Biochemical and Structural Studies of the Role of FKBP(L)s in the fly and Mammalian Circadian Cycle

FK506 binding-like proteins (FKBPLs) are proteins that structurally resemble the immunophilins. Their functions in cell signaling include protein folding, molecular trafficking, RNA splicing and cell cycle regulation among others. Bride of Doubletime (BDBT) previously designated as CG17282 in the flybase was identified as a circadian clock component that stimulates DBT’s circadian function. Here, we have used biochemical and structural approaches to characterize BDBT. We determined the x-ray crystal structure of BDBT and the analysis showed that it is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that interacts with DOUBLETIME and tetratricopeptide repeats at its C-terminal region that may mediate protein-protein interaction. We used similar techniques to identify BDBT vertebrate functional homologues FKBP51, FKBP52 and FKBPL. In vitro kinase assays showed that all three proteins stimulate CK1δ phosphorylation activity towards casein. Among these three proteins, FKBP51 was seen to bind mammalian casein kinase 1 delta (CK1δ) with much stronger affinity than the other FKBPs. From genetic studies, ShRNA to fkbpl in mammalian U20S cell model showed reduced amplitude and longer circadian period. Further site-directed mutagenesis revealed to us that certain residues, which are important for nuclear localization of CK1δ affect its interaction with FKBP51. Altogether, our work illustrates novel role for FKBP(L)s as molecular clock components in flies and mammals.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Molecular Biology and Biochemistry (UMKC)

Grantor dc:publisher

University of Missouri -- Kansas City

Year dc:date.issued

2017

Author dc:creator

• Agyekum, Kwaku Boadi

Advisor dc:contributor.advisor

• Bouyain, Samuel

Language dc:language.iso

en_US

Handle dc:identifier.uri

https://hdl.handle.net/10355/66803

OAI identifier oai:identifier

oai:mospace.umsystem.edu:10355/66803