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Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B)

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Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B). / Brehm, Maria A; Schreiber, Isabell; Bertsch, Uwe; Wegner, Albrecht; Mayr, Georg W.

In: BIOCHEM J, Vol. 382, No. 1, 1, 15.08.2004, p. 353-362.

Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review

Harvard

Brehm, MA, Schreiber, I, Bertsch, U, Wegner, A & Mayr, GW 2004, 'Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B)', BIOCHEM J, vol. 382, no. 1, 1, pp. 353-362. https://doi.org/10.1042/BJ20031751

APA

Brehm, M. A., Schreiber, I., Bertsch, U., Wegner, A., & Mayr, G. W. (2004). Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B). BIOCHEM J, 382(1), 353-362. [1]. https://doi.org/10.1042/BJ20031751

Vancouver

Brehm MA, Schreiber I, Bertsch U, Wegner A, Mayr GW. Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B). BIOCHEM J. 2004 Aug 15;382(1):353-362. 1. https://doi.org/10.1042/BJ20031751

Bibtex

@article{9eed171441cb4e2a9cbfe242b076037a,
title = "Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B)",
abstract = "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.",
keywords = "Actin Cytoskeleton, Actins, Animals, Blotting, Western, