Identification of a high affinity binding protein for the regulatory subunit RII beta of cAMP-dependent protein kinase in Golgi enriched membranes of human lymphoblasts.
Immunocytochemical evidence of an association between the regulatory subunit RII of the cAMP-dependent protein kinase (cAMP-PK) and the Golgi apparatus in several cell types has been reported. In order to identify endogenous Golgi proteins binding RII, a fraction enriched in Golgi vesicles was isolated from human lymphoblasts. Only the RII beta isoform was detected in the Golgi-rich fraction, although RII alpha has also been found to be present in these cells. A 85 kDa RII-binding protein was identified in Golgi vesicles using a [32P]RII overlay of Western blots. The existence of an endogenous RII beta-p85 complex in isolated Golgi vesicles was demonstrated by two independent means: (i) co-immunoprecipitation of both proteins under non-denaturing conditions with an antibody against RII beta and (ii) co-purification of RII beta-p85 complexes on a cAMP-analogue affinity column. p85 was phosphorylated by both endogenous and purified catalytic subunits of cAMP-pKII. Extraction experiments and protease protection experiments indicated that p85 is an integral membrane protein although it partitioned atypically during Triton X-114 phase separation. We propose that p85 anchors RII beta to the Golgi apparatus of human lymphoblasts and thereby defines the Golgi substrate targets most accessible to phosphorylation by C subunit. This mechanism may be relevant to the regulation of processes involving the Golgi apparatus itself, such as membrane traffic and secretion, but also relevant to nearby nuclear events dependent on C subunit.
Rios
R M
RM
Centre de Genetique Moleculaire, Centre National de la Recherche Scientifique, Gif/Yvette, France.
Celati
C
C
Lohmann
S M
SM
Bornens
M
M
Keryer
G
G
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002417:Cattle; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D008214:Lymphocytes / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D011494:Protein Kinases