Characterization of RII(beta) and D-AKAP1 in differentiated adipocytes.
A-kinase anchoring proteins (AKAPs) have been proposed to regulate cAMP-dependent signaling in the cell by targeting RII subunits of protein kinase A (PKA) to specific subcellular compartments. RII(beta) is the predominant PKA subtype in adipose tissue. In gel overlay assays of C3H/10T1/2 adipocytes and adipose tissue, RII(beta) bound to several proteins including a prominent 132-kDa band, which was strongly induced upon differentiation of C3H/10T1/2 cells into adipocytes. Immunoblotting and nuclease protection analysis of C3H/10T1/2 cellular extracts identified this band as D-AKAP1/S-AKAP84, a putative AKAP. Immunocytochemical analysis of C3H/10T1/2 adipocytes revealed that most of D-AKAP1/S-AKAP84, but not RII(beta), was colocalized with a mitochondrial-selective dye, MitoTracker red. These findings were further confirmed in studies where D-AKAP1/ S-AKAP84, but not RII(beta), were localized in purified mitochondria made from C3H/10T1/2 adipocytes. Moreover, D-AKAP1, which is upregulated after differentiation, did not recruit RII(beta) to membrane fractions enriched in mitochondria. These results demonstrate that D-AKAP1/S-AKAP84 does not interact with PKA in differentiated C3H/10T1/2 adipocytes under the conditions tested.
Chaudhry
Archana
A
CNS Molecular Sciences, Pfizer Global Research and Development, Ann Arbor, Michigan 48105, USA. archana.chaudry@pfizer.com
Zhang
Chen
C
Granneman
James G
JG
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000249:Adenosine Monophosphate / Q000378:metabolism; D017667:Adipocytes / Q000166:cytology* / Q000201:enzymology*; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D008809:Mice, Inbred C3H; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; C486333:Akap1 protein, mouse; D002352:Carrier Proteins; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D010761:Phosphorus Radioisotopes; C517838:Prkar2b protein, mouse; D000249:Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases