Kuret, J., Johnson, K. E., Nicolette, C., Zoller, M. J. (July 1988) Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit. J Biol Chem, 263 (19). pp. 9149-54. ISSN 0021-9258
Abstract
Oligonucleotide-directed mutagenesis was used to produce mutants in the hinge region of the regulatory subunit (R) of the Saccharomyces cerevisiae cAMP-dependent protein kinase. The mutant proteins were expressed in Escherichia coli, purified, urea treated to produce cAMP-free regulatory (R), and analyzed in vitro for catalytic (C) subunit inhibitory activity in the presence and absence of cAMP. When assayed in the absence of cAMP, wild type R dimer inhibited C with an IC50 of 40 nM. Replacement of amino acid residue Ser-145 (the autophosphorylation site of yeast R) with Ala or Gly produced mutants which were 2-10-fold better inhibitors of C, while replacement with Glu, Asp, Lys, or Thr produced mutants which were 2-5-fold worse inhibitors of C relative to wild type R. When assayed in the presence of cAMP, all R subunits had a decreased affinity for C subunit, with Ser-145 and Thr-145 undergoing autophosphorylation. These results suggest that the amino acid at position 145 of R contributes to R-C interaction and therefore influences the equilibrium of yeast protein kinase subunits in vitro.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Amino Acid Sequence Binding Sites Carrier Proteins/*genetics/metabolism Cloning, Molecular Escherichia coli/genetics Genes, Fungal Genes, Structural *Intracellular Signaling Peptides and Proteins Molecular Sequence Data *Mutation Plasmids Protein Binding Protein Kinases/*genetics Research Support, U.S. Gov't, P.H.S. Saccharomyces cerevisiae/enzymology/*genetics Templates, Genetic |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > mutations > mutagenesis bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > kinase > Protein kinase C |
| CSHL Authors: | |
| Communities: | CSHL labs |
| Depositing User: | Gail Sherman |
| Date: | 5 July 1988 |
| Date Deposited: | 28 Sep 2017 18:54 |
| Last Modified: | 28 Sep 2017 18:54 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/35171 |
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