- Title
- Effects of multi-site phosphorylation on CaMKII function
- Creator
- Abdul Majeed, A'qilah Banu binte
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2012
- Description
- Masters Research - Master of Philosophy (Medical Biochemistry)
- Description
- CaMKII, or calcium/calmodulin stimulated protein kinase II, is a multifunctional Serine/Threonine protein kinase. CaMKII regulates a number of cell processes including synaptic function and plasticity, learning and memory and cell proliferation. CaMKII is ubiquitously expressed and it is crucial to understand how the enzyme is regulated to produce various functional outcomes in the body. CaMKII is regulated in three ways: an increase in intracellular Ca²⁺increases the binding of Ca²⁺/CaM to CaMKII and activates the enzyme, multi-site autophosphorylation and targeting. The activated enzyme can phosphorylate a number of residues on CaMKII itself (autophosphorylation). Phosphorylation of these residues can alter the proteins that CaMKII binds to (targeting). CaMKII can autophosphorylate at several sites such as T253, T286 and T305/306. Phosphorylation of CaMKII at T286 induces autonomous activity (activity in the absence of Ca²⁺/CaM), whereas phosphorylation of CaMKII at T305/306 prevents activation of CaMKII by preventing Ca²⁺/CaM from binding. Phosphorylation of CaMKII at T253 on the contrary has no direct effect on CaMKII activity. Phosphorylation of CaMKII at T253, T286 and T305/306 can change the targeting of CaMKII, and alter the proteins CaMKII binds to. There is evidence that phosphorylation of CaMKII at more than one site can produce different functional outcomes. It is not known how double phosphorylation produces the effects of altered CaMKII function. Therefore, the aim of this project is to investigate if the functional properties of doubly phosphorylated CaMKII will be different from the sum total of the functional properties of the corresponding singly phosphorylated CaMKII. In order to address this aim the functional properties of αCaMKII with phosphomimic mutations at T253, T286 and T305, were compared with αCaMKII containing phosphomimic mutations of two of these sites in various combinations (T253/T286, T286/T305). The comparison was first addressed in vitro by examining the effects of the T253D/T286D, and the T286D/T305D-αCaMKII double phosphomimic mutants compared to the corresponding single phosphomimic mutants on CaM binding, kinase activity and protein binding (targeting). In terms of CaM binding, the results obtained with the double phosphomimic mutants directly reflected the effects of the single phosphomimic mutations. The effects of the T253D/T286D-αCaMKII double phosphomimic mutant on kinase activity reflected that of the combination of the single phosphomimic mutants. In contrast, the effect of the T286D/T305D-αCaMKII double phosphomimic was different to that which would be expected from the sum of the effects of the individual sites. In terms of targeting, both of the double phosphomimics bound a subset of proteins which did not interact with either of the corresponding single phosphomimics. The comparison was then addressed in situ by examining the effects of the T253D/T286D, and T286D/T305D-αCaMKII double phosphomimic mutants on cellular proliferation and cell cycle progression. The effects of expression of the T253D/T286D-αCaMKII, T253D/T305D-αCaMKII and T286D/T305D-αCaMKII double phosphomimic on cellular proliferation/cell cycle were completely different from that of the sum of the effects of its corresponding single phosphomimic mutants. T253D/T286D-αCaMKII expressing cells had a higher proliferation as compared to cells expressing the corresponding individual phosphomimics, T253D-αCaMKII and T286D-αCaMKII. Our results examining CaMKII activity, targeting, and effects on cell cycle progression indicate that phosphorylation of CaMKII at mutiple sites regulate CaMKII function in a co-operative manner.
- Subject
- CaMKII; posphorylation; protein interactions; targeting; T253; T286; T305/T306
- Identifier
- http://hdl.handle.net/1959.13/936838
- Identifier
- uon:12418
- Rights
- Copyright 2012 A'qilah Banu binte Abdul Majeed
- Language
- eng
- Full Text
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