Human regulatory subunit RI beta of cAMP-dependent protein kinases: expression, holoenzyme formation and microinjection into living cells.
The human regulatory subunit RI beta of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin. The human recombinant RI beta protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RI beta antiserum. Furthermore, the purified recombinant RI beta protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit C alpha. The rate of RI beta 2C alpha 2 holoenzyme formation was faster in the presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active C alpha-subunit, due to cAMP binding to RI beta. Compared to a RI alpha 2C alpha 2 holoenzyme, the RI beta 2C alpha 2 holoenzyme exhibited a more than twofold higher sensitivity to cAMP. The subcellular localization of RI beta was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm. A cytoplasmic distribution was observed when free RI beta was injected, whereas free C alpha injected into the cytoplasm appeared in the nucleus. When holoenzymes with labeled RI beta and unlabeled C alpha, or unlabeled RI beta and labeled C alpha, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RI beta was the labeled subunit of the in vivo dissociated holoenzyme. In contrast, nuclear fluorescence was evident from the release and translocation of labeled C alpha from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.
Solberg
R
R
Institute of Medical Biochemistry, University of Oslo, Norway.
Taskén
K
K
Wen
W
W
Coghlan
V M
VM
Meinkoth
J L
JL
Scott
J D
JD
Jahnsen
T
T
Taylor
S S
SS
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D002417:Cattle; D002451:Cell Compartmentation; D000242:Cyclic AMP / Q000494:pharmacology; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008845:Microinjections; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism
D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; C517766:PRKAR1B protein, human; C517769:Prkar1b protein, rat; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases