Regulation of expression and activity of the yeast transcription factor ADR1.

Disruption of ADR1, a positive regulatory gene in the yeast Saccharomyces cerevisiae, abolished derepression of ADH2 but did not affect glucose repression of ADH2 or cell viability. The ADR1 mRNA was 5 kilobases long and had an unusually long leader containing 509 nucleotides. ADR1 mRNA levels were regulated by the carbon source in a strain-dependent fashion. beta-Galactosidase levels measured in strains carrying an ADR1-lacZ gene fusion paralleled ADR1 and ADR1-lacZ mRNA levels, indicating a lack of translational regulation of ADR1 mRNA. ADH2 was regulated by the carbon source to the same extent in all strains examined and showed complete dependence on ADR1 as well. The expression of ADR1 mRNA and an ADR1-beta-galactosidase fusion protein during glucose repression suggested that the activity of the ADR1 protein is regulated at the posttranslational level to properly regulate ADH2 expression. The ADR1-beta-galactosidase fusion protein was able to activate ADH2 expression during glucose repression but showed significantly higher levels of activation upon derepression. A similar result was obtained when ADR1 was present on a multicopy plasmid. These results suggest that low-level expression of ADR1 is required to maintain glucose repression of ADH2 and are consistent with the hypothesis that ADR1 is regulated at the posttranslational level.     

Relationship of the Camp-Dependent Protein Kinase Pathway to the Snf1 Protein Kinase and Invertase Expression in Saccharomyces Cerevisiae

The SNF1 protein kinase and the associated SNF4 protein are required for release of glucose repression in Saccharomyces cerevisiae. To identify functionally related proteins, we selected genes that in multicopy suppress the raffinose growth defect of snf4 mutants. Among the nine genes recovered were two genes from the cAMP-dependent protein kinase (cAPK) pathway, MSI1 and PDE2. Increased dosage of these genes partially compensates for defects in nutrient utilization and sporulation in snf1 and snf4 null mutants, but does not restore invertase expression. These results suggest that SNF1 and cAPK affect some of the same cellular responses to nutrients. To examine the role of the cAPK pathway in regulation of invertase, we assayed mutants in which the cAPK is not modulated by cAMP. Expression of invertase was regulated in response to glucose and was dependent on SNF1 function. Thus, a cAMP-responsive cAPK is dispensable for regulation of invertase.     

Feedback inhibition on cell wall integrity signaling by Zds1 involves Gsk3 phosphorylation of a cAMP-dependent protein kinase regulatory subunit

We report here that budding yeast cAMP-dependent protein kinase (cAPK) is controlled by heat stress. A rise in temperature from 30 to 37 degrees C was found to result in both a higher expression and an increased cytoplasmic localization of its regulatory subunit Bcy1. Both of these effects required phosphorylation of serines located in its localization domain. Surprisingly, classic cAPK-controlled processes were found to be independent of Bcy1 phosphorylation, indicating that these modifications do not affect cAPK activity as such. Alternatively, phosphorylation may recruit cAPK to, and thereby control, a specific subset of (perhaps novel) cAPK targets that are presumably localized extranuclearly. Zds1 and Zds2 may play a role in this process, since these were found required to retain hyperphosphorylated Bcy1 in the cytoplasm at 37 degrees C. Mck1, a homologue of mammalian glycogen synthase kinase 3 and a downstream component of the heat-activated Pkc1-Slt2/Mpk1 cell wall integrity pathway, is partly responsible for hyperphosphorylations of Bcy1. Remarkably, Zds1 appears to act as a negative regulator of cell wall integrity signaling, and this activity is dependent in part on the phosphorylation status of Bcy1. Thus, Mck1 phosphorylation of Bcy1 and Zds1 may constitute an unprecedented negative feedback control on the cell wall integrity-signaling pathway.     

Overexpression of Dd PK2 protein kinase causes rapid development and affects the intracellular cAMP pathway of Dictyostelium discoideum.

The Dd PK2 gene codes for a putative protein of 648 amino acids with a C-terminal half sharing high homology with protein kinase A catalytic subunits from other organisms. In order to find out more about the physiological role of the Dd PK2 kinase, its gene, and a version having a frame shift mutation in the middle of the catalytic region, were overexpressed in developing Dictyostelium cells. Both the intact gene (K-) and the frame shift mutant (Kdel-) caused rapid development with spores formed in 16-18 hours compared to the 24 hours required by their parent. This result was confirmed by the pattern of expression of some developmentally regulated genes. Other rapid developing strains (rde) are activated in the cAMP second messenger system. Both K- and Kdel-containing strains have lower cAMP levels than the parental strain during late development, thus resembling rdeC mutants. K-cells (but not Kdel-cells) produced bizarre fruiting bodies with many prostrate forms. The parallel with rde mutants was confirmed by demonstrating that K-cells are able to form spores in submerged monolayer culture. Furthermore, K-cells have about four times more protein kinase A (cAPK) activity than wild-type cells. These results indicate that the N-terminal domain of Dd PK2 is sufficient to influence cAMP levels and to provoke rapid development, whereas kinase activity seems to be required for the sporogenous phenotype. The association between elevated cAPK and Dd PK2 overexpression phenotype further indicates a role for cAPK in the formation of spores.     

A three-dimensional model of the Cdc2 protein kinase: localization of cyclin- and Suc1-binding regions and phosphorylation sites.

The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Suc1. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A- and B- and Suc1-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991). Those sites were mapped by building a Cdc2 model based on the crystallographic coordinates of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (D. R. Knighton, J. Zheng, L. F. Ten Eyck, V. A. Ashford, N.-H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253:407-414, 1991). On the basis of this model, additional mutations were made and tested for cyclin A and Suc1 binding and for kinase activity. Mutations that interfere with cyclin A binding are localized primarily on the small lobe near its interface with the cleft and include an acidic patch on the B helix and R-50 in the highly conserved PSTAIRE sequence. Two residues in the large lobe, R-151 and T-161, influence cyclin binding, and both are at the surface of the cleft near its interface with the PSTAIRE motif. Cyclin-dependent phosphorylation of T-161 in Cdc2 is essential for activation, and the model provides insights into the importance of this site. T-161 is equivalent to T-197, a stable phosphorylation site in cAPK. On the basis of the model, cyclin binding very likely alters the surface surrounding T-161 to allow for T-161 phosphorylation. The two major ligands to T-197 in cAPK are conserved as R-127 and R-151 in Cdc2. The equivalent of the third ligand, H-87, is T-47 in the PSTAIRE sequence motif. Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Suc1 binding; however, the Suc1-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Suc1-binding site maps to the large lobe near a 25-residue insert and includes R-215.     

Functional Analysis of the Yeast Glc7-Binding Protein Reg1 Identifies a Protein Phosphatase Type 1-Binding Motif as Essential for Repression of ADH2 Expression

In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose. Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2. Constitutive ADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2 expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells. To further elucidate the functional role of Reg1 in repressing ADH2 expression, deletions scanning the entire length of the protein were analyzed. Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression. Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional. Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses. Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex. Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source. When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression. These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression. This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm.