Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1999 Apr 1;18(7):1858–1868. doi: 10.1093/emboj/18.7.1858

Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450.

O Witczak 1, B S Skålhegg 1, G Keryer 1, M Bornens 1, K Taskén 1, T Jahnsen 1, S Orstavik 1
PMCID: PMC1171271  PMID: 10202149

Abstract

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A-kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21-22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled-coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII-binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA-buffer extracts of HeLa cells demonstrated co-precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co-purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.

Full Text

The Full Text of this article is available as a PDF (515.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailly E., Dorée M., Nurse P., Bornens M. p34cdc2 is located in both nucleus and cytoplasm; part is centrosomally associated at G2/M and enters vesicles at anaphase. EMBO J. 1989 Dec 20;8(13):3985–3995. doi: 10.1002/j.1460-2075.1989.tb08581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bischoff J. R., Anderson L., Zhu Y., Mossie K., Ng L., Souza B., Schryver B., Flanagan P., Clairvoyant F., Ginther C. A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J. 1998 Jun 1;17(11):3052–3065. doi: 10.1093/emboj/17.11.3052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bloom G. S., Luca F. C., Vallee R. B. Microtubule-associated protein 1B: identification of a major component of the neuronal cytoskeleton. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5404–5408. doi: 10.1073/pnas.82.16.5404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bornens M., Paintrand M., Berges J., Marty M. C., Karsenti E. Structural and chemical characterization of isolated centrosomes. Cell Motil Cytoskeleton. 1987;8(3):238–249. doi: 10.1002/cm.970080305. [DOI] [PubMed] [Google Scholar]
  6. Bouckson-Castaing V., Moudjou M., Ferguson D. J., Mucklow S., Belkaid Y., Milon G., Crocker P. R. Molecular characterisation of ninein, a new coiled-coil protein of the centrosome. J Cell Sci. 1996 Jan;109(Pt 1):179–190. doi: 10.1242/jcs.109.1.179. [DOI] [PubMed] [Google Scholar]
  7. Bregman D. B., Bhattacharyya N., Rubin C. S. High affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II-B. Cloning, characterization, and expression of cDNAs for rat brain P150. J Biol Chem. 1989 Mar 15;264(8):4648–4656. [PubMed] [Google Scholar]
  8. Carr D. W., Stofko-Hahn R. E., Fraser I. D., Bishop S. M., Acott T. S., Brennan R. G., Scott J. D. Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J Biol Chem. 1991 Aug 5;266(22):14188–14192. [PubMed] [Google Scholar]
  9. De Camilli P., Moretti M., Donini S. D., Walter U., Lohmann S. M. Heterogeneous distribution of the cAMP receptor protein RII in the nervous system: evidence for its intracellular accumulation on microtubules, microtubule-organizing centers, and in the area of the Golgi complex. J Cell Biol. 1986 Jul;103(1):189–203. doi: 10.1083/jcb.103.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Doxsey S. J., Stein P., Evans L., Calarco P. D., Kirschner M. Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell. 1994 Feb 25;76(4):639–650. doi: 10.1016/0092-8674(94)90504-5. [DOI] [PubMed] [Google Scholar]
  11. Dransfield D. T., Yeh J. L., Bradford A. J., Goldenring J. R. Identification and characterization of a novel A-kinase-anchoring protein (AKAP120) from rabbit gastric parietal cells. Biochem J. 1997 Mar 15;322(Pt 3):801–808. doi: 10.1042/bj3220801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eide T., Coghlan V., Orstavik S., Holsve C., Solberg R., Skâlhegg B. S., Lamb N. J., Langeberg L., Fernandez A., Scott J. D. Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95. Exp Cell Res. 1998 Feb 1;238(2):305–316. doi: 10.1006/excr.1997.3855. [DOI] [PubMed] [Google Scholar]
  13. Fernandez-Silva P., Martinez-Azorin F., Micol V., Attardi G. The human mitochondrial transcription termination factor (mTERF) is a multizipper protein but binds to DNA as a monomer, with evidence pointing to intramolecular leucine zipper interactions. EMBO J. 1997 Mar 3;16(5):1066–1079. doi: 10.1093/emboj/16.5.1066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fernandez A., Cavadore J. C., Demaille J., Lamb N. Implications for cAMP-dependent protein kinase in the maintenance of the interphase state. Prog Cell Cycle Res. 1995;1:241–253. doi: 10.1007/978-1-4615-1809-9_19. [DOI] [PubMed] [Google Scholar]
  15. Francis S. H., Corbin J. D. Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol. 1994;56:237–272. doi: 10.1146/annurev.ph.56.030194.001321. [DOI] [PubMed] [Google Scholar]
  16. Fry A. M., Mayor T., Meraldi P., Stierhof Y. D., Tanaka K., Nigg E. A. C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2. J Cell Biol. 1998 Jun 29;141(7):1563–1574. doi: 10.1083/jcb.141.7.1563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fry A. M., Meraldi P., Nigg E. A. A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators. EMBO J. 1998 Jan 15;17(2):470–481. doi: 10.1093/emboj/17.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gavet O., Ozon S., Manceau V., Lawler S., Curmi P., Sobel A. The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network. J Cell Sci. 1998 Nov;111(Pt 22):3333–3346. doi: 10.1242/jcs.111.22.3333. [DOI] [PubMed] [Google Scholar]
