Dewaste et al. [Dewaste, Moreau, De Smedt, Bex, De Smedt, Wuytaack, Missiaen and Erneux (2003) Biochem. J. 374, 41–49] showed that over-expressed EGFP (enhanced green fluorescent protein) fused to Ins(1,4,5)P3 3-kinase B (IP3K-B) co-localizes with the cytoskeleton, as well as with the endoplasmic reticulum and the plasma membrane. The domains responsible for these subcellular localizations are not yet identified. For the endogenous enzyme, we confirmed both actin and endoplasmic reticulum localization by employing a high affinity antibody against IP3K-B. F-actin targeting is exclusively dependent on the non-catalytic N-terminal region of IP3K-B. By expressing fragments of this N-terminal domain as EGFP-fusion proteins and inspecting transfected cells by confocal microscopy, we characterized a distinct 63-amino-acid domain comprising amino acids 108–170 of the enzyme which is responsible for F-actin targeting. A truncation of this fragment from both sides revealed that the full size of this segment is essential for this function. Deletion of this segment in a full-length over-expressed IP3K-B–EGFP-fusion protein completely abolished F-actin interaction. Direct interaction of this actin-binding segment with only F-actin, but not with G-actin, was observed in vitro using a bacterially expressed, affinity-purified GST (glutathione S-transferase)–Rattus norvegicus IP3K (aa 108–170) fusion protein. Helix-breaking mutations within this isolated segment abolished the F-actin binding properties both in vitro and when over-expressed in cells, indicating that an intact secondary structure is essential for actin targeting. The segment shows sequence similarities to the actin-binding region in IP3K-A, but no similarity to other actin-binding domains.
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Research Article|
August 10 2004
Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B)
Maria A. BREHM;
Maria A. BREHM
1
*Institut für Biochemie and Molekularbiologie I: Zelluläre Signaltransduktion, Zentrum für Experimentelle Medizin, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany
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Isabell SCHREIBER;
Isabell SCHREIBER
1
*Institut für Biochemie and Molekularbiologie I: Zelluläre Signaltransduktion, Zentrum für Experimentelle Medizin, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany
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Uwe BERTSCH;
Uwe BERTSCH
†Institut für Neuropathologie, Ludwig-Maximilians Universität, Zentrum f. Neuropathologie und Prionforschung, Feodor-Lynen-Strasse 23, München 81377, Germany
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Albrecht WEGNER;
Albrecht WEGNER
‡Institute of Physiological Chemistry, Ruhr University, Universitaetsstr. 150, Bochum 44780, Germany
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Georg W. MAYR
Georg W. MAYR
2
*Institut für Biochemie and Molekularbiologie I: Zelluläre Signaltransduktion, Zentrum für Experimentelle Medizin, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany
2To whom correspondence should be addressed (email mayr@uke.uni-hamburg.de).
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Publisher: Portland Press Ltd
Received:
November 17 2003
Revision Received:
March 30 2004
Accepted:
May 06 2004
Accepted Manuscript online:
May 06 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2004
Biochem J (2004) 382 (1): 353–362.
Article history
Received:
November 17 2003
Revision Received:
March 30 2004
Accepted:
May 06 2004
Accepted Manuscript online:
May 06 2004
Citation
Maria A. BREHM, Isabell SCHREIBER, Uwe BERTSCH, Albrecht WEGNER, Georg W. MAYR; Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B). Biochem J 15 August 2004; 382 (1): 353–362. doi: https://doi.org/10.1042/BJ20031751
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