Karakterisering av A-kinase lokaliserende protein, AKAP450

• Main Author: Bornstedt, Mette Eskild
• Format: Dissertation
• Language: Norwegian
• Subjects: ernæringslære

Protein kinase A, PKA, regulates a number of cellular processes like metabolism, cell growth and differentiation and gene transcription. PKA becomes activated upon binding of cAMP to its regulatory subunits thus releasing the catalytic subunit. Four isoforms of the regulatory subunit have been described and are designated RI α and β and RII α and β. Cyclic AMP is generated from ATP by the action of adenylyl cyclase after hormonal triggering of sevenspan receptors coupled to heterotrimeric G proteins. PKA changes the phosphorylation level of certain substrates in the cell by phosphorylating either serine or threonine residues. The specificity of cAMP and thus the hormonal signal is partly determined by cellular targeting of PKA by A-Kinase Anchoring Proteins, AKAPs. AKAPs constitute a family of functionally related proteins which locate PKA and other signal molecules important for signal transduction to various subcellular structures including the Golgi-centrosomal complex. Previously an AKAP of 453 kDa designated AKAP450 was identified. AKAP450 contains two binding sites for the R-subunit of PKA. In order to characterize PKA-dependent features associated with AKAP450, wild type and mutated forms of the AKAP450 R-binding domains fused to GST were used in pull-down assays. Furthermore, Surface Plasmon Resonance was applied to describe the binding qualitatively and quantitatively. Together the results implied that RII has higher affinity for the C-terminal RII-binding site than the N-terminal binding site and that the α subtype of RII exhibits stronger affinity for the RII binding motive compared to RII β. Furthermore, AKAP450 and several AKAP450 related splice variants are transcribed from the AKAP450 gene which constitutes 51 exons. Immunofluorescence studies with antibodies against N-terminal, middle and C-terminal part of AKAP450, imply that AKAP450 locates PKA to the Golgi apparatus and the centrosome. Overlapping constructs of AKAP450 fused to GFP which were transfected into HeLa cells suggested that the N-terminal part, aa 1-1029 of AKAP450 may contain a Golgi targeting domain. Transfections of the N-terminal deletion fragment of AKAP450 destroyed the distinct staining of the Golgi apparatus. This may suggest that the endogenous AKAP450 plays an important role in sustaining the morphology of the Golgi.