Regulation of yeast glycogen phosphorylase by the cyclin-dependent protein kinase Pho85p

Yeast accumulate glycogen in response to nutrient limitation. The key enzymes of glycogen synthesis and degradation, glycogen synthase, and phosphorylase, are regulated by reversible phosphorylation. Phosphorylation inactivates glycogen synthase but activates phosphorylase. The kinases and phosphatases that control glycogen synthase are well characterized whilst the enzymes modifying phosphorylase are poorly defined. Here, we show that the cyclin-dependent protein kinase, Pho85p, which we have previously found to regulate glycogen synthase also controls the phosphorylation state of phosphorylase.     

Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p

In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.     

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.     

Structure and function of cyclin-dependent Pho85 kinase of Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae has five cyclin-dependent protein kinases (Cdks), Cdc28, Srb10, Kin28, Ctk1, and Pho85. Any of these Cdks requires a cyclin partner for its kinase activity and a Cdk/cyclin complex, thus produced, phosphorylates a set of specific substrate proteins to exert its function. The cyclin partners of Srb10, Kin28, and Ctk1 are Srb11, Ccl1, and Ctk2, respectively. In contrast to the fact that each of Srb10, Kin28, and Ctk1 has a single cyclin partner, Cdc28 and Pho85 are polygamous; Cdc28 has 9 cyclins and Pho85 has 10 cyclins. Among these Cdks, Kin28 and Cdc28 are essential Cdks and it is well known that Cdc28 kinase plays a major role in regulating cell cycle progression. Pho85 is a non-essential Cdk but its absence causes a broad spectrum of phenotypes such as constitutive expression of PHO5, inability to utilize non-fermentable carbon sources, defects in cell cycle progression, and so on. Pho85 homologues are expanding to higher eukaryotes. Pho85 is most closely related with Cdk5 in terms of the amino acid sequence. The functional analysis of the domains of Pho85 also supports the close relationship between Pho85 and Cdk5, in which it was shown that the method of regulation of these two kinases is similar. Furthermore, forced expression of the mammalian CDK5 gene in a pho85Delta strain canceled a part of the pho85 defects. In this review, we summarize the functions of both Pho85/cyclin kinase and emphasize yeast Pho85 as valuable model systems to elucidate the functions of their homologues in other organisms.     

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.     

Genetic evidence for a morphogenetic function of the Saccharomyces cerevisiae Pho85 cyclin-dependent kinase

The Saccharomyces cerevisiae PHO85 gene encodes a nonessential cyclin-dependent kinase that associates with 10 cyclin subunits. To survey the functions provided by Pho85, we identified mutants that require PHO85 for viability. We identified mutations that define seven Pho Eighty-Five Requiring or Efr loci, six of which are previously identified genes-BEM2 (YER155C), SPT7 (YBR081C), GCR1 (YPL075W), SRB5 (YGR104C), HFI1 (YPL254W), and BCK1 (YJL095W)-with one novel gene (YMR212C). We found that mutations in the EFR genes involved in morphogenesis are specifically inviable when the Pho85-associated G1 cyclins encoded by PCL1 and PCL2 are absent. pcl1 Delta bem2, pcl1 Delta pcl2 Delta cla4 Delta, and pcl1 Delta pcl2 Delta cdc42-1 strains are inviable. pcl1 Delta pcl2 Delta mpk1 Delta, pcl1 Delta pcl2 Delta bck1, and pcl1 Delta pcl2 Delta cln1 Delta cln2 Delta strains are also inviable, but are rescued by osmotic stabilization with 1 m sorbitol. We propose that the G1 cyclins encoded by PCL1 and PCL2 positively regulate CDC42 or another morphogenesis promoting function.     

Expression of a Pho85 cyclin-dependent kinase is repressed during the dimorphic transition in Sporothrix schenckii

Sporothrix schenckii is a pathogenic fungus that undergoes a dimorphic transition from yeast to mycelium in response to environmental conditions such as cell density, temperature, and calcium. We identified a homolog of the Pho85 cyclin-dependent kinase (Cdk) that mediates cellular responses to environmental conditions in other organisms. By Western blot, three proteins containing the PSTAIRE motif, which characterize the cyclin-dependent protein kinases, were identified in S. schenckii. The gene encoding a Pho85 homolog, PhoSs, was identified and sequenced. The phoSs gene consists of 990bp, contains one intron, and encodes a protein of 306 amino acids. The S. schenckii Pho85 homolog shares features with Cdks, including the PSTAIRE motif, an ATP binding domain, and a serine-threonine kinase domain. By quantitative competitive RT-PCR, expression of the phoSs gene was found to decrease 30-fold during the yeast to mycelium transition. The addition of extracellular calcium accelerated the dimorphic transition and restored phoSs expression. These findings suggest PhoSs may participate in the control of the yeast to mycelium transition in S. schenckii.     

Identification of Xenopus cyclin-dependent kinase inhibitors, p16Xic2 and p17Xic3

The Cip/Kip family of mammalian cyclin-dependent kinase (cdk) inhibitors plays important roles in development, particularly in cell fate determination and differentiation, in addition to their function of blocking cell cycle progression. We have identified two novel members of the Kip/Cip cdk inhibitor family, p16Xic2 and p17Xic3, from Xenopus laevis. Sequence analysis revealed that p16Xic2 and p17Xic3 are orthologues of mammalian p21Cip1 and p27Kip1, respectively. Overexpression of these inhibitors results in cell cycle arrest by inhibition of cdk2 activity. Interestingly, the expression of these inhibitors is highly developmentally regulated. p16Xic2 is highly expressed in differentiating somite, tail bud, lens, and cement gland, while p17Xic3 is expressed in the central nervous system. In a retinal cell fate determination assay, both p16Xic2 and p17Xic3 have an activity that influences cell fate determination. These observations suggest that p16Xic2 and p17Xic3 might be involved in cell fate determination in a tissue-specific manner by coordinating proliferation and differentiation as observed with p27Xic1.     

The absence of cyclin-dependent protein kinase Pho85 affects stability of mitochondrial DNA in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The cyclin-dependent protein kinase Pho85 is involved in the regulation of phosphate metabolism in yeast Saccharomyces cerevisiae. Mutations in the PHO85 gene lead to constitutive synthesis of Pho5 acid phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, and other pleiotropic effects. In this work, it was shown that the accumulation of respiratory incompetent cells occurs with high frequency in strains carrying pho85 mutations as early as during the first cell divisions, and the number of these cells at the early logarithmic growth phase of the culture promptly reaches virtually 100%. Cytological analysis revealed a high accumulation rate of [rho⁰] cells in the background of gene pho85 that may be related to disturbances in the distribution of mitochondrial nucleoids rather than to changes in morphology of mitochondria and a delay in their transport into the bud. Genetic analysis revealed that secondary mutations pho4, pho81, pho84, and pho87 stabilize nucleoids and prevent the loss of mitochondrial DNA caused by pho85. These results provide an evidence for the influence of intracellular phosphate concentration on the inheritance of mitochondrial nucleoids, but do not exclude the possibility that the occurrence of mutation pho4 in the background of gene pho85 may change the expression level of other genes required for the stabilization of mitochondrial functions.     

The extracellular matrix and mitogenic growth factors control G1 phase cyclins and cyclin-dependent kinase inhibitors

Most cell types require both mitogenic growth factors and cell adhesion to the extracellular matrix (ECM) for proliferation. Over the past few years, these growth requirements have received renewed attention and can now be explained by studies showing that signals provided by growth factors and the ECM are jointly required to stimulate the cyclin-dependent kinases (CDKs) that mediate cell-cycle progression through G1 phase. This article summarizes our current understanding of the control of G1 cyclins and CDK inhibitors by growth factors and the ECM. In addition, we have highlighted one or two signal-transduction pathways that presently seem closely linked to regulation of the G1 phase cyclin-CDK system.     

Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25.

Paullones constitute a new family of benzazepinones with promising antitumoral properties. They were recently described as potent, ATP-competitive, inhibitors of the cell cycle regulating cyclin-dependent kinases (CDKs). We here report that paullones also act as very potent inhibitors of glycogen synthase kinase-3beta (GSK-3beta) (IC50: 4-80 nM) and the neuronal CDK5/p25 (IC50: 20-200 nM). These two enzymes are responsible for most of the hyperphosphorylation of the microtubule-binding protein tau, a feature observed in the brains of patients with Alzheimer's disease and other neurodegenerative 'taupathies'. Alsterpaullone, the most active paullone, was demonstrated to act by competing with ATP for binding to GSK-3beta. Alsterpaullone inhibits the phosphorylation of tau in vivo at sites which are typically phosphorylated by GSK-3beta in Alzheimer's disease. Alsterpaullone also inhibits the CDK5/p25-dependent phosphorylation of DARPP-32 in mouse striatum slices in vitro. This dual specificity of paullones may turn these compounds into very useful tools for the study and possibly treatment of neurodegenerative and proliferative disorders.     

The cyclin-dependent kinase Cdc28p regulates multiple aspects of Kar9p function in yeast

During mitosis in the yeast Saccharomyces cerevisiae, Kar9p directs one spindle pole body (SPB) toward the incipient daughter cell by linking the associated set of cytoplasmic microtubules (cMTs) to the polarized actin network on the bud cortex. The asymmetric localization of Kar9p to one SPB and attached cMTs is dependent on its interactions with microtubule-associated proteins and is regulated by the yeast Cdk1 Cdc28p. Two phosphorylation sites in Kar9p were previously identified. Here, we propose that the two sites are likely to govern Kar9p function through separate mechanisms, each involving a distinct cyclin. In the first mechanism, phosphorylation at serine 496 recruits Kar9p to one SPB. A phosphomimetic mutation at serine 496 bypasses the requirement of BIK1 and CLB5 in generating Kar9p asymmetry. In the second mechanism, Clb4p may target serine 197 of Kar9p for phosphorylation. This modification is required for Kar9p to direct cMTs to the bud. Two-hybrid analysis suggests that this phosphorylation may attenuate the interaction between Kar9p and the XMAP215-homologue Stu2p. We propose that phosphorylation at serine 197 regulates the release of Kar9p from Stu2p at the SPB, either to clear it from the mother-SPB or to allow it to travel to the plus end.     

Activation of the Cdc42p GTPase by cyclin-dependent protein kinases in budding yeast

Cyclin-dependent kinases (CDKs) trigger essential cell cycle processes including critical events in G1 phase that culminate in bud emergence, spindle pole body duplication, and DNA replication. Localized activation of the Rho-type GTPase Cdc42p is crucial for establishment of cell polarity during G1, but CDK targets that link the Cdc42p module with cell growth and cell cycle commitment have remained largely elusive. Here, we identify the GTPase-activating protein (GAP) Rga2p as an important substrate related to the cell polarity function of G1 CDKs. Overexpression of RGA2 in the absence of functional Pho85p or Cdc28p CDK complexes is toxic, due to an inability to polarize growth. Mutation of CDK consensus sites in Rga2p that are phosphorylated both in vivo and in vitro by Pho85p and Cdc28p CDKs results in a loss of G1 phase-specific phosphorylation. A failure to phosphorylate Rga2p leads to defects in localization and impaired polarized growth, in a manner dependent on Rga2p GAP function. Taken together, our data suggest that CDK-dependent phosphorylation restrains Rga2p activity to ensure appropriate activation of Cdc42p during cell polarity establishment. Inhibition of GAPs by CDK phosphorylation may be a general mechanism to promote proper G1-phase progression.