  19. Gosti-Testu F., Marty M. C., Berges J., Maunoury R., Bornens M. Identification of centrosomal proteins in a human lymphoblastic cell line. EMBO J. 1986 Oct;5(10):2545–2550. doi: 10.1002/j.1460-2075.1986.tb04533.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gotoh Y., Nishida E., Matsuda S., Shiina N., Kosako H., Shiokawa K., Akiyama T., Ohta K., Sakai H. In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase. Nature. 1991 Jan 17;349(6306):251–254. doi: 10.1038/349251a0. [DOI] [PubMed] [Google Scholar]
  21. Gradin H. M., Larsson N., Marklund U., Gullberg M. Regulation of microtubule dynamics by extracellular signals: cAMP-dependent protein kinase switches off the activity of oncoprotein 18 in intact cells. J Cell Biol. 1998 Jan 12;140(1):131–141. doi: 10.1083/jcb.140.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hausken Z. E., Scott J. D. Properties of A-kinase anchoring proteins. Biochem Soc Trans. 1996 Nov;24(4):986–991. doi: 10.1042/bst0240986. [DOI] [PubMed] [Google Scholar]
  23. Keryer G., Rios R. M., Landmark B. F., Skalhegg B., Lohmann S. M., Bornens M. A high-affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II in the centrosome of human cells. Exp Cell Res. 1993 Feb;204(2):230–240. doi: 10.1006/excr.1993.1029. [DOI] [PubMed] [Google Scholar]
  24. Keryer G., Yassenko M., Labbé J. C., Castro A., Lohmann S. M., Evain-Brion D., Taskén K. Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase. J Biol Chem. 1998 Dec 18;273(51):34594–34602. doi: 10.1074/jbc.273.51.34594. [DOI] [PubMed] [Google Scholar]
  25. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  26. Lane M. E., Kalderon D. RNA localization along the anteroposterior axis of the Drosophila oocyte requires PKA-mediated signal transduction to direct normal microtubule organization. Genes Dev. 1994 Dec 15;8(24):2986–2995. doi: 10.1101/gad.8.24.2986. [DOI] [PubMed] [Google Scholar]
  27. Lin J. W., Wyszynski M., Madhavan R., Sealock R., Kim J. U., Sheng M. Yotiao, a novel protein of neuromuscular junction and brain that interacts with specific splice variants of NMDA receptor subunit NR1. J Neurosci. 1998 Mar 15;18(6):2017–2027. doi: 10.1523/JNEUROSCI.18-06-02017.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lohmann S. M., DeCamilli P., Einig I., Walter U. High-affinity binding of the regulatory subunit (RII) of cAMP-dependent protein kinase to microtubule-associated and other cellular proteins. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6723–6727. doi: 10.1073/pnas.81.21.6723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  30. Mayer L., Fu S. M., Kunkel H. G. Human T cell hybridomas secreting factors for IgA-specific help, polyclonal B cell activation, and B cell proliferation. J Exp Med. 1982 Dec 1;156(6):1860–1865. doi: 10.1084/jem.156.6.1860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nigg E. A., Schäfer G., Hilz H., Eppenberger H. M. Cyclic-AMP-dependent protein kinase type II is associated with the Golgi complex and with centrosomes. Cell. 1985 Jul;41(3):1039–1051. doi: 10.1016/s0092-8674(85)80084-2. [DOI] [PubMed] [Google Scholar]
  32. Rubin C. S. A kinase anchor proteins and the intracellular targeting of signals carried by cyclic AMP. Biochim Biophys Acta. 1994 Dec 30;1224(3):467–479. [PubMed] [Google Scholar]
  33. Schwartz D. A., Rubin C. S. Identification and differential expression of two forms of regulatory subunits (RII) of cAMP-dependent protein kinase II in Friend erythroleukemic cells. Differentiation and 8-bromo-cAMP elicit a large and selective increase in the rate of biosynthesis of only one type of RII. J Biol Chem. 1985 May 25;260(10):6296–6303. [PubMed] [Google Scholar]
  34. Scott J. D. Cyclic nucleotide-dependent protein kinases. Pharmacol Ther. 1991;50(1):123–145. doi: 10.1016/0163-7258(91)90075-w. [DOI] [PubMed] [Google Scholar]
  35. Skålhegg B. S., Landmark B., Foss K. B., Lohmann S. M., Hansson V., Lea T., Jahnsen T. Identification, purification, and characterization of subunits of cAMP-dependent protein kinase in human testis. Reverse mobilities of human RII alpha and RII beta on sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared with rat and bovine RIIs. J Biol Chem. 1992 Mar 15;267(8):5374–5379. [PubMed] [Google Scholar]
  36. Skålhegg B. S., Taskén K., Hansson V., Huitfeldt H. S., Jahnsen T., Lea T. Location of cAMP-dependent protein kinase type I with the TCR-CD3 complex. Science. 1994 Jan 7;263(5143):84–87. doi: 10.1126/science.8272870. [DOI] [PubMed] [Google Scholar]
  37. Taskén K., Skålhegg B. S., Solberg R., Andersson K. B., Taylor S. S., Lea T., Blomhoff H. K., Jahnsen T., Hansson V. Novel isozymes of cAMP-dependent protein kinase exist in human cells due to formation of RI alpha-RI beta heterodimeric complexes. J Biol Chem. 1993 Oct 5;268(28):21276–21283. [PubMed] [Google Scholar]
  38. Zhou H., Kuang J., Zhong L., Kuo W. L., Gray J. W., Sahin A., Brinkley B. R., Sen S. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet. 1998 Oct;20(2):189–193. doi: 10.1038/2496. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES