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.     

Lcb4p sphingoid base kinase localizes to the Golgi and late endosomes

Sphingoid long chain base phosphates (LCBPs) regulate cell proliferation, survival and motility in mammals. To learn more about LCBPs in Saccharomyces cerevisiae, we determined the cellular location of Lcb4p, the major enzyme catalyzing LCBP synthesis. By indirect immunofluorescence microscopy and subcellular fractionation, Lcb4p localizes to the trans-Golgi network and late endosomes and cycles between these compartments. Lcb4p faces the cytosol and is probably bound to membranes by protein-protein interactions. These results indicate that LCBs made in the endoplasmic reticulum must transit to the Golgi to be converted into LCBPs, which must then return to the endoplasmic reticulum to be degraded.     

Intracellular trafficking pathway of yeast long-chain base kinase Lcb4, from its synthesis to its degradation

Sphingoid long-chain base 1-phosphates act as bioactive lipid molecules in eukaryotic cells. In budding yeast, long-chain base 1-phosphates are synthesized mainly by the long-chain base kinase Lcb4. We recently reported that, soon after yeast cells enter into the stationary phase, Lcb4 is rapidly degraded by being delivered to the vacuole in a palmitoylation- and phosphorylation-dependent manner. In this study, we investigated the complete trafficking pathway of Lcb4, from its synthesis to its degradation. After membrane anchoring by palmitoylation at the Golgi apparatus, Lcb4 is delivered to the plasma membrane (PM) through the late Sec pathway and then to the endoplasmic reticulum (ER). The yeast ER consists of a cortical network juxtaposed to the PM (cortical ER) with tubular connections to the nuclear envelope (nuclear ER). Remarkably, the localization of Lcb4 is restricted to the cortical ER. As the cells reach the stationary phase, G(1) cell cycle arrest initiates Lcb4 degradation and its delivery to the vacuole via the Golgi apparatus. The protein transport pathway from the PM to the ER found in this study has not been previously reported. We speculate that this novel pathway is mediated by the PM-ER contact.     

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.     

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.     

Lcb4p is a key regulator of ceramide synthesis from exogenous long chain sphingoid base in Saccharomyces cerevisiae

Long chain sphingoid bases (LCBs) and their phosphates (LCBPs) are not only important intermediates in ceramide biosynthesis but also signaling molecules in the yeast, Saccharomyces cerevisiae. Their cellular levels, which control multiple cellular events in response to external and intrinsic signals, are tightly regulated by coordinated action of metabolic enzymes such as LCB kinase and LCBP phosphatase. However, little is known about the mechanisms by which the two enzymes generate biosynthetic or signaling outputs. It has been shown that the LCBP phosphatase, Lcb3p, is required for efficient ceramide synthesis from exogenous LCB. Here we present direct evidence that the major LCB kinase, Lcb4p, but not the minor kinase, Lcb5p, regulates synthesis of ceramide from exogenously added LCB. Surprisingly, our biochemical evidence suggests that the LCBP used for ceramide synthesis must be generated on the membrane. Our data show that Lcb4p is tightly associated with membranes and is localized to the endoplasmic reticulum where it can work in concert with Lcb3p. These results raise the conceptually attractive possibility that membrane-associated and cytosolic Lcb4p play distinct roles to differentially generate biosynthetic and signaling pools of LCBP.     

The yeast cyclins Pc16p and Pc17p are involved in the control of glycogen storage by the cyclin-dependent protein kinase Pho85p

Pho85p is a yeast cyclin-dependent protein kinase (Cdk) that can interact with 10 cyclins (Pcls) to form multiple protein kinases. The functions of most of the Pcls, including Pc16p and Pc17p, are poorly defined. We report here that Pc16p and Pc17p are involved in the metabolism of the branched storage polysaccharide glycogen under certain conditions and deletion of PCL6 and PCL7 restores glycogen accumulation to a snf1 pcl8 pcl10 triple mutant, paradoxically activating both glycogen synthase and phosphorylase. Pho85p thus affects glycogen accumulation through multiple Cdks composed of different cyclin partners.     

Long-chain base kinase Lcb4 Is anchored to the membrane through its palmitoylation by Akr1

Sphingoid long-chain base kinase Lcb4 catalyzes the production of the bioactive lipid molecules the long-chain base 1-phosphates. Although Lcb4 has no apparent transmembrane-spanning domain, it is tightly associated with the membrane. Here, we demonstrate that Lcb4 is modified by palmitoylation. This modification was greatly reduced in mutants for AKR1, which was recently identified as encoding a protein acyltransferase. In vitro experiments revealed that Akr1 indeed acts as a protein acyltransferase for Lcb4. Studies using site-directed mutagenesis indicated that Cys-43 and Cys-46 are palmitoylated. The loss of palmitoylation on Lcb4 caused several effects, including mislocalization of the protein to the cytosol, reduced phosphorylation, and loss of downregulation during the stationary phase. Although Akr2 is highly homologous to Akr1, the deletion of AKR2 did not result in any remarkable phenotypes. However, overproduction of Akr2 resulted in reduced amounts of Lcb4. We demonstrated that Akr2 is an unstable protein and is degraded in the vacuole. Akr2 exhibits high affinity for Lcb4, and in Akr2-overproducing cells this interaction caused unusual delivery of Lcb4 to the vacuole and degradation.     

Phosphorylation of the eukaryotic initiation factor 3f by cyclin-dependent kinase 11 during apoptosis

eIF3f is a subunit of eukaryotic initiation factor 3 (eIF3). We previously showed that eIF3f is phosphorylated by cyclin dependent kinase 11 (CDK11^p46) which is an important effector in apoptosis. Here, we identified a second eIF3f phosphorylation site (Thr119) by CDK11^p46 during apoptosis. We demonstrated that eIF3f is directly phosphorylated by CDK11^p46 in vivo. Phosphorylation of eIF3f plays an important role in regulating its function in translation and apoptosis. Phosphorylation of eIF3f enhances the association of eIF3f with the core eIF3 subunits during apoptosis. Our data suggested that eIF3f may inhibit translation by increasing the binding to the eIF3 complex during apoptosis.

Structured summary

MINT-6948874: EIF3b (uniprotkb:P55884) physically interacts (MI:0218) with EIF3f (uniprotkb:O00303) by anti bait coimmunoprecipitation (MI:0006)MINT-6948891: EIF3b (uniprotkb:P55884) physically interacts (MI:0218) with EIF3c (uniprotkb:Q99613), EIF3a (uniprotkb:Q14152) and EIF3f (uniprotkb:O00303) by anti bait coimmunoprecipitation (MI:0006)MINT-6948836, MINT-6948849, MINT-6948862: CDK11p46 (uniprotkb:P21127) phosphorylates (MI:0217) EIF3f (uniprotkb:O00303) by protein kinase assay (MI:0424)     

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.