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: email@example.com.
34. Toda, T., S. Cameron, P. Sass, M. Zoller, and M. Wigler. 1987. Three
different genes in S. cerevisiae encode the catalytic subunits of the cAMP-
dependent protein kinase. Cell 50:277–287.
35. Toda, T., I. Uno, T. Ishikawa, S. Powers, T. Kataoka, D. Broek, S. Cameron,
J. Broach, K. Matsumoto, and M. Wigler. 1985. In yeast, RAS proteins are
controlling elements of adenylate cyclase. Cell 40:27–36.
36. Xue, Y., M. Batlle, and J. P. Hirsch. 1998. GPR1 encodes a putative G
protein-coupled receptor that associates with the Gpa2p G?subunit and
functions in a Ras-independent pathway. EMBO J. 17:1996–2007.
37. Yun, C.-W., H. Tamaki, R. Nakayama, K. Yamamoto, and H. Kumagai. 1997.
G-protein coupled receptor from yeast Saccharomyces cerevisiae. Biochem.
Biophys. Res. Commun. 240:287–292.
1800 LU AND HIRSCHEUKARYOT. CELL