? tpk2? strain HS232-2B, tpk1? tpk3? strain HS230-15B, gpb1?
gpb2? tpk1? tpk3? strain HS229-11D, tpk2? tpk3? strain HS233-8B,
and gpb1? gpb2? tpk2? tpk3? strain HS231-29A. (B) RNA isolated
from the log-phase cultures of the strains described in panel A was
transferred to nylon membrane, hybridized with a FLO11 probe, and
rehybridized with an ACT1 probe.
FIG. 2. Phosphorylation of in vivo PKA substrates is increased by
gpb1? gpb2? mutations. (A) Extracts made from the following strains
expressing Sfl1p-myc from plasmid pXP181 (27) (provided by X. Pan
and J. Heitman) were analyzed by SDS-PAGE and immunoblotting
with anti-myc antibody 9E10: wild-type strain SKY762 (lane 1), gpb1?
gpb2? strain HS182-3B (lane 2), tpk2? strain HS192-2C (lane 3), and
gpb1? gpb2? tpk2? strain HS196-5A (lane 4). (B) Extracts made from
the following strains expressing Msn2p-GFP fusion protein from plas-
mid pAdh1-Msn2-GFP (12) (provided by C. Schu ¨ller and W. Reiter)
were analyzed by SDS-PAGE and immunoblotting with antibodies to
P-CREB (S133) and GFP: wild-type strain SKY762 (lane 1), gpb1?
gpb2? strain HS182-3B (lane 2), SKY762 carrying vector YCplac33
(lane 3), and SKY762 carrying plasmid YCp50-RAS2ala18val19(lane 4).
1796LU AND HIRSCHEUKARYOT. CELL
gpb2? mutations cause an increase in phosphorylation of PKA
substrates in vivo at sites that are specifically phosphorylated
Effect of gpb1? gpb2? mutations is independent of adenylyl
cyclase and the high- and low-affinity cAMP phosphodiester-
ases. The observation that gpb1? gpb2? mutations affect the
phosphorylation level of PKA substrates raises the question of
whether Gpb1p and Gpb2p function by inhibiting the activity
of adenylyl cyclase. Adenylyl cyclase activates PKA by produc-
ing cAMP, which binds to the PKA regulatory subunit, result-
ing in the release of active PKA catalytic subunits. The PKA
regulatory subunit, Bcy1p, is common to all three PKA cata-
lytic isoforms (33). Therefore, if Gpb1p and Gpb2p were to
function by inhibiting production of cAMP, all three PKA
catalytic isoforms would be expected to be affected by deletion
of GPB1 and GPB2. To test this idea directly, the effect of
deleting GPB1 and GPB2 was determined in a strain contain-
ing a deletion of the CYR1 gene, which encodes adenylyl cy-
clase. The viability of cells containing a cyr1? mutation can be
maintained by deletion of PDE2, a gene encoding a high-
affinity cAMP phosphodiesterase (2). In these cells, viability is
maintained by supplementing the medium with exogenous
cAMP. Cells containing cyr1? pde2? mutations were viable
when supplied with 1.5 mM cAMP but did not grow on 0.75 or
0.5 mM cAMP (Fig. 3, top panels). In contrast, cells lacking
adenylyl cyclase and carrying gpb1? gpb2? mutations were
viable on 0.75 mM cAMP. However, cyr1? pde2? gpb1? gpb2?
cells were inviable on 0.5 mM cAMP. Wild-type and pde2?
cells were viable at all concentrations of cAMP (data not
shown). Therefore, gpb1? gpb2? mutations do not confer via-
bility to cells containing very low levels of cAMP but are able
to remediate the loss of viability at an intermediate concentra-
tion of cAMP. cyr1? pde2? cells containing either an activated
allele of RAS2 or empty vector were viable when supplied with
1.5 mM cAMP but did not grow on 0.75 mM or 0.5 mM cAMP
(Fig. 3, bottom panels). Therefore, activation of Ras has no
effect in cells lacking adenylyl cyclase, a finding consistent with
previous results showing that Ras2p directly activates adenylyl
cyclase (35). The finding that deletion of GPB1 and GPB2 has
an effect on cells that cannot synthesize cAMP eliminates the
possibility that the inhibitory function of Gpb1p and Gpb2p
acts solely on adenylyl cyclase.
The level of cAMP in cells results from the combined effect
of adenylyl cyclase-mediated synthesis of cAMP and phos-
phodiesterase-mediated degradation of cAMP. The results
shown above demonstrate that gpb1? gpb2? mutations have an
effect on cells that lack adenylyl cyclase and the high-affinity
cAMP phosphodiesterase Pde2p. In addition to these enzymes,
the level of cellular cAMP is regulated by the low-affinity
cAMP phosphodiesterase Pde1p (23). To test the involvement
of Pde1p in mediating the effect of the kelch repeat proteins,
strains containing a pde1? mutation in combination with other
mutations that affect cAMP levels were constructed. However,
strains containing cyr1? pde1? pde2? mutations grew ex-
tremely slowly in several different cAMP concentrations, and
they rapidly generated faster growing variants that were likely
to contain suppressor mutations. In addition, strains contain-
FIG. 3. Effect of gpb1? gpb2? mutations is independent of adenylyl
cyclase and the high-affinity cAMP phosphodiesterase. Tenfold dilu-
tions of the following strains were spotted onto medium containing the
indicated concentrations of cAMP: HS222-3A (cyr1? pde2?) and
HS224-3D (gpb1? gpb2? cyr1? pde2?) grown in synthetic complete
medium containing 2 mM cAMP (upper panel), and HS222-3A.T
pde2?) carrying vector
RAS2ala18val19grown in medium lacking uracil and containing 2 mM
cAMP (lower panel). Plates were photographed after 2 days of growth.
YEp352or plasmid YCp50-
FIG. 4. Effect of gpb1? gpb2? mutations is independent of the
low-affinity cAMP phosphodiesterase. (A) Extracts made from the
following strains expressing Msn2p-GFP fusion protein from plasmid
pAdh1-Msn2-GFP were analyzed by SDS-PAGE and immunoblotting
with antibodies to P-CREB (S133) and GFP: wild-type strain SKY762
(lane 1), gpb1? gpb2? strain HS182-3B (lane 2), pde1? strain SKY.p1k
(lane 3), gpb1? gpb2? pde1? strain HS182-3B.p1k (lane 4). (B) The
strains described in panel A were grown to saturation for 2 days in
synthetic complete medium, incubated at 50°C for 15 min, and diluted
and plated to determine the percent survival. Values for GPB1 GPB2
strains are represented by the open bars; values for gpb1? gpb2?
strains are represented by the filled bars. Values shown are the mean
and standard deviation from three independent experiments.
VOL. 4, 2005Gpb1p AND Gpb2p CONTROL PKA ACTIVITY 1797
ing cyr1? pde1? mutations were inviable at all concentrations
of cAMP tested (data not shown). Therefore, the effect of
gpb1? gpb2? mutations was tested in a strain containing only a
In strains containing a pde1? mutation, Msn2p phosphory-
lation at serine 620 was substantially increased in gpb1? gpb2?
cells compared to GPB1 GPB2 cells (Fig. 4A, lanes 3 and 4).
This effect is similar to that conferred by gpb1? gpb2? muta-
tions in a wild-type strain (Fig. 4A, lanes 1 and 2). Therefore,
Pde1p is not required for the decrease in Msn2p phosphory-
lation caused by the presence of Gpb1p and Gpb2p. To deter-
mine whether PDE1 is involved in the effect of gpb1? gpb2?
mutations on a physiological phenotype that is controlled by
PKA, a heat shock experiment was performed. Deletion of
GPB1 and GPB2 results in a very large increase in heat shock
sensitivity in a wild-type background (1). In strains containing
a pde1? mutation, the effect of gpb1? gpb2? mutations on heat
shock sensitivity was the same as that seen in wild-type strains
(Fig. 4B). These results demonstrate that the inhibitory func-
tion of Gpb1p and Gpb2p does not act solely on the low-affinity
cAMP phosphodiesterase Pde1p.
The kelch repeat-containing proteins Gpb1p and Gpb2p are
negative regulators of the cAMP/PKA pathway that were iden-
tified on the basis of their interaction with the G protein
?-subunit Gpa2p (1, 14). Here we show that deletion of GPB1
and GPB2 causes an increase in the phosphorylation level of
physiologically relevant PKA substrates. These effects corre-
late with changes in PKA-dependent characteristics such as
invasive growth and heat shock sensitivity. However, the in-
crease in signaling conferred by gpb1? gpb2? mutations does
not require any of the three enzymes known to be involved in
cAMP metabolism, which include adenylyl cyclase and the
high- and low-affinity cAMP phosphodiesterases. The most
straightforward interpretation of these results is that Gpb1p
and Gpb2p either directly or indirectly inhibit a component of
the cAMP/PKA pathway that acts downstream of cAMP. In
fact, signaling through Gpb1p and Gpb2p could account for
the observation that there is a cAMP-independent mechanism
for regulating PKA-dependent phenotypes (4). Potential tar-
gets of Gpb1p and Gpb2p include the PKA regulatory subunit,
PKA catalytic subunits, and phosphatases that act on PKA
These results appear to contradict an earlier report showing
that mutation of GPB1 and GPB2 causes changes in the intra-
cellular concentration of cAMP, which would suggest that
Gpb1p and Gpb2p modulate the activity of enzymes involved
in cAMP metabolism (14). In the previous report, it was shown
that wild-type cells and cells containing either a gpb1? or
gpb2? mutation display a twofold increase in cAMP levels
upon readdition of glucose to starved cells. However, whereas
in wild-type cells the concentration of cAMP returns to its
basal level after one minute, in mutant cells it remains at the
elevated level for several minutes. In contrast, cells containing
double gpb1? gpb2? mutations do not display a twofold in-
crease in cAMP levels upon readdition of glucose to starved
cells. Therefore, deletion of either GPB1 or GPB2 results in
slightly higher cAMP levels, and deletion of both genes results
in slightly lower cAMP levels. These observations are some-
what unexpected given that the defects conferred by individual
gpb1? and gpb2? mutations are similar to, though less severe
than, the defects conferred by double gpb1? gpb2? mutations
for all other phenotypes tested, including pseudohyphal
growth, invasive growth, FLO11 expression, heat shock sensi-
tivity, sporulation efficiency, and glycogen accumulation (1,
14). Moreover, all of the observed phenotypes of gpb1? gpb2?
cells are consistent with an increase in PKA activity, rather
than a decrease. Given that the glucose-induced spike in
cAMP levels is extremely sensitive to the nutritional status of
the cells (5), it is possible that the reported differences between
wild-type, gpb1?, gpb2?, and gpb1? gpb2? cells were caused by
differences in the nutritional state of the cells in the culture
sample used for the assay. Alternatively, the effects of GPB1
and GPB2 mutant alleles on cAMP levels could be due to a
feedback mechanism that regulates the concentration of
cAMP in response to PKA activity (25). In any case, the finding
that loss of Gpb1p and Gpb2p causes significant effects in
cyr1? pde2? and pde1? strains indicates that the kelch repeat
proteins can function independently of cAMP metabolism.
The G protein ?-subunit Gpa2p has been thought to medi-
ate signaling through direct activation of adenylyl cyclase, like
mammalian G?s, based on the following observations. First,
the glucose-induced spike in cAMP concentration is increased
in cells that overexpress GPA2, and it is eliminated in cells that
contain a gpa2? mutation when the cells are grown under
certain nutritional conditions (5, 22, 24). Second, the addition
of cAMP compensates for the defect in pseudohyphal growth
conferred by a gpa2? mutation (17, 21). However, here we
show that the Gpa2p-interacting proteins Gpb1p and Gpb2p
affect signaling through the cAMP/PKA pathway by a process
that is independent of cAMP metabolism. These observations
can be reconciled if Gpa2p and the kelch repeat proteins
impinge on the cAMP/PKA pathway at different points. Such a
situation might be expected based on the complex relationship
between the functions of Gpb1p, Gpb2p, and Gpa2p. For ex-
ample, although it has been proposed that Gpb1p and Gpb2p
bind to Gpa2p in a manner that mimics G protein ?-subunits
(14), deletion of GPB1 and GPB2 in either wild-type cells or in
gpa2? cells causes a substantial increase in PKA-dependent
phenotypes (1, 14). These results suggest that Gpb1p and
Gpb2p inhibit PKA signaling in both the presence and the
absence of Gpa2p. Therefore, one possibility is that the kelch
repeat proteins act as effectors of the G protein. However,
deletion of GPA2 in gpb1? gpb2? cells causes a two- to three-
fold decrease in FLO11 RNA abundance compared to that
seen in gpb1? gpb2? cells. Similarly, gpa2? gpb1? gpb2? cells
display a decreased level of pseudohyphal growth compared to
gpb1? gpb2? cells. Therefore, part of the signal present in
gpb1? gpb2? cells is dependent on Gpa2p. One possible model
to explain these results is that Gpb1p and Gpb2p negatively
regulate both Gpa2p and another component of the PKA
signaling pathway that is downstream of adenylyl cyclase. De-
letion of GPB1 and GPB2 activates both Gpa2p and the other
target, resulting in very high signal generation. In a gpb1?
gpb2? mutant background, deletion of GPA2 eliminates the
part of the signal that originates with Gpa2p but leaves intact
the part of the signal resulting from the other target of Gpb1p
and Gpb2p. Direct activation of adenylyl cyclase by Gpa2p
1798LU AND HIRSCHEUKARYOT. CELL
would be consistent with this model, if the other target of
Gpb1p and Gpb2p acts downstream of adenylyl cyclase.
The relationship between Gpa2p and the kelch repeat pro-
teins has also been investigated by using an assay that detects
changes in the subcellular location of GFP-Gpb2p. A recent
study showed that overexpression of GPA2 causes GFP-Gpb2p
to accumulate at the cell periphery and that overexpression of
the nonactivatable GPA2G299Aallele has a similar effect on
GFP-Gpb2p localization (15). These results are not consistent
with the idea that Gpb1p and Gpb2p act as effectors of Gpa2p,
because an effector would be expected to bind the GTP-bound
form of a G protein ?-subunit and the G299A version of
Gpa2p is expected to be present predominantly in the GDP-
bound form. However, that study did not investigate whether a
constitutively active allele of GPA2 has any effect on GFP-
Gpb2p localization. A comparison between the effects of non-
activatable and constitutively active versions of Gpa2p on
Gpb2p localization would provide information about whether
the function of Gpb1p and Gpb2p is more similar to that of
Gpa2p effectors or G protein ?-subunits.
Signaling through the Gpa2p pathway displays several un-
usual features. In contrast to essentially all known G protein-
mediated systems, the Gpa2p ?-subunit does not appear to
form a heterotrimer with classical ??-subunits. Moreover, here
we show that the Gpa2p-interacting proteins Gpb1p and
Gpb2p affect cAMP/PKA signaling at a step downstream of
adenylyl cyclase. Gpb1p and Gpb2p may also have a function
that negatively regulates Gpa2p activity. Given that kelch re-
peat-containing proteins of unknown function are present in
all eukaryotes, it will be of interest to determine whether the
alternative signaling mechanisms used in the Gpa2p pathway
are also seen in other G protein-mediated pathways.
This study was supported by Grant-In-Aid 9951029T from the
American Heart Association, Heritage Affiliate, and by grant
GM60332 from the National Institutes of Health.
We thank S. Palecek, S. Kron, X. Pan, J. Heitman, C. Schu ¨ller,
W. Reiter, and S. Garrett for plasmids and strains used in this study.
We also thank S. Pin ˜ol-Roma for critical comments on the manuscript.
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