A processive phosphorylation circuit with multiple kinase inputs and mutually diversional routes controls G1/S decision.

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.

Comparative study of microbiological, chemical and sensory properties of kefirs produced in Estonia, Latvia and Lithuania.

In the current study the microbiological, sensory and chemical properties of 24 kefirs (12 producers) from Estonian, Latvian and Lithuanian retail market were determined using gas chromatography (GC), high performance liquid chromatography (HPLC-MS/MS-Q-TOF and LC-ion trap MS/MS), spectrophotometry and other methods. Antihypertensive, angiotensin-converting enzyme (ACE) inhibiting, antioxidant and antibacterial peptides were found in the kefir samples. According to the results of principal component analysis of 200 most abundant compounds obtained with HPLC-MS/MS-Q-TOF analysis, Estonian kefirs differed from the rest. Kefirs of Latvian and Lithuanian origin showed similarities in several characteristics, probably related to the starter cultures and technological processes. The fatty acids composition of all Baltic kefirs was uniform. The antioxidant capacity of the kefirs varied slightly, whereas intermediate positive correlation (r = 0.32, P < 0.05) was found between antioxidativity and total bacterial count. The lipid oxidation level, estimated as the content of linoleic and oleic acid primary oxidation products, oxylipins, was very low in all studied kefirs. Only one third of analysed kefirs met the requirements of the minimum sum of viable microorganisms, indicated in the Codex Standard for Fermented Milks.

Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase.

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.

Dynamics of Cdk1 substrate specificity during the cell cycle.

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.

Ozone-triggered rapid stomatal response involves the production of reactive oxygen species, and is controlled by SLAC1 and OST1.

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.

Reversible and irreversible components of [(3)H]-N-propylnorapomorphine interaction with rat striatal membranes.

The kinetics of L-(-)-[N-propyl-(3)H(N)]-norapomorphine ([(3)H]NPA) interactions with rat striatal membranes were studied. The analysis revealed that in addition to specific dopaminergic binding a substantial part of the radioligand was bound irreversibly to heterogeneous populations of non-specific binding sites of these membranes. The specific binding of [(3)H]NPA with dopamine receptors, determined from the differences of kinetic curves of total and non-specific binding, was fast, reversible, and revealed high affinity. The irreversible component was heterogeneous and seems to be related to oxidative degradation of the radioligand, as the rate of this process was substantially reduced by antioxidants like ascorbic acid and dithiothreitol.