HUMAN REGULATORY SUBUNIT RI-BETA OF CAMP-DEPENDENT PROTEIN-KINASES - EXPRESSION, HOLOENZYME FORMATION AND MICROINJECTION INTO LIVING CELLS

被引:36
作者
SOLBERG, R
TASKEN, K
WEN, W
COGHLAN, VM
MEINKOTH, JL
SCOTT, JD
JAHNSEN, T
TAYLOR, SS
机构
[1] UNIV CALIF SAN DIEGO,DEPT CHEM,LA JOLLA,CA 92037
[2] UNIV CALIF SAN DIEGO,DEPT MED,LA JOLLA,CA 92037
[3] UNIV CALIF SAN DIEGO,CTR CANC,LA JOLLA,CA 92037
[4] VOLLUM INST ADV BIOMED RES,PORTLAND,OR
关键词
D O I
10.1006/excr.1994.1297
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
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(2)C 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(2)C alpha(2) holoenzyme, the RI beta(2)C 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. (C) 1994 Academic Press, Inc.
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页码:595 / 605
页数:11
相关论文
共 58 条
[1]  
Adams S.R., 1993, FLUORESCENT PROBES B, P133
[2]   FLUORESCENCE RATIO IMAGING OF CYCLIC-AMP IN SINGLE CELLS [J].
ADAMS, SR ;
HAROOTUNIAN, AT ;
BUECHLER, YJ ;
TAYLOR, SS ;
TSIEN, RY .
NATURE, 1991, 349 (6311) :694-697
[3]   CULTURE OF HORMONE-DEPENDENT FUNCTIONAL EPITHELIAL-CELLS FROM RAT THYROIDS [J].
AMBESIIMPIOMBATO, FS ;
PARKS, LAM ;
COON, HG .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1980, 77 (06) :3455-3459
[4]   MOLECULAR-CLONING OF A TISSUE-SPECIFIC PROTEIN-KINASE (C-GAMMA) FROM HUMAN TESTIS - REPRESENTING A 3RD ISOFORM FOR THE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE [J].
BEEBE, SJ ;
OYEN, O ;
SANDBERG, M ;
FROYSA, A ;
HANSSON, V ;
JAHNSEN, T .
MOLECULAR ENDOCRINOLOGY, 1990, 4 (03) :465-475
[5]   CORRELATION OF PHOTOLABELING WITH OCCUPANCY OF CAMP BINDING-SITES IN THE REGULATORY SUBUNIT OF CAMP-DEPENDENT PROTEIN KINASE-I [J].
BUBIS, J ;
TAYLOR, SS .
BIOCHEMISTRY, 1987, 26 (12) :3478-3486
[6]   COVALENT MODIFICATION OF BOTH CAMP BINDING-SITES IN CAMP-DEPENDENT PROTEIN KINASE-I BY 8-AZIDOADENOSINE 3',5'-MONOPHOSPHATE [J].
BUBIS, J ;
TAYLOR, SS .
BIOCHEMISTRY, 1985, 24 (09) :2163-2170
[7]   DISTINCT PATTERNS OF CAMP-DEPENDENT PROTEIN-KINASE GENE-EXPRESSION IN MOUSE-BRAIN [J].
CADD, G ;
MCKNIGHT, GS .
NEURON, 1989, 3 (01) :71-79
[8]  
CADD GG, 1990, J BIOL CHEM, V265, P19502
[9]   GENETIC-CHARACTERIZATION OF A BRAIN-SPECIFIC FORM OF THE TYPE-I REGULATORY SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE [J].
CLEGG, CH ;
CADD, GG ;
MCKNIGHT, GS .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (11) :3703-3707
[10]  
Cohen P, 1978, Curr Top Cell Regul, V14, P117