Kelch-repeat proteins interacting with the Galpha protein Gpa2 bypass adenylate cyclase for direct regulation of protein kinase A in yeast.
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 glucose/sucrose-sensitive seven-transmembrane domain receptor, Gpr1, that was proposed to activate adenylate cyclase through the G(alpha) protein Gpa2. Consistently, we show here that adenylate cyclase binds only to active, GTP-bound Gpa2. Two related kelch-repeat proteins, Krh1/Gpb2 and Krh2/Gpb1, are associated with Gpa2 and were suggested to act as G(beta) mimics for Gpa2, based on their predicted seven-bladed beta-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 Krh1/2 stimulate the association between the catalytic and regulatory subunits in vivo. Indeed, both a constitutively active GPA2 allele and deletion of KRH1/2 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 Krh1/2 also enhance the association between mouse R and C subunits, suggesting that Krh control of PKA has been evolutionarily conserved.
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