Protein kinase A (PKA) or cAMP-dependent protein kinase, is one of the most well studied kinases in eukaryotic biology. The inactive form of PKA consists of a complex of two catalytic subunits bound to two regulatory subunits.1
The classic mechanism of protein kinase A activation, which is highly conserved in eukaryotes, involves binding of cAMP to the regulatory subunit, resulting in the release of free, active catalytic subunits. Because PKA was first characterized more than 40 years ago,2
it is generally thought that its regulation is now very well understood. However, recent studies in yeast have revealed that there may be something new to learn about this venerable enzyme.
In the yeast Saccharomyces cerevisiae
, PKA is required for normal growth, regulation of energy metabolism and control of stress responses.3
PKA is also thought to be involved in the response to nutrients, based on the observation that addition of a fermentable sugar to yeast cells that have been starved for a carbon source results in a spike of cAMP. In yeast, adenylate cyclase is activated by the heterotrimeric G protein β-subunit Gpa2, as well as by Ras proteins (). Gpa2 is coupled to a cell surface receptor, Gpr1, that may directly detect nutrients in the extracellular environment.4–8
The way in which Ras is activated by nutrient signals is not known. Once activated, Gpa2 and Ras stimulate adenylate cyclase to produce cAMP, which activates PKA by the classic mechanism. The yeast PKA regulatory subunit is called Bcy1 and the three isoforms of the PKA catalytic subunit are called Tpk1, Tpk2 and Tpk3. The PKA catalytic subunits phosphorylate transcription factors that regulate growth, energy metabolism and stress responses.
The PKA pathway in Saccharomyces cerevisiae.
A novel regulatory process that affects signaling through the PKA pathway involves a pair of proteins, Gpb1 and Gpb2, that contain several copies of a domain that was originally identified in the Drosophila protein kelch
. Sequence analysis of Gpb1 (also called Krh2) and Gpb2 (also called Krh1) has shown that their C-terminal region contains either six9
kelch repeats. Based on the structure of other kelch repeat proteins, this sequence would predict that they fold into a six- or seven-bladed β-propeller,12
a structure that is known to participate in protein-protein interactions. Gpb1 and Gpb2 also contain a large region of sequence at their N-terminal region that is not homologous to other proteins.
The way in which Gpb1 and Gpb2 regulate signal transduction has been controversial since these proteins were first described. Gpb2 was originally identified based on its ability to interact with the G protein β-subunit Gpa2 in a two-hybrid screen, and Gpb1 was found by its homology to Gpb2.9–11
Proposed mechanisms to explain their effects on signaling include acting as G protein β-subunit mimics10
or G protein effectors,9
functioning to inhibit receptor-G protein coupling,13
to positively regulate Ras GTPase activating proteins,14
and to stimulate the association between PKA regulatory and catalytic subunits.11
One characteristic of Gpb1 and Gpb2 that has been confirmed by several different methods is their ability to bind Gpa2.9–11,15
However, the functional significance of this binding is not well understood.
In the present study, we have focused on the predominant function of Gpb1 and Gpb2, which involves inhibition of PKA activity by a mechanism that does not involve changes in cAMP.11,16,17
Previous work by others has shown that cells lacking Gpb1 and Gpb2 display decreased binding of the PKA regulatory subunit to the catalytic subunits.11
This phenotype could be due to direct effects of Gpb1 and Gpb2 on either the regulatory subunit or the catalytic subunits. Here we investigate the effects of Gbp1 and Gpb2 on the PKA regulatory subunit Bcy1.
Our recent study has shown that Gpb1 and Gpb2 affect both the abundance and phosphorylation state of Bcy1 in response to nutrients.18
In low glucose concentrations, Bcy1 abundance increases due to a process that requires the kelch repeat proteins. The increase in Bcy1 abundance that occurs when nutrients are scarce is due to an increase in protein stability. Stability of Bcy1 is controlled by phosphorylation at serine-145, the site of Bcy1 phosphorylation by PKA catalytic subunits. Phosphorylation of Bcy1 at serine-145 appears to target it for degradation in cells that lack Gpb1 and Gpb2. In addition to the effect of Gpb1 and Gpb2 on Bcy1 stability, the kelch repeat proteins also promote phosphorylation of Bcy1 at an unknown site when cells are grown in low glucose concentrations. The effects of Gpb1 and Gpb2 on Bcy1 stability and phosphorylation are eliminated when the kinase activity of PKA catalytic subunits is inhibited using variants of the Tpk proteins that are sensitive to an ATP analog. Therefore, we undertook to determine whether the effects of Gpb1 and Gpb2 on Bcy1 abundance and phosphorylation are a direct effect on the regulatory subunit or are an indirect effect resulting from their regulation of the catalytic subunits. Regulation of the catalytic subunits could affect Bcy1 abundance and phosphorylation indirectly by several different mechanisms. For example, Gpb1 and Gpb2 could alter the catalytic subunits in a way that results in an increase in the amount of Bcy1 bound to them. Binding of Tpk proteins to Bcy1 could cause Bcy1 to be protected from degradation and to be phosphorylated by another kinase.