Cloning and characterization of A-kinase anchor protein 100 (AKAP100). A protein that targets A-kinase to the sarcoplasmic reticulum.
Differential localization of the type II cAMP-dependent protein kinase (PKA) is achieved by interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Anchoring is a likely means to adapt PKA for regulation of cAMP-responsive events through colocalization of the kinase with preferred substrates. Using an interaction cloning strategy with an RII alpha protein probe, we have identified a 655-amino acid protein (named AKAP100). Recombinant AKAP100, expressed in Escherichia coli, binds RII alpha in a solid-phase overlay assay. The cellular and subcellular distribution of AKAP100 was analyzed by various methods. Northern blot analysis with the AKAP100 cDNA as a probe detected an 8-kilobase message in some human tissues including various brain regions; however, the message was predominately expressed in cardiac and skeletal muscle. Anti-AKAP100 antibodies confirmed expression in the rat cardiac and skeletal muscle cell lines, H9c2 and L6P, whereas immunohistochemical analysis revealed that AKAP100 was localized to the sarcoplasmic reticulum of both cell types. RII was also detected in these regions. AKAP100 was detected in preparations of RII purified from L6P cell extracts by cAMP-agarose affinity chromatography. Collectively, these results suggest that AKAP100 functions to maintain the type II PKA at the sarcoplasmic reticulum.
McCartney
S
S
Vollum Institute, Oregon Health Sciences University, Portland, 97201, USA.
Little
B M
BM
Langeberg
L K
LK
Scott
J D
JD
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; C484293:Akap6 protein, rat; D002352:Carrier Proteins; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; C517819:PRKAR2A protein, human; C517822:Prkar2a protein, rat; D011506:Proteins; D012333:RNA, Messenger; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases