The role of two novel kelch proteins Krh1 and Krh2 in the Gpa2p-cAMP/PKA signaling pathway in Saccharomyces cerevisiae

The G protein Gpa2p transmits signals from its cognate receptor Gpr1p to downstream effectors to regulate cell proliferation and differentiation. Gpr1p and Gpa2p are required for the transition to filamentous growth. The Gpr1p-Gpa2p pathway is known to activate cAMP/PKA signaling, but the downstream components and the way by which Gpa2p stimulates PKA activity are not understood. A two-hybrid screen using a constitutively active allele of GPA2 in the laboratory identified a novel kelch repeat-containing protein Krh1p. Here we report that Krh1p and its homolog Krh2p act downstream of Gpa2p to negatively regulate yeast vegetative and filamentous growth. Cells containing krh1Deltakrh2Delta mutations display phenotypes resembling that of cells expressing a constitutively active Gpa2p. These phenotypes include increased expression of FLO11 mRNA and invasive growth, decreased heat resistance and efficiency of sporulation. The filamentous defect of a gpa2Delta strain is suppressed by further deleting KRH1 and KRH2, suggesting Krh1p and Krh2p exert their inhibition at a step downstream of Gpa2p.; Furthermore, we identified the catalytic subunits of PKA as the potential targets of Krh1p and Krh2p. Deletion of KRH1 and KRH2 results in a high PKA activity. krh1Deltakrh2Delta mutations confer a growth advantage to cyr1Deltapde2Delta cells, indicating Krh1p and Krh2p are not involved in synthesizing cAMP. Importantly, we show that deletion of KRH1 and KRH2 leads to nuclear accumulation of PKA catalytic subunits, and overexpression of Krh1p causes an opposite effect. Krh1p and Krh2p are localized to both the cytoplasm and nucleus, and physically associate with PKA catalytic subunits. Altogether, we propose that binding of Krh1p and Krh2p to PKA catalytic subunits results in cytoplasmic sequestration of PKA, and thus nuclear PKA activity is low in wild type cells.; Finally, we show that Krh1p and Krh2p also regulate the expression of the PKA regulatory subunit Bcy1p. Deletion of KRH1 and KRH2 dramatically decreases Bcy1p expression, which causes a substantial increase of free catalytic subunits. Therefore, both nuclear accumulation of the catalytic subunits and a decrease of Bcy1p abundance lead to a dramatic increase of the nuclear ratio of catalytic to regulatory subunits and result in high nuclear PKA activity in krh1Deltakrh2Delta cells.