Functional,genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in <Emphasis Type="Italic">Sporothrix schenckii</Emphasis>

Background  

Sporothrix schenckiiis a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism inS. schenckiiresponds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene inS. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle.     

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.     

New Structural Insights into Phosphorylation-free Mechanism for Full Cyclin-dependent Kinase (CDK)-Cyclin Activity and Substrate Recognition

Pho85 is a versatile cyclin-dependent kinase (CDK) found in budding yeast that regulates a myriad of eukaryotic cellular functions in concert with 10 cyclins (called Pcls). Unlike cell cycle CDKs that require phosphorylation of a serine/threonine residue by a CDK-activating kinase (CAK) for full activation, Pho85 requires no phosphorylation despite the presence of an equivalent residue. The Pho85-Pcl10 complex is a key regulator of glycogen metabolism by phosphorylating the substrate Gsy2, the predominant, nutritionally regulated form of glycogen synthase. Here we report the crystal structures of Pho85-Pcl10 and its complex with the ATP analog, ATPγS. The structure solidified the mechanism for bypassing CDK phosphorylation to achieve full catalytic activity. An aspartate residue, invariant in all Pcls, acts as a surrogate for the phosphoryl adduct of the phosphorylated, fully activated CDK2, the prototypic cell cycle CDK, complexed with cyclin A. Unlike the canonical recognition motif, SPX(K/R), of phosphorylation sites of substrates of several cell cycle CDKs, the motif in the Gys2 substrate of Pho85-Pcl10 is SPXX. CDK5, an important signal transducer in neural development and the closest known functional homolog of Pho85, does not require phosphorylation either, and we found that in its crystal structure complexed with p25 cyclin a water/hydroxide molecule remarkably plays a similar role to the phosphoryl or aspartate group. Comparison between Pho85-Pcl10, phosphorylated CDK2-cyclin A, and CDK5-p25 complexes reveals the convergent structural characteristics necessary for full kinase activity and the variations in the substrate recognition mechanism.     

Pho85 and signaling environmental conditions

Through its association with a family of ten cyclins, the Pho85 cyclin-dependent kinase is involved in several signal transduction pathways in the yeast Saccharomyces cerevisiae. The responses mediated by Pho85 include cell-cycle progression and metabolism of nutrients such as phosphate and carbon sources. Although these responses require the phosphorylation of different substrates, and have different mechanistic consequences as a result of this phosphorylation, all appear to be involved in responses to changes in environmental conditions. Few of the activating signals or regulated targets have been unambiguously identified, but the kinase activity of Pho85 appears to inform the cell that the current environment is satisfactory.     

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.     

Mouse cyclin-dependent kinase (Cdk) 5 is a functional homologue of a yeast Cdk, pho85 kinase

Mouse cyclin-dependent kinase (Cdk) 5 and yeast Pho85 kinase share similarities in structure as well as in the regulation of their activity. We found that mouse Cdk5 kinase produced in pho85Delta mutant cells could suppress some of pho85Delta mutant phenotypes including failure to grow on nonfermentable carbon sources, morphological defects, and growth defect caused by Pho4 or Clb2 overproduction. We also demonstrated that Cdk5 coimmunoprecipitated with Pho85-cyclins including Pcl1, Pcl2, Pcl6, Pcl9, and Pho80, and that the immunocomplex could phosphorylate Pho4, a native substrate of Pho85 kinase. Thus mouse Cdk5 is a functional homologue of yeast Pho85 kinase.     

Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii

Protein kinase C (PKC) plays an important role in the control of proliferation and differentiation of a wide range of cell types, and fungi are no exception. Previous results reported by us on the effects of the phorbol ester, 12-myristate-13-acetate phorbol (PMA) and other PKC effector molecules, on dimorphism in Sporothrix schenckii suggested the presence of this enzyme in the fungus and its involvement in the control of morphogenetic transitions. The work summarized here confirms the presence of PKC in yeast and mycelium extracts of S. schenckii. Different isoforms of this enzyme were found to be present in the yeast and mycelium forms of the fungus and were identified by Western blot analysis using affinity purified anti-PKC isoforms specific antibodies: the γ and ζ isoforms were detected in both the yeast and mycelium forms of the fungus, while the β isoform was only detected in the yeast form. The presence of PKC was confirmed biochemically by measuring total enzyme activity in both forms of the fungus. No significant differences were observed for the PKC activity level recorded for both the mycelium and yeast forms of the fungus (p ≤ 0.05). These data confirm the presence of PKC activity in Sporothrix schenckii and constitutes the first evidence concerning the differential expression of PKC isoforms in the mycelium and yeast forms of a dimorphic fungus, supporting the possible involvement of this important signal transduction enzyme in the control of morphogenesis in this fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic analysis of pleiotropic effects of pho85 mutations in yeast Saccharomyces cerevisiae

The cyclin-dependent phosphoprotein kinase Pho85p is involved in the regulation of metabolism and cell cycle in the yeast Saccharomyces cerevisiae. It is known that mutations in the PHO85 gene lead to constitutive synthesis of Pho5p acidic phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, sensitivity to high temperature, and other phenotypic effects. A lack of growth at 37 degrees C and on a medium with alcohol as the carbon source was shown to be associated with the rapid accumulation of nuclear ts and mitochondrial [rho-] mutations occurring in the background of gene PHO85 inactivation. Thus, Pho85p seems to play an important role in the maintenance of yeast genome stability.     

Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor

Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase.     

Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.     

Genetic Analysis of Pleiotropic Effects of pho85 Mutations in Yeast Saccharomyces cerevisiae

The cyclin-dependent phosphoprotein kinase Pho85p is involved in the regulation of metabolism and cell cycle in the yeast Saccharomyces cerevisiae. It is known that mutations in the PHO85gene lead to constitutive synthesis of Pho5p acidic phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, sensitivity to high temperature, and other phenotypic effects. A lack of growth at 37°C and on a medium with alcohol as the carbon source was shown to be associated with the rapid accumulation of nuclear ts and mitochondrial [rho ^– ] mutations occurring in the background of gene PHO85 inactivation. Thus, Pho85p seems to play an important role in the maintenance of yeast genome stability.     

Phosphorylation by Pho85 cyclin-dependent kinase acts as a signal for the down-regulation of the yeast sphingoid long-chain base kinase Lcb4 during the stationary phase

Sphingoid long-chain base 1-phosphates (LCBPs) act as bioactive lipid molecules in eukaryotic cells. In yeast, LCBPs are synthesized mainly by the long-chain base kinase Lcb4p. Until now, the regulatory mechanism for Lcb4p has been unclear. In the present study, we found that Lcb4p is post-translationally modified by phosphorylation. Using a protein kinase mutant yeast collection, we further demonstrated that the cyclin-dependent kinase Pho85p is involved in this phosphorylation. Pho85p functions in a number of cellular processes, especially in response to environmental changes. Two of 10 Pho85p cyclins, Pcl1p and Pcl2p had overlapping functions in the phosphorylation of Lcb4p. Site-directed mutagenesis identified the phosphorylation sites in Lcb4p as Ser(451) and Ser(455). Additionally, pulse-chase experiments revealed that Lcb4p is degraded via the ubiquitin-dependent pathway. The protein was stabilized in Deltapho85 cells, suggesting that phosphorylation acts as a signal for the degradation. Lcb4p is down-regulated in the stationary phase of cell growth, and both phosphorylation and ubiquitination appear to be important for this process. Moreover, we demonstrated that Lcb4p is delivered to the vacuole for degradation via the multivesicular body. Since forced accumulation of LCBPs results in prolonged growth during the stationary phase, down-regulation of Lcb4p may be physiologically important for proper cellular responses to nutrient deprivation.