Abstract/Details

Dissecting the allosteric regulation of PKA-I alpha activation


2009 2009

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Abstract (summary)

Cyclic adenosine monophosphate (cAMP) signaling through cAMP-dependent protein kinase (PKA) is a ubiquitous mammalian signaling pathway involved in metabolism, cell proliferation, and cell death. While the PKA catalytic (C) subunit has served as a prototype for the protein kinase superfamily, the regulatory (R) subunit defines the mechanism whereby cAMP translates an extracellular signal into an intracellular biological response.

This dissertation investigates three major areas of PKA research: (1) defining the molecular features that govern RIα:C complex formation as a means to understand the allosteric regulation of cAMP-induced PKA activation; (2) elucidating the molecular rules that govern substrate recognition; and (3) understanding the molecular basis for isoform-specific activation by cAMP derivatives.

A structure of a PKA RIα:C holoenzyme was solved with a RIα deletion mutant that contains both cAMP binding domains. This structure revealed the extraordinary conformational range that the R-subunit can adopt as it toggles between binding the C-subunit and cAMP. Mutational analysis explains how Domain B is a "gate-keeper" for Domain A. A critical salt bridge links the two hydrophobic capping residues that stack against cAMP (for Domains A and B), such that binding of cAMP to Domain B can release the capping residue for Domain A. Small angle X-ray analysis of various mutant RIα-subunits revealed that Domain B is also highly dynamic. Dissection of the inhibitor sites from both cAMP binding domains shows that binding is only preserved for RII subunits. The differences observed between the RI and RII subunits suggest why the RII subunits, but not RI, can bind to the C-subunit in the absence of ATP.

A crystal structure of a complex between PKA and a specific substrate, phospholamban, was solved, revealing an overall common docking mode as the protein inhibitors at this site. Peptide array methods, site-directed mutagenesis, and biochemical analysis combined defines a unique consensus substrate recognition motif for PKA substrates as R-Xy-R-R-X-S/T-ϕ, where y is 0-4 residues.

Finally, the crystal structure of RIα bound to a cAMP analog, HE-33, was solved, revealing the structural basis of how cAMP analogs result in selective activation of Type Iα versus Type IIβ isoforms.

Indexing (details)


Subject
Biochemistry
Classification
0487: Biochemistry
Identifier / keyword
Pure sciences; Allosteric regulation; Allostery; Crystallography; Protein kinase A; Small angle x-ray scattering; cAMP
Title
Dissecting the allosteric regulation of PKA-I alpha activation
Author
Cheng, Cecilia Yuen-Man
Number of pages
236
Publication year
2009
Degree date
2009
School code
0033
Source
DAI-B 70/05, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9781109149234
Advisor
Taylor, Susan S.
Committee member
Baker, Timothy; Handel, Tracy M.; Joseph, Simpson; Sailor, Michael
University/institution
University of California, San Diego
Department
Chemistry
University location
United States -- California
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3355645
ProQuest document ID
304853287
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/304853287
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