Membrane-targeting sequences on AKAP79 bind phosphatidylinositol-4, 5-bisphosphate.
Protein kinases and phosphatases are targeted through association with anchoring proteins that tether the enzymes to subcellular structures and organelles. Through in situ fluorescent techniques using a Green Fluorescent Protein tag, we have mapped membrane-targeting domains on AKAP79, a multivalent anchoring protein that binds the cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and protein phosphatase 2B, calcineurin (CaN). Three linear sequences termed region A (residues 31-52), region B (residues 76-101) and region C (residues 116-145) mediate targeting of AKAP79 in HEK-293 cells and cortical neurons. Analysis of these targeting sequences suggests that they contain putative phosphorylation sites for PKA and PKC and are rich in basic and hydrophobic amino acids similar to a class of membrane-targeting domains which bind acidic phospholipids and calmodulin. Accordingly, the AKAP79 basic regions mediate binding to membrane vesicles containing acidic phospholipids including phosphatidylinositol-4, 5-bisphosphate [PtdIns(4,5)P2] and this binding is regulated by phosphorylation and calcium-calmodulin. Finally, AKAP79 was shown to be phosphorylated in HEK-293 cells following stimulation of PKA and PKC, and activation of PKC or calmodulin was shown to release AKAP79 from membrane particulate fractions. These findings suggest that AKAP79 might function in cells not only as an anchoring protein but also as a substrate and effector for the anchored kinases and phosphatases.
Dell'Acqua
M L
ML
Howard Hughes Medical Institute, Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, USA.
Faux
M C
MC
Thorburn
J
J
Thorburn
A
A
Scott
J D
JD
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002352:Carrier Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002147:Calmodulin; D002352:Carrier Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011493:Protein Kinase C; D019703:Calcineurin; D002118:Calcium