Domain shuffling as a tool for investigation of protein function: substitution of the cysteine-rich region of Raf kinase and PKC eta for that of yeast Pkc1p.

With the completion of the sequences of entire genomes, the need for functional characterisation of proteins and their domains is becoming acute. Conserved regions within proteins often share overlapping functions but despite this conservation may fulfil quite different tasks in different species. In this work, we investigated the cysteine-rich motif (C1 domain) of yeast protein kinase C (Pkc1p) as a model to establish a test system for domain function. C1 domains activate kinases through binding of either diacylglycerol and/or phosphatidylserine, as in many members of the protein kinase C (PKC) family, or by binding small GTPases, as in Raf kinase. In contrast to other members of the protein kinase C superfamily, Pkc1p of Saccharomyces cerevisiae is activated via binding of the small G-protein Rho1p to its C1 domain. We developed a system for domain shuffling to establish the function of C1 domains from human Raf kinase and rat PKC eta in yeast. Only the C1 domain from Raf kinase enabled the chimeric enzyme to bind Rho1p when substituted for the native yeast domain. Accordingly, a chimeric Pkc1p carrying the C1 from Raf kinase, but not that from PKC eta, was able to partially complement the phenotypes of a yeast pkc1 deletion mutant. We interpret these data as further evidence that interaction with a small GTPase is the main regulatory function of the C1 domain in yeast.

Comparative genetic and physiological studies of the MAP kinase Mpk1p from Kluyveromyces lactis and Saccharomyces cerevisiae.

MAP kinases are essential components of signal transduction pathways in yeasts and higher eukaryotes. Here, we report on the isolation of the gene encoding the MAP kinase KlMpk1p by complementation of the respective Saccharomyces cerevisiae deletion mutant with a genomic library from Kluyveromyces lactis. Sequencing revealed the presence of an open reading frame capable of encoding a protein of 520 amino acid residues with a deduced molecular mass of 59.726 Da. The deduced protein sequence displayed a high degree of similarity to known MAP kinases from yeast to man, with an overall identity of 70 % to ScMpk1p. One-hybrid analysis demonstrated the presence of a cryptic transcriptional activation domain in the C-terminal part of the protein. Deletion of this sequence in ScMpk1p resulted in a reduced MAP kinase activity (measured by an indirect assay), an increased sensitivity towards caffeine and an increased resistance against Calcofluor white. Complete deletion mutants of Klmpk1 display an osmo-remedial phenotype on rich medium, but are capable of growth in the absence of osmotic stabilization on synthetic medium. As Scmpk1 deletion mutants, they are sensitive to cell surface destabilizing agents such as Calcofluor white and SDS, and growth is inhibited in the presence of 5 mM caffeine. Overexpression of KlMPK1 did not produce a growth defect in S. cerevisiae or in K. lactis.

Mutants affected in the putative diacylglycerol binding site of yeast protein kinase C.

In an attempt to study the functional similarities between protein kinase C from the yeast Saccharomyces cerevisiae and its human homologues we have started in vitro mutagenesis to alter specific domains. Here we report on the exchange of four cysteine residues by serines in yeast Pkc1p that have been shown to be essential for diacylglycerol (DAG) binding and activation by this compound in humans. The mutant yeast protein leads to sensitivity to caffeine and low concentrations of SDS when expressed in a pkc1 deletion strain. However, sensitivity to staurosporine was not affected. Our data indicate that the conserved DAG binding domain serves an important function in yeast Pkc1p.

The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae.

Signal transduction mediated by the single yeast isozyme of protein kinase C (Pkc1p) is essential for the maintenance of cellular integrity in this model eukaryote. The past few years have seen a dramatic increase in our knowledge of the upstream regulatory factors that modulate Pkc1p activity (e.g. Tor2p, Rom1p, Rom2p, Rho1p, Slg1p, Mid2p) and of the downstream targets of the MAP kinase cascade triggered by it (e.g. Rlm1p, SBF complex). The picture that has emerged connects this pathway to a variety of other cellular processes, such as cell cycle progression (Cdc28p, Swi4p), mating (Ste20p), nutrient sensing (Ira1p), calcium homeostasis (calcineurin, Mid2p, Fks2p) and the structural dynamics of the cytoskeleton (Spa1p, Bni1p).

The PH domain of the yeast GEF Rom2p serves an essential function in vivo.

In a screen designed to identify new upstream components of the Pkc1p-MAP kinase signal transduction pathway that responds to cell wall damage in yeast, we identified a new mutant allele of the ROM2 gene, which encodes a GDP/GTP exchange factor that acts on Rho1p. This allele, originally termed ubk1 (upstream of Bck1p) encodes a truncated protein that lacks the putative PH domain. Complementation experiments showed that genes coding for several known components of the pathway are able to suppress the ubk1 mutation to various degrees when introduced on low- or high-copy-number vectors. Analysis of several rom2 mutants showed that mutants in which the PH domain is deleted result in a phenotype indistinguishable from that of a strain deleted for the entire gene, indicating that this domain fulfills an essential function in vivo. Furthermore, we found that the growth phenotype of rom2 mutants is highly dependent on the strain background. Surprisingly, analysis of the phosphorylation status of Mpk1p in these mutants showed an elevated level of doubly phosphorylated Mpk1 protein, indicating that the growth defect of rom2 mutants is not due to an inability to activate the MAP kinase module, but rather to lack of a function of the Rom2 protein that has yet to be identified precisely.

Lrg1p functions as a putative GTPase-activating protein in the Pkc1p-mediated cell integrity pathway in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the ROM2 gene encodes a GDP/GTP exchange factor for the small G-protein Rho1p, a known activator of protein kinase C. In a screen designed to isolate suppressors of a rom2 mutant allele, we identified a mutant defective in the gene coding for the putative GTPase-activating protein Lrg1p. This protein was previously suggested to be involved in sporulation and mating. Here we provide evidence for its role in Pkc1p-mediated signal transduction based on the following results. (1) Deletion of LRG1 suppresses the growth phenotypes associated with mutations in SLG1 (which codes for a putative sensor of cell wall damage). (2) Using two-hybrid assays an interaction between the GAP domain of Lrg1p and Rho1p was demonstrated. (3) The lrg1 mutant shows enhanced activity of the Pkc1p pathway. (4) Overexpression of LRG1 leads to a cell lysis defect that can be suppressed by the addition of osmotic stabilizers. Phenotypic comparison of lrg1 mutants with mutants defective in other GTPase-activating proteins (Sac7p, Bem2p, Bag7p) presumed to act on Rho1p revealed that deletion of SAC7, but not BEM2 or BAG7, suppresses the phenotype of rom2 mutants. Pairwise combination of mutations in all these genes showed that the simultaneous deletion of SAC7 and LRG1 is synthetically lethal. We therefore suggest that Lrg1p acts as a negative regulator of the Pkc1p pathway in conjunction with its known homologue Sac7p.