Abstract
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 NH2 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 α-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.
| Original language | English |
|---|---|
| Pages (from-to) | 663-675 |
| Number of pages | 13 |
| Journal | Journal of Molecular Biology |
| Volume | 320 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2002 |
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Keywords
- A-kinase anchor proteins
- Mitochondria
- Mitotic spindles
- S-AKAP84/AKAP121
ASJC Scopus subject areas
- Virology
Cite this
A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs. / Cardone, Luca; De Cristofaro, Tiziana; Affaitati, Adelina; Garbi, Corrado; Ginsberg, Michael D.; Saviano, Michele; Varrone, Stelio; Rubin, Charles S.; Gottesman, Max E.; Avvedimento, Enrico V.; Feliciello, Antonio.
In: Journal of Molecular Biology, Vol. 320, No. 3, 2002, p. 663-675.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs
AU - Cardone, Luca
AU - De Cristofaro, Tiziana
AU - Affaitati, Adelina
AU - Garbi, Corrado
AU - Ginsberg, Michael D.
AU - Saviano, Michele
AU - Varrone, Stelio
AU - Rubin, Charles S.
AU - Gottesman, Max E.
AU - Avvedimento, Enrico V.
AU - Feliciello, Antonio
PY - 2002
Y1 - 2002
N2 - 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 NH2 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 α-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.
AB - 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 NH2 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 α-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.
KW - A-kinase anchor proteins
KW - Mitochondria
KW - Mitotic spindles
KW - S-AKAP84/AKAP121
UR - http://www.scopus.com/inward/record.url?scp=18444375552&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=18444375552&partnerID=8YFLogxK
U2 - 10.1016/S0022-2836(02)00479-5
DO - 10.1016/S0022-2836(02)00479-5
M3 - Article
VL - 320
SP - 663
EP - 675
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 3
ER -