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 glutathion e 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 display ed immunoreactivity with an anti-human RI beta antiserum. Furthermore, the purified recombinant RI beta protein was shown to exist as a dime r that was able to form holoenzyme with the catalytic subunit C alpha. The rate of RI beta(2)C alpha(2) holoenzyme formation was faster in t he presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presen ce 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 (2)C alpha(2) holoenzyme, the RI beta(2)C alpha(2) holoenzyme exhibite d a more than twofold higher sensitivity to cAMP. The subcellular loca lization of RI beta was analyzed in quiescent REF-52 fibroblasts and W istar rat thyroid (WRT) cells after microinjection of fluorescently la beled proteins into the cytoplasm. A cytoplasmic distribution was obse rved when free RI beta was injected, whereas free C alpha injected int o the cytoplasm appeared in the nucleus. When holoenzymes with labeled RI beta and unlabeled C alpha, or unlabeled RI beta and labeled C alp ha, were injected, unstimulated cells showed fluorescence in the cytop lasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8- Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluoresce nce mainly in the cytoplasm when RI beta was the labeled subunit of th e in vivo dissociated holoenzyme. In contrast, nuclear fluorescence wa s evident from the release and translocation of labeled C alpha from t he holoenzyme complex after stimulation with 8-Br-cAMP or TSH. (C) 199 4 Academic Press, Inc.