Kelch-repeat proteins interacting with the G _α protein Gpa2 bypass adenylate cyclase for direct regulation of protein kinase A in yeast

Abstract

The cAMP-PKA pathway consists of an extracellular ligand-sensitive G protein-coupled receptor, a G protein signal transmitter, and the effector, adenylate cyclase, of which the product, cAMP, acts as an intracellular second messenger. cAMP activates PKA by dissociating the regulatory subunit from the catalytic subunit. Yeast cells (Saccharomyces cerevisiae) contain a glucose͞sucrose-sensitive seven-transmembrane domain receptor, Gpr1, that was proposed to activate adenylate cyclase through the G␣ protein Gpa2. Consistently, we show here that adenylate cyclase binds only to active, GTP-bound Gpa2. Two related kelch-repeat proteins, Krh1͞ Gpb2 and Krh2͞Gpb1, are associated with Gpa2 and were suggested to act as G␤ mimics for Gpa2, based on their predicted seven-bladed ␤-propeller structure. However, we find that although Krh1 associates with both GDP and GTP-bound Gpa2, it displays a preference for GTP-Gpa2. The strong down-regulation of PKA targets by Krh1 and Krh2 does not require Gpa2 but is strictly dependent on both the catalytic and the regulatory subunits of PKA. Krh1 directly interacts with PKA by means of the catalytic subunits, and Krh1͞2 stimulate the association between the catalytic and regulatory subunits in vivo. Indeed, both a constitutively active GPA2 allele and deletion of KRH1͞2 lower the cAMP requirement of PKA for growth. We propose that active Gpa2 relieves the inhibition imposed by the kelch-repeat proteins on PKA, thereby bypassing adenylate cyclase for direct regulation of PKA. Importantly, we show that Krh1͞2 also enhance the association between mouse R and C subunits, suggesting that Krh control of PKA has been evolutionarily conserved.Saccharomyces cerevisiae ͉ signal transduction ͉ glucose

Environmental Sensing and Signal Transduction Pathways Regulating Morphopathogenic Determinants of Candida albicans

“…Apart from the receptor, ScGpa2 also interacts with ScGbp1/ScKrh2 and ScGbp2/ScKrh1, two proteins that appear to act as G␤-mimicking subunits, based on structural resemblance with classical G␤ proteins (24,117). Recently, two different molecular mechanisms by which these kelch repeat proteins function were described (116,233). It seems that activated ScGpa2 relieves the inhibition imposed by the kelch repeat proteins on PKA, thereby bypassing adenylate cyclase for direct regulation of PKA.…”

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…This probably ensures that the cAMP-PKA pathway is only fully activated when glucose levels are high enough to switch easily from respiration to fermentation. GTP-bound Gpa2 activates the cAMP-PKA pathway and this is most probably through stimulation of adenylate cyclase (Nakafuku et al 1988;Kubler et al 1997;Lorenz and Heitman 1997;Colombo et al 1998;Rolland et al 2000;Peeters et al 2006). Gpa2 interacts with Rgs2, a member of the family of regulators of G protein signalling (RGS), that negatively regulates the Gpa2-GTP signal by stimulating the intrinsic GTPase activity of Gpa2 (Versele et al 1999).…”

“…One report suggests that the kelch-repeat proteins inhibit Gpr1-Gpa2 coupling (Harashima and Heitman 2005). Alternatively, the kelch-repeat proteins were reported to stimulate the interaction between the catalytic and the regulatory subunits of PKA (Peeters et al 2006). Activated, GTP-bound Gpa2 would relieve this inhibition of PKA by inhibiting the kelch-proteins, thereby bypassing adenylate cyclase to regulate PKA.…”

Genetic Identification of Factors That Modulate Ribosomal DNA Transcription in Saccharomyces cerevisiae

“…IRA1 and IRA2 encode GTPase-activating proteins (GAPs) that negatively regulate RAS by converting it from the GTP-to the GDP-bound inactive form, and mutations in these genes result in constitutive activation of RAS and, therefore, of the cAMP/PKA pathway (Broach 1991). GPB1 encodes a protein that inhibits the cAMP/PKA pathway by binding and stabilizing Ira1 and Ira2 (Harashima et al 2006;Peeters et al 2006), and GPG1 encodes the gamma subunit required for Gpb1 or Gpb2 to interact with Gpa2 as a heterotrimeric G-protein complex (Harashima and Heitman 2002). Constitutive activation of RAS and the cAMP/PKA pathway prevents the rapamycin-induced repression of Pol I and Pol III transcription (Schmelzle et al 2004) and the starvation-induced repression of ribosomal protein genes (Klein and Struhl 1994).…”