A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs.
A-kinase anchor proteins (AKAPs) assemble multi-enzyme signaling complexes in proximity to substrate/effector proteins, thus directing and amplifying membrane-generated signals. S-AKAP84 and AKAP121 are alternative splicing products with identical NH(2) termini. These AKAPs bind and target protein kinase A (PKA) to the outer mitochondrial membrane. Tubulin was identified as a binding partner of S-AKAP84 in a yeast two-hybrid screen. Immunoprecipitation and co-sedimentation experiments in rat testis extracts confirmed the interaction between microtubules and S-AKAP84. In situ immunostaining of testicular germ cells (GC2) shows that AKAP121 concentrates on mitochondria in interphase and on mitotic spindles during M phase. Purified tubulin binds directly to S-AKAP84 but not to a deletion mutant lacking the mitochondrial targeting domain (MT) at residues 1-30. The MT is predicted to form a highly hydrophobic alpha-helical wheel that might also mediate interaction with tubulin. Disruption of the wheel by site-directed mutagenesis abolished tubulin binding and reduced mitochondrial attachment of an MT-GFP fusion protein. Some MT mutants retain tubulin binding but do not localize to mitochondria. Thus, the tubulin-binding motif lies within the mitochondrial attachment motif. Our findings indicate that S-AKAP84/AKAP121 use overlapping targeting motifs to localize signaling enzymes to mitochondrial and cytoskeletal compartments.
Cardone
Luca
L
Dipartimento di Biologia e Patologia Molecolare e Cellulare, BioGem Consortium, Instituto di Endocrinologia ed Oncologia Sperimentale CNR, Universitá Federico II, via S. Pansini 5, 80131 Naples, Italy.
de Cristofaro
Tiziana
T
Affaitati
Adelina
A
Garbi
Corrado
C
Ginsberg
Michael D
MD
Saviano
Michele
M
Varrone
Stelio
S
Rubin
Charles S
CS
Gottesman
Max E
ME
Avvedimento
Enrico V
EV
Feliciello
Antonio
A
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013094:Spermatozoa / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; C486333:Akap1 protein, mouse; C486332:Akap1 protein, rat; D002352:Carrier Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D017868:Cyclic AMP-Dependent Protein Kinases