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.

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.

Structured education can improve primary-care management of headache: the first empirical evidence, from a controlled interventional study.

BACKGROUND: Headache disorders are under-recognized and under-diagnosed. A principal factor in their suboptimal management is lack of headache-related training among health-care providers, especially in primary care. In Estonia, general practitioners (GPs) refer many headache patients to neurological specialist services, mostly unnecessarily. GPs request "diagnostic" investigations, which are usually unhelpful and therefore wasteful. GP-made headache diagnoses are often arcane and non-specific, and treatments based on these are inappropriate. The aim of this study was to develop, implement and test an educational model intended to improve headache-related primary health care in Estonia. METHODS: This was a controlled study consisting of baseline observation, intervention and follow-up observation using the same measures of effect. It involved six GPs in Põlva and the surrounding region in Southern Estonia, together with their future patients presenting consecutively with headache as their main complaint, all with their consent. The primary outcome measure was referral rate (RR) to neurological specialist services. Secondary measures included number of GP-requested investigations, GP-made headache diagnoses and how these conformed to standard terminology (ICD-10), and GP-recommended or initiated treatments. RESULTS: RR at baseline (n = 490) was 39.5 %, falling to 34.7 % in the post-intervention group (n = 295) (overall reduction 4.8 %; p = 0.21). In the large subgroup of patients (88 %) for whom GPs made clearly headache-related ICD-10 diagnoses, RR fell by one fifth (from 40 to 32 %; p = 0.08), but the only diagnosis-related RR that showed a statistically significant reduction was (pericranial) myalgia (19 to 3 %; p = 0.03). There was a significant increase towards use of more specific diagnoses. Use of investigations in diagnosing headache reduced from 26 to 4 % (p < 0.0001). Initiation of treatment by GPs increased from 58 to 81 % (p < 0.0001). CONCLUSIONS: These were modest changes in GPs' entrenched behaviour. Nevertheless they were empirical evidence that GPs' practice in the field of headache could be improved by structured education. Furthermore, the changes were likely to be cost-saving. To our knowledge this study is the first to produce such evidence.

Properties and use of novel replication-competent vectors based on Semliki Forest virus.

BACKGROUND: Semliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important. RESULTS: We analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture. CONCLUSION: The properties of replication-competent vectors of alphaviruses depend on the details of their construction. In the case of SFV4, such vectors should contain the SG promoter with structural characteristics for this isolate. The main factor for instability of SFV4-based replication-competent vectors was the deletion of genes of interest, since the resulting shorter genomes had a growth advantage over the original vector.

Multisite phosphorylation networks as signal processors for Cdk1.

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.