ABSTRACT
Cells make accurate decisions in the face of molecular noise and environmental fluctuations by relying not only on present pathway activity, but also on their memory of past signaling dynamics. Once a decision is made, cellular transitions are often rapid and switch-like due to positive feedback loops in the regulatory network. While positive feedback loops are good at promoting switch-like transitions, they are not expected to retain information to inform subsequent decisions. However, this expectation is based on our current understanding of network motifs that accounts for temporal, but not spatial, dynamics. Here, we show how spatial organization of the feedback-driven yeast G1/S switch enables the transmission of memory of past pheromone exposure across this transition. We expect this to be one of many examples where the exquisite spatial organization of the eukaryotic cell enables previously well-characterized network motifs to perform new and unexpected signal processing functions.
Subject(s)
Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/physiology , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , Cyclins/metabolism , Cytoplasm/metabolism , Feedback, Physiological , Guanine Nucleotide Exchange Factors/metabolism , Pheromones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal TransductionABSTRACT
Cyclin-dependent kinases (CDKs) coordinate hundreds of molecular events during the cell cycle. Multiple cyclins are involved, but the global role of cyclin-specific phosphorylation has remained unsolved. We uncovered a cyclin docking motif, LxF, that mediates binding of replication factor Cdc6 to mitotic cyclin. This interaction leads to phospho-adaptor Cks1-mediated inhibition of M-CDK to facilitate Cdc6 accumulation and sequestration in mitosis. The LxF motif and Cks1 also mediate the mutual inhibition between M-CDK and the tyrosine kinase Swe1. Additionally, the LxF motif is critical for targeting M-CDK to phosphorylate several mitotic regulators; for example, Spo12 is targeted via LxF to release the phosphatase Cdc14. The results complete the full set of G1, S, and M-CDK docking mechanisms and outline the unified role of cyclin specificity and CDK activity thresholds. Cooperation of cyclin and Cks1 docking creates a variety of CDK thresholds and switching orders, including combinations of last in, first out (LIFO) and first in, first out (FIFO) ordering.
Subject(s)
Cell Cycle Proteins/genetics , Cyclins/genetics , G1 Phase Cell Cycle Checkpoints/genetics , Gene Expression Regulation, Fungal , M Phase Cell Cycle Checkpoints/genetics , S Phase Cell Cycle Checkpoints/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs , Binding Sites , Cell Cycle Proteins/metabolism , Cyclins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphorylation , Protein Binding , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal TransductionABSTRACT
Cyclin-dependent kinases (CDKs), the master regulators of cell division, are activated by different cyclins at different cell cycle stages. In addition to being activators of CDKs, cyclins recognize various linear motifs to target CDK activity to specific proteins. We uncovered a cyclin docking motif, NLxxxL, that contributes to phosphorylation-dependent degradation of the CDK inhibitor Far1 at the G1/S stage in the yeast Saccharomyces cerevisiae. This motif is recognized exclusively by S-phase CDK (S-CDK) Clb5/6-Cdc28 and is considerably more potent than the conventional RxL docking motif. The NLxxxL and RxL motifs were found to overlap in some target proteins, suggesting that cyclin docking motifs can evolve to switch from one to another for fine-tuning of cell cycle events. Using time-lapse fluorescence microscopy, we show how different docking connections temporally control phosphorylation-driven target degradation. This also revealed a differential function of the phosphoadaptor protein Cks1, as Cks1 docking potentiated degron phosphorylation of RxL-containing but not of NLxxxL-containing substrates. The NLxxxL motif was found to govern S-cyclin-specificity in multiple yeast CDK targets including Fin1, Lif1, and Slx4, suggesting its wider importance.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , S Phase/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Signal Transduction/physiology , Amino Acid Sequence , Cell Cycle Proteins/metabolism , Phosphorylation/physiologyABSTRACT
Cyclins are the activators of cyclin-dependent kinase (CDK) complex, but they also act as docking scaffolds for different short linear motifs (SLiMs) in CDK substrates and inhibitors. According to the unified model of CDK function, the cell cycle is coordinated by CDK both via general CDK activity thresholds and cyclin-specific substrate docking. Recently, it was found that the G1-cyclins of S. cerevisiae have a specific function in promoting polarization and growth of the buds, making the G1 cyclins essential for cell survival. Thus, while a uniform CDK specificity of a single cyclin can be sufficient to drive the cell cycle in some cells, such as in fission yeast, cyclin specificity can be essential in other organisms. However, the known G1-CDK specific LP docking motif, was not responsible for this essential function, indicating that G1-CDKs use yet other unknown docking mechanisms. Here we report a discovery of a G1 cyclin-specific (Cln1,2) lysine-arginine-rich helical docking motif (the K/R motif) in G1-CDK targets involved in the mating pathway (Ste7), transcription (Xbp1), bud morphogenesis (Bud2) and spindle pole body (Spc29, Spc42, Spc110, Sli15) function of S. cerevisiae. We also show that the docking efficiency of K/R motif can be regulated by basophilic kinases such as protein kinase A. Our results further widen the list of cyclin specificity mechanisms and may explain the recently demonstrated unique essential function of G1 cyclins in budding yeast.
Subject(s)
CDC2 Protein Kinase/chemistry , Multienzyme Complexes/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Amino Acid Motifs , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Phosphorylation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Activation of the guard cell S-type anion channel SLAC1 is important for stomatal closure in response to diverse stimuli, including elevated CO2 The majority of known SLAC1 activation mechanisms depend on abscisic acid (ABA) signaling. Several lines of evidence point to a parallel ABA-independent mechanism of CO2-induced stomatal regulation; however, molecular details of this pathway remain scarce. Here, we isolated a dominant mutation in the protein kinase HIGH LEAF TEMPERATURE1 (HT1), an essential regulator of stomatal CO2 responses, in an ozone sensitivity screen of Arabidopsis thaliana The mutation caused constitutively open stomata and impaired stomatal CO2 responses. We show that the mitogen-activated protein kinases (MPKs) MPK4 and MPK12 can inhibit HT1 activity in vitro and this inhibition is decreased for the dominant allele of HT1. We also show that HT1 inhibits the activation of the SLAC1 anion channel by the protein kinases OPEN STOMATA1 and GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) in Xenopus laevis oocytes. Notably, MPK12 can restore SLAC1 activation in the presence of HT1, but not in the presence of the dominant allele of HT1. Based on these data, we propose a model for sequential roles of MPK12, HT1, and GHR1 in the ABA-independent regulation of SLAC1 during CO2-induced stomatal closure.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Mitogen-Activated Protein Kinases/metabolism , Plant Stomata/metabolism , Protein Kinases/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Carbon Dioxide/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinases/genetics , Mutation/genetics , Plant Stomata/genetics , Protein Kinases/genetics , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2 for photosynthesis, loss of water, and entrance of air pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata, and stomatal CO2-insensitivity phenotypes of the Arabidopsis thaliana accession Cvi-0 to a single amino acid substitution in MITOGEN-ACTIVATED PROTEIN (MAP) KINASE 12 (MPK12). In parallel, we showed that stomatal CO2-insensitivity phenotypes of a mutant cis (CO2-insensitive) were caused by a deletion of MPK12. Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HIGH LEAF TEMPERATURE 1 (HT1)-a central node in guard cell CO2 signaling-and that MPK12 functions as an inhibitor of HT1. These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2 signaling controls plant water management.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Carbon Dioxide/metabolism , Genetic Variation , Mitogen-Activated Protein Kinases/metabolism , Signal Transduction , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Chromosome Mapping , Ozone/metabolism , Photosynthesis , Quantitative Trait Loci , WaterABSTRACT
Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.
Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Motifs , Binding Sites , CDC2 Protein Kinase/chemistry , Consensus Sequence , Hydrophobic and Hydrophilic Interactions , Models, Biological , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/chemistry , Substrate SpecificityABSTRACT
The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.
Subject(s)
CDC2 Protein Kinase/metabolism , Kinesins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Anaphase/physiology , Catalytic Domain , Cyclin B/metabolism , Kinesins/chemistry , Kinesins/genetics , Microtubules/metabolism , Mutagenesis, Site-Directed , Phosphorylation , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Spindle Apparatus/metabolismABSTRACT
Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.
Subject(s)
Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , S Phase/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Binding Sites , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cyclin B/metabolism , Cyclins/metabolism , Phosphorylation , ProteolysisABSTRACT
Protein synthesis underpins cell growth and controls when cells commit to a new round of cell division at a point in late G1 of the cell cycle called Start. Passage through Start also coincides with the duplication of the microtubule-organizing centers, the yeast spindle pole bodies, which will form the 2 poles of the mitotic spindle that segregates the chromosomes in mitosis. The conserved Mps1p kinase governs the duplication of the spindle pole body (SPB) in Saccharomyces cerevisiae. Here, we show that the MPS1 transcript has a short upstream open reading frame (uORF) that represses the synthesis of Mps1p. Mutating the MPS1 uORF makes the cells smaller, accelerates the appearance of Mps1p in late G1, and promotes completion of Start. Monitoring the SPB in the cell cycle using structured illumination microscopy revealed that mutating the MPS1 uORF enabled cells to duplicate their SPB earlier at a smaller cell size. The accelerated Start of MPS1 uORF mutants depends on the G1 cyclin Cln3p and the transcriptional repressor Whi5p but not on the Cln1,2p G1 cyclins. These results identify growth inputs in mechanisms that control duplication of the microtubule-organizing center and implicate these processes in the coupling of cell growth with division.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Spindle Pole Bodies , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Spindle Pole Bodies/metabolism , Spindle Pole Bodies/genetics , Cyclins/metabolism , Cyclins/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Open Reading Frames , Protein Biosynthesis , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Division/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Gene Expression Regulation, FungalABSTRACT
Since the discovery of cyclin-dependent kinases (CDKs), it has been perceived as a dogma that CDK signaling in the cell cycle is mediated via targeting the CDK consensus sites: the optimal and the minimal motifs S/T-P-x-K/R and S/T-P, respectively. However, more recent evidence suggests that often the CDK phosphorylation events of regulatory importance are mediated via nonconventional CDK sites that lack the required +1Pro of the consensus site motif. In these cases, the loss of specificity seems to be compensated via distant docking interactions facilitated by 1) phosphorylated priming sites binding to phospho-adaptor Cks1 and/or 2) cyclin-specific docking interactions via Short Linear Motifs (SLiMs) in substrates. This Perspective discusses the possible reasons why nonconventional CDK sites are used for CDK signaling. First, the nonconventional CDK sites can act as specificity filters to recognize and distinguish the CDK signal from many other proline-directed kinases in cells. Second, the nonconventional CDK sites in combination with the docking mechanisms provide a much wider range of phosphorylation rates, and thus, also a wider range of CDK thresholds during the accumulation and decline of CDK activity during the cell cycle. As a large number of Cks1-dependent nonconventional CDK sites have been discovered recently, past studies focusing on mutating only the consensus sites should likely be critically reexamined. It is also very likely that phosphorylation of nonconventional sites is crucial in many other kinase-signaling networks.
Subject(s)
Saccharomyces cerevisiae Proteins , Phosphorylation , Saccharomyces cerevisiae Proteins/metabolism , Cyclin-Dependent Kinases/metabolism , Signal Transduction , Cyclins/metabolismABSTRACT
The air pollutant ozone can be used as a tool to unravel in planta processes induced by reactive oxygen species (ROS). Here, we have utilized ozone to study ROS-dependent stomatal signaling. We show that the ozone-triggered rapid transient decrease (RTD) in stomatal conductance coincided with a burst of ROS in guard cells. RTD was present in 11 different Arabidopsis ecotypes, suggesting that it is a genetically robust response. To study which signaling components or ion channels were involved in RTD, we tested 44 mutants deficient in various aspects of stomatal function. This revealed that the SLAC1 protein, essential for guard cell plasma membrane S-type anion channel function, and the protein kinase OST1 were required for the ROS-induced fast stomatal closure. We showed a physical interaction between OST1 and SLAC1, and provide evidence that SLAC1 is phosphorylated by OST1. Phosphoproteomic experiments indicated that OST1 phosphorylated multiple amino acids in the N terminus of SLAC1. Using TILLING we identified three new slac1 alleles where predicted phosphosites were mutated. The lack of RTD in two of them, slac1-7 (S120F) and slac1-8 (S146F), suggested that these serine residues were important for the activation of SLAC1. Mass-spectrometry analysis combined with site-directed mutagenesis and phosphorylation assays, however, showed that only S120 was a specific phosphorylation site for OST1. The absence of the RTD in the dominant-negative mutants abi1-1 and abi2-1 also suggested a regulatory role for the protein phosphatases ABI1 and ABI2 in the ROS-induced activation of the S-type anion channel.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Membrane Proteins/metabolism , Ozone/pharmacology , Plant Stomata/physiology , Protein Kinases/metabolism , Reactive Oxygen Species/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant , Membrane Proteins/genetics , Mutagenesis, Site-Directed , Phosphorylation , Protein Kinases/geneticsABSTRACT
Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) within proteins have attracted considerable attention in recent years. Several important biological signaling mechanisms including protein-protein interactions and post-translational modifications can be easily mediated by IDPs and IDRs due to their flexible structure. These regions can encode linear sequences that are indispensable in cell-signaling networks and circuits. For example, the linear multisite phosphorylation networks encoded in disordered protein sequences play a key role in cell-cycle regulation where the phosphorylation of proteins controls the orchestration of all major mechanisms. While elucidating a systems-level understanding of this process and other multisite phosphorylation processes, we extensively used mass-spectrometry and found it to be an ideal tool to identify, characterize, and quantify phosphorylation dynamics within IDPs. Here, we describe a quantitative proteomics method, together with a detailed protocol to analyze dynamic multisite phosphorylation processes within IDPs using an in vitro protein phosphorylation assay with "light" gamma-16O ATP and "heavy" gamma-18O ATP, combined with liquid chromatography mass spectrometry.
Subject(s)
Chromatography, Liquid/methods , Intrinsically Disordered Proteins/chemistry , Isotope Labeling/methods , Protein Processing, Post-Translational , Spectrometry, Mass, Electrospray Ionization/methods , Adenosine Triphosphate/chemistry , Amino Acid Sequence , Autoradiography/methods , Cell Cycle Proteins/analysis , Cell Cycle Proteins/chemistry , Electrophoresis, Polyacrylamide Gel/methods , Intrinsically Disordered Proteins/analysis , Molecular Structure , Oxygen , Oxygen Isotopes , Phosphorylation , Rosaniline Dyes , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/chemistry , Spectrometry, Mass, Electrospray Ionization/instrumentation , Staining and Labeling/methodsABSTRACT
Studies on multisite phosphorylation networks of cyclin-dependent kinase (CDK) targets have opened a new level of signaling complexity by revealing signal processing routes encoded into disordered proteins. A model target, the CDK inhibitor Sic1, contains linear phosphorylation motifs, docking sites, and phosphodegrons to empower an N-to-C terminally directed phosphorylation process. Here, we uncover a signal processing mechanism involving multi-step competition between mutually diversional phosphorylation routes within the S-CDK-Sic1 inhibitory complex. Intracomplex phosphorylation plays a direct role in controlling Sic1 degradation, and provides a mechanism to sequentially integrate both the G1- and S-CDK activities while keeping S-CDK inhibited towards other targets. The competing phosphorylation routes prevent premature Sic1 degradation and demonstrate how integration of MAPK from the pheromone pathway allows one to tune the competition of alternative phosphorylation paths. The mutually diversional phosphorylation circuits may be a general way for processing multiple kinase signals to coordinate cellular decisions in eukaryotes.
Subject(s)
G1 Phase/physiology , S Phase/radiation effects , Signal Transduction/physiology , Blotting, Western , Cell Division/genetics , Cell Division/physiology , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , G1 Phase/genetics , Immunoprecipitation , Mass Spectrometry , Phosphorylation , S Phase/genetics , Signal Transduction/geneticsABSTRACT
The hydrophobic patch (hp), a docking pocket on cyclins of CDKs (cyclin-dependent kinases), has been thought to accommodate a single short linear motif (SLiM), the "RxL or Cy" docking motif. Here we show that hp can bind different motifs with high specificity. We identify a PxxPxF motif that is necessary for G2-cyclin Clb3 function in S. cerevisiae, and that mediates Clb3-Cdk1 phosphorylation of Ypr174c (proposed name: Cdc5 SPB anchor-Csa1) to regulate the localization of Polo kinase Cdc5. Similar motifs exist in other Clb3-Cdk1 targets. Our work completes the set of docking specificities for the four major cyclins: LP, RxL, PxxPxF, and LxF motifs for G1-, S-, G2-, and M-phase cyclins, respectively. Further, we show that variations in motifs can change their specificity for human cyclins. This diversity could provide complexity for the encoding of CDK thresholds to achieve ordered cell-cycle phosphorylation.
Subject(s)
Cell Cycle Checkpoints/physiology , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinases/metabolism , Proline/metabolism , Cell Cycle/physiology , Humans , Phosphorylation , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Substrate Specificity/physiologyABSTRACT
BACKGROUND AND OBJECTIVE: Activated bronchial epithelial cells exert considerable potential to maintain a microenvironment in the airway wall that promotes airway inflammation and remodelling. Cysteinyl leucotrienes (CysLT) and transforming growth factor-beta(1) (TGF-beta(1)) are both increased in asthmatic airways and may influence the pathophysiology of disease. However, the consequences of activation of bronchial epithelial cells by these mediators are not fully understood. A proteomic-based approach was used to characterize the inflammatory pathways in bronchial epithelial cells after stimulation with CysLT and TGF-beta(1). METHODS: Human bronchial epithelial cells (BEAS-2B) were stimulated with 1 ng/mL TGF-beta(1) and 50 nmol/L leucotriene E(4) (LTE(4)) for 48 h and whole-cell lysates were subjected to two-dimensional gel electrophoresis. Proteins showing statistically significant differential expression were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searching. RESULTS: Stimulation with LTE(4) increased the expression of three proteins and five proteins showed decreased expression. Of the latter group, two were definitively identified as heat shock protein (Hsp90 alpha) and stress-70 protein. Hsp90 alpha forms a heterocomplex with the glucocorticoid receptor (GR) and a significant decrease in GR following LTE(4) stimulation was confirmed. TGF-beta(1) downregulated 18 intracellular proteins, including lamin A/C, glyceraldehyde-3-phosphate dehydrogenase, protein DJ-1, voltage-dependent calcium channel gamma-7 subunit, heterogeneous nuclear ribonucleoprotein A2/B1 and stress-70 protein. CONCLUSIONS: The current findings suggest that by downregulating GR and Hsp90 alpha, CysLT may interfere with the action of glucocorticoids. Overall, the results confirm the complex role of bronchial epithelium in aspects of airway inflammation and remodelling.
Subject(s)
Asthma/physiopathology , Bronchi/cytology , Leukotriene E4/metabolism , Proteome/biosynthesis , Respiratory Mucosa/metabolism , Transforming Growth Factor beta1/metabolism , Cells, Cultured , Down-Regulation , Electrophoresis, Gel, Two-Dimensional , Heat-Shock Proteins/biosynthesis , Humans , Proteomics , Receptors, Glucocorticoid/biosynthesisABSTRACT
Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions, but some substrates maintain precise phosphosite positions over billions of years. Here, we examined the phosphoregulation of the kinesin-5, Cin8, using synthetic Cdk1-sites. We first analyzed the three native Cdk1 sites within the catalytic motor domain. Any single site conferred regulation, but to different extents. Synthetic sites were then systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites, 8 disrupted function; 19 were neutral, similar to the phospho-deficient variant; and only two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one was immediately adjacent to a native Cdk1 site. Only one novel site position resulted in phospho-regulation. This site was sampled elsewhere in evolution, but the synthetic version was inefficient in S. cerevisiae. This study shows that a single phosphorylation site can modulate complex spindle dynamics, but likely requires further evolution to optimally regulate the precise reaction cycle of a mitotic motor.
Subject(s)
Evolution, Molecular , Kinesins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , CDC2 Protein Kinase/metabolism , CDC2 Protein Kinase/physiology , Kinesins/chemistry , Models, Molecular , Phosphorylation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Sequence Analysis, Protein , Spindle Apparatus/metabolism , Spindle Apparatus/ultrastructureABSTRACT
The quantitative model of cyclin-dependent kinase (CDK) function states that cyclins temporally order cell cycle events at different CDK activity levels, or thresholds. The model lacks a mechanistic explanation, as it is not understood how different thresholds are encoded into substrates. We show that a multisite phosphorylation code governs the phosphorylation of CDK targets and that phosphorylation clusters act as timing tags that trigger specific events at different CDK thresholds. Using phospho-degradable CDK threshold sensors with rationally encoded phosphorylation patterns, we were able to predictably program thresholds over the entire range of the Saccharomyces cerevisiae cell cycle. We defined three levels of CDK multisite phosphorylation encoding: (i) serine-threonine swapping in phosphorylation sites, (ii) patterning of phosphorylation sites, and (iii) cyclin-specific docking combined with modulation of CDK activity. Thus, CDK can signal via hundreds of differentially encoded targets at precise times to provide a temporally ordered phosphorylation pattern required for cell division.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Cycle , Models, Biological , Phosphorylation , Saccharomyces cerevisiae/cytologyABSTRACT
BACKGROUND: Eukaryotic cell division is driven by cyclin-dependent kinases (CDKs). Distinct cyclin-CDK complexes are specialized to drive different cell-cycle events, though the molecular foundations for these specializations are only partly understood. In budding yeast, the decision to begin a new cell cycle is regulated by three G1 cyclins (Cln1-Cln3). Recent studies revealed that some CDK substrates contain a novel docking motif that is specifically recognized by Cln1 and Cln2, and not by Cln3 or later S- or M-phase cyclins, but the responsible cyclin interface was unknown. RESULTS: Here, to explore the role of this new docking mechanism in the cell cycle, we first show that it is conserved in a distinct cyclin subtype (Ccn1). Then, we exploit phylogenetic variation to identify cyclin mutations that disrupt docking. These mutations disrupt binding to multiple substrates as well as the ability to use docking sites to promote efficient, multi-site phosphorylation of substrates in vitro. In cells where the Cln2 docking function is blocked, we observed reductions in the polarized morphogenesis of daughter buds and reduced ability to fully phosphorylate the G1/S transcriptional repressor Whi5. Furthermore, disruption of Cln2 docking perturbs the coordination between cell size and division, such that the G1/S transition is delayed. CONCLUSIONS: The findings point to a novel substrate interaction interface on cyclins, with patterns of conservation and divergence that relate to functional distinctions among cyclin subtypes. Furthermore, this docking function helps ensure full phosphorylation of substrates with multiple phosphorylation sites, and this contributes to punctual cell-cycle entry.
Subject(s)
Cell Cycle/physiology , Cyclins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Amino Acid Sequence , Binding Sites/genetics , Cyclins/genetics , Flow Cytometry , Molecular Sequence Data , Phosphorylation , Protein Binding , Real-Time Polymerase Chain Reaction , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Time-Lapse ImagingABSTRACT
The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, distribution of serines and threonines as phosphoacceptors and positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phosphoadaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within networks of phosphorylation sites in different targets provides a wide range of possibilities for differential amplification of Cdk1 signals, thus providing a mechanism to generate a wide range of thresholds in the cell cycle.