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Structural characterization of human protein kinase CaMKK2 and its interactions with binding partners
Koupilová, Nicola ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) belongs to the serine/ threonine protein kinase family, which is involved in the calcium signaling pathway. The increase of intracellular calcium concentration induces the activation of calmodulin (CaM), which then activates its binding partners including CaMKII, CaMKIII, CaMKK1 and CaMKK2. CaMKK2 activates CaMKI, CaMKIV and AMP-dependent kinase, AMPK, by phosphorylation. CaMKK2 is naturally present in cells in an autoinhibited state, which is caused by the steric hindrance of the active site by the autoinhibitory domain. When calmodulin binds to the calmodulin-binding domain, the autoinhibitory domain is removed and the active site becomes accessible. Upon activation, CaMKK2 undergoes autophosphorylation, which increases its enzyme activity. Negative regulation of CaMKK2 is mediated by cAMP-dependent protein kinase A (PKA)- and GSK3-dependent phosphorylation. Sites phosphorylated by PKA have been identified for both CaMKK1 and CaMKK2. Two of them are also motifs recognized by scaffolding 14-3-3 proteins. Previous studies have shown that the 14-3-3 protein binding maintains phosphorylated CaMKK2 in an inhibited state by blocking the dephosphorylation of S495, which prevents the binding to calmodulin. However, it is unclear if it is the...
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Preparation of human Ca2+/calmodulin-dependent protein kinase kinase 2 phosphorylated at Ser100 and Ser511
Koupilová, Nicola ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
5 Abstract Ca2+ /calmodulin-dependent protein kinase kinases (CaMKK) are serine/threonine kinases involved in the calcium signaling pathway. Two CaMKK isoforms were described in mammals: CaMKK1 and CaMKK2. The increase in calcium concentrations induces Ca2+ /CaM binding to the C-terminal segment of CaMKK, thus relieving autoinhibition by disrupting the interaction between the autoinhibitory segment and the kinase domain. Active CaMKK then phosphorylate and activate their downstream kinases CaMK1 and CaMK4, and in the case of CaMKK2 also AMPK. The activity of CaMKK is also regulated by phosphorylation mediated by cAMP-dependent protein kinase A (PKA). This phosphorylation creates two binding motifs recognized by the regulatory 14-3-3 proteins. Previous studies have suggested that the 14-3-3 protein keeps phos- phorylated CaMKK1 in the inhibited state by blocking the dephosphorylation of the inhibitory phosphorylation site and it has been speculated that CaMKK2 is regulated in a similar manner. However, the role of 14-3-3 protein in the regulation of CaMKK2 is unclear. In order to study this protein complex, it is necessary to prepare recombinant CaMKK2 fully phosphorylated at both 14-3-3 binding motifs. The main aim of this bachelor thesis was to optimize the protocol for the phosphorylation of human CaMKK2...
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