Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate

When Saccharomyces cerevisiae cells are starved of inorganic phosphate, the Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) is inactivated by the Pho81 CDK inhibitor (CKI). The regulation of Pho80-Pho85 is distinct from previously characterized mechanisms of CDK regulation: the Pho81 CKI is constitutively associated with Pho80-Pho85, and a small-molecule ligand, inositol heptakisphosphate (IP7), is required for kinase inactivation. We investigated the molecular basis of the IP7- and Pho81-dependent Pho80-Pho85 inactivation using electrophoretic mobility shift assays, enzyme kinetics and fluorescence spectroscopy. We found that IP7 interacts noncovalently with Pho80-Pho85-Pho81 and induces additional interactions between Pho81 and Pho80-Pho85 that prevent substrates from accessing the kinase active site. Using synthetic peptides corresponding to Pho81, we define regions of Pho81 responsible for constitutive Pho80-Pho85 binding and IP7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action.     

Genetic analysis of spontaneous suppressors of the pho85 mutation in the yeast Saccharomyces cerevisiae

Chaperones are known to play an important role in complexation of cyclin-dependent kinases with cyclins. In yeast cells growing in the presence of phosphate, cyclin-dependent kinase Pho85p and cyclin Pho80p form a complex and phosphorylate activator Pho4p. As a result, Pho4p is exported from the nucleus, and the PHO5 gene is not transcribed. The mutations suppressing the pho85 mutation were analyzed in order to identify genes which code for chaperones involved in the formation of the Pho80p-Pho85p complex in the presence of environmental phosphate. Dominant mutations DSP1, DSP2, and DSP4-6 were found. It is shown that the DSP1 gene is 2.1 cM away from the PHO85 gene on chromosome XVI and probably coincides with the EGD1 gene coding for a chaperone.     

Genetic Analysis of Spontaneous Suppressors of the pho85 Mutation in Yeast Saccharomyces cerevisiae

Chaperones are known to play an important role in complexation of cyclin-dependent kinases with cyclins. In yeast cells growing in the presence of phosphate, cyclin-dependent kinase Pho85p and cyclin Pho80p form a complex and phosphorylate activator Pho4p. As a result, Pho4p is exported from the nucleus, and the PHO5 gene is not transcribed. The mutations suppressing thepho85 mutation were analyzed in order to identify genes which code for chaperones involved in the formation of the Pho80p–Pho85p complex in the presence of environmental phosphate. Dominant mutations DSP1, DSP2, and DSP4–6 were found. It is shown that the DSP1gene is 2.1 cM away from thePHO85 gene on chromosome XVI and probably coincides with the EGD1 gene coding for a chaperone.     

Regulation of G0 entry by the Pho80-Pho85 cyclin-CDK complex

Eukaryotic cell proliferation is controlled by growth factors and essential nutrients. In their absence, cells may enter into a quiescent state (G0). In Saccharomyces cerevisiae, the conserved protein kinase A (PKA) and rapamycin-sensitive TOR (TORC1) pathways antagonize G0 entry in response to carbon and/or nitrogen availability primarily by inhibiting the PAS kinase Rim15 function. Here, we show that the phosphate-sensing Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) complex also participates in Rim15 inhibition through direct phosphorylation, thereby effectively sequestering Rim15 in the cytoplasm via its association with 14-3-3 proteins. Inactivation of either Pho80-Pho85 or TORC1 causes dephosphorylation of the 14-3-3-binding site in Rim15, thus enabling nuclear import of Rim15 and induction of the Rim15-controlled G0 program. Importantly, we also show that Pho80-Pho85 and TORC1 converge on a single amino acid in Rim15. Thus, Rim15 plays a key role in G0 entry through its ability to integrate signaling from the PKA, TORC1, and Pho80-Pho85 pathways.