Characterization of in‐vitro‐translated human regulatory and catalytic subunits of cAMP‐dependent protein kinases
Authors:
- 1 February 1994
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 220 (1) , 217-223
Abstract
Full-length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases (PKA) were transcribed and translated in a cell-free in vitro system. The resulting proteins were characterized with respect to molecular size, isoelectric focusing, immunoreactivity, cAMP binding, and to what extent the RII protein subunits revealed mobility shifts upon phosphorylation by catalytic subunit of PKA. We were able to express cDNAs for all the human R (RI alpha, RI beta, RII alpha and RII beta) and C (C alpha, C beta and C gamma) subunits in a wheat-germ extract. [35S]Methionine-labelled in-vitro-translated products were analyzed by SDS/PAGE and revealed distinct protein bands with apparent molecular masses of 49 (RI alpha), 54-55 (RI beta), 51 (RII alpha) and 53 kDa (RII beta) for the R subunits. In vitro transcription/translation of the cDNAs for the C subunits of PKA gave proteins with molecular masses of approximately 40 kDa for all the different C subunits. Phosphorylation of RII alpha and RII beta by the C subunit of PKA, revealed a distinct mobility shift of the RII alpha subunit on one-dimensional SDS/PAGE (51-54 kDa), but not of RII beta (53 kDa). Further characterization of the R subunits by two-dimensional SDS/PAGE revealed that RI alpha was more acidic than RI beta, with pIs of 6.1-6.0 and 6.4-6.2, respectively. Furthermore, the RII alpha protein was more basic than RII beta, with pIs of approximately 5.4-5.3 and 5.3-5.1, respectively. All the in-vitro-translated R subunits could be photoaffinity labelled by the cAMP-analog 8-azido-[32P]cAMP and were also detected by immunoprecipitation with subunit-specific antibodies.Keywords
This publication has 21 references indexed in Scilit:
- Molecular cloning, cDNA structure and tissue-specific expression of the human regulatory subunit RIβ of cAMP-dependent protein kinasesBiochemical and Biophysical Research Communications, 1991
- cAMP-DEPENDENT PROTEIN KINASE: FRAMEWORK FOR A DIVERSE FAMILY OF REGULATORY ENZYMESAnnual Review of Biochemistry, 1990
- Human testis cDNA for the regulatory subunit RIIα of CAMP‐dependent protein kinase encodes an alternate amino‐terminal regionFEBS Letters, 1989
- Molecular cloning, cDNA structure and deduced amino acid sequence for a type I regulatory subunit of cAMP-dependent protein kinase from human testisBiochemical and Biophysical Research Communications, 1987
- The molecular cloning of a type II regulatory subunit of the cAMP-dependent protein kinase from rat skeletal muscle and mouse brain.Proceedings of the National Academy of Sciences, 1987
- 3 Cyclic Nucleotide-Dependent Protein Kinases1986
- Amino acid sequence of the catalytic subunit of bovine type II adenosine cyclic 3',5'-phosphate-dependent protein kinaseBiochemistry, 1983
- [15] Photoaffinity labeling of the regulatory subunit of cAMP-dependent protein kinase1983
- Phosphorylation-Dephosphorylation of EnzymesAnnual Review of Biochemistry, 1979
- Analytical techniques for cell fractionsAnalytical Biochemistry, 1978