Journal
BIOCHEMICAL JOURNAL
Volume 382, Issue -, Pages 353-362Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20031751
Keywords
actin-binding domain; F-actin; Ins(1,4,5)P-3 3-kinase B; subcellular localization
Categories
Ask authors/readers for more resources
Dewaste et al. [Dewaste, Moreau, De Smedt, Bex, De Smedt, Wuytaack, Missiaen and Emeux (2003) Biochem. J. 374, 41-49] showed that over-expressed EGFP (enhanced green fluorescent protein) fused to Ins(1,4,5)P-3 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available