Objective
Smooth muscle contractility is controlled by regulatory proteins associated with the myosin and acting filaments. Whilst activation of myosin by Ca2+ dependent phosphorylation is the primary regulator in all smooth muscles, tonic smooth muscles (e.g. vascular smooth muscle, airway smooth muscle) are also regulated by the 'chemoreceptor' pathway, which involves cell signalling kinases and phosphorylation of target nuclear and cytoplasmic proteins. Caldesmon is the regulatory protein of the thin filaments; it inhibits contractile activity and it is regulated by Ca2+-calmodulin and also by phosphorylation. The Moscow laboratory has recently established that at least 90% of caldesmon phosphorylation in vivo is due to MAP kinase. In mammalian caldesmon there are three potential MAP kinase sites: Thr730, Ser759 and Ser799 (absent in chicken). Whilst much investigation has been concentrated on the effects of Ser759 phosphorylation, recent results form the Moscow lab indicate that in vivo the predominant site of phosporylation in PdBu-stimulated arterial smooth muscle is probably Thr730, however there may be more additional N-terminal sites.
It is proposed to study the physiological, functional and structural consequences of caldesmon phosphorylation. The initial task of this study will be to determine accurately the in vivo phosphorylation sites of caldesmon under physiological stimulation.
The assignment of sites will be confirmed by phosphorylating deletion mutants of caldesmon containing just one of the identified sites with MAP kinase in vitro. It is then planned to develop a phosphospecific antibody to each site to monitor intracellular interactions of caldesmon and the level of agonist-stimulated phosphorylation of caldesmon. This will be used to explore the contribution of site-specific phosphorylation in regulation of smooth muscle contraction/relaxation by caldesmon.
The functional effects of Thr730 phosphorylation on actomyosin interaction will be investigated in vitro by ATPase and in vitro motility assays using well-characterised deletion mutants of caldesmon. Specific phosphorylation will be achieved by making Ser/Thr>Ala point mutants. It is expected that phosphorylation will reduce inhibitory function and interaction with Ca2+-calmodulin and that this will primarily affect the cooperativity of the regulated thin filament. The effects of phosphorylation on the structure of caldesmon domain 4 will be determined by high-resolution protein NMR and the effect on caldesmon-actin interaction will be studied by heteronuclear NMR, SPR and by the unique method of Dr Borovikov, which detects conformational changes in actin molecules in ghost fibres, which have an intact actin filament lattice. The functional effects of caldesmon phosphorylation on contractility in a more physiological system will be tested in chemically skinned smooth muscle fibres by replacing native caldesmon with recombinant caldesmon deletion mutants and specifically phosphorylated caldesmon mutants.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
This project has not yet been classified with EuroSciVoc.
Be the first one to suggest relevant scientific fields and help us improve our classification service
You need to log in or register to use this function
Programme(s)
Multi-annual funding programmes that define the EU’s priorities for research and innovation.
Multi-annual funding programmes that define the EU’s priorities for research and innovation.
Topic(s)
Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.
Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.
Call for proposal
Procedure for inviting applicants to submit project proposals, with the aim of receiving EU funding.
Data not available
Procedure for inviting applicants to submit project proposals, with the aim of receiving EU funding.
Funding Scheme
Funding scheme (or “Type of Action”) inside a programme with common features. It specifies: the scope of what is funded; the reimbursement rate; specific evaluation criteria to qualify for funding; and the use of simplified forms of costs like lump sums.
Funding scheme (or “Type of Action”) inside a programme with common features. It specifies: the scope of what is funded; the reimbursement rate; specific evaluation criteria to qualify for funding; and the use of simplified forms of costs like lump sums.
Data not available
Coordinator
N6 6NT London
United Kingdom
The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.