Structural studies of the dimerization/docking domain of the type la regulatory subunit of PKA

P. Banky, J. L. Huang, S. S. Taylor

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Abstract

The regulatory subunits ( R) of c AM P dependent protein kinase are modular. multi-functional proteins. Within the family of R-subunits, the N-terminal regions are the most sequentially variable and in fact are antigenic. The two general (lasses arc designated as RI and RII, and within each class there are at least two variants, o- and ;. At the amino terminus, a dimerization domain maintains the R-subunits as an asymmetric dimer. For the RII-subunit, this region has been implicated in a critical biological function, namely, interaction with A-Kinase-Anchoring Proteins (AKAPs). While anchoring proteins have been associated primarily with RII subum'ts. a recent finding has shown that a novel family of anchoring proteins, (D-AKAPs) recognizes Rl as well as RII. This finding suggests thaï the RI subunit may also play a role in the subcellutar targetthig of PKA. N-terminal deletion mutants were constructed to determine the minimum sequence requirement for dimerization. In addi tion, aianirie-scanning mutagenesis was used to assess the role of particular hydrophobic amino acids in dimerization. In order to obtain a high résolu tion NMR structure of RIa( 1-61 ). two-dimensional homonulear NMR experi ments were performed. Most of the expected cross-peaks were observed in the TOCSY finger-print region. Substantial overlap of peaks in the amide region justified isotopic labeling. Minima] media expression was optimized for 15N labeling. 2D heteronuclear experiments were done to ease the assignment pro cess. All expected peaks were observed well dispersed in a 2D HSQC, and the determination of the secondary structure of this domain is underway-.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997

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ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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