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Title: Targeting AGC protein kinases
Author: Gold, Matthew
ISNI:       0000 0004 2678 9302
Awarding Body: Institute of Cancer Research (University Of London)
Current Institution: Institute of Cancer Research
Date of Award: 2008
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A-kinase anchoring proteins (AKAPs) restrict the action of the broad-specificity cyclic AMP-dependent protein kinase (PKA) to discrete subcellular locations. The highresolution crystal structure of the docking and dimerisation (D/D) domain of the RIIa regulatory Bubunit of PKA in complex with the high-affinity anchoring peptide AKAP-IS explains the molecular basis of AKAP specificity for PKA regulatory subunits. AKAPIS folds into an amphipathic a-helix that engages an essentially preformed shallow groove on the surface of the RUa dimer D/D domains. Conserved AKAP aiiphatic residues dominate interactions to RUa at the predominantly hydrophobic interface, whereas polar residues are important in conferring regulatory subunit isoform specificity. Structural information for the AKAP family was previously limited to studies of the PKA-AKAP interface. To address this deficiency, a bioinformatic screen of AKAPs was performed to identify domains within AKAPs that might be suitable for structural investigation. A central domain in AKAP18 was identified, and its crystal structure was solved. The domain is structurally similar to 2H phosphoesterase enzymes, which catalyse the hydrolysis of cyclic nucleotides, with a central groove at the base of which two His-x-Thr motifs are positioned. The domain binds specifically to 5' AMP/CMP, with a dissociation constant for AMP in the physiological range, and the molecular basis for nucleotide specificity has been established. No catalytic activity was associated with the domain, so it may function as an AMP sensor. AKAP79 is a prototypical mammalian scaffold protein, which nucleates mUlti-protein kinase-phosphatase complexes, and localises at the cell membrane under the control of calmodulin. A system for expression and purification of AKAP79 has been developed enabling sufficient production of pure AKAP79 complexes for structural and biochemical investigation. Imaging of an AKAP79-PKA-calmodulin complex by negative-staining transmission electron microscopy indicates that the first three-dimen'sional reconstruction of this complex may be possible in the near future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available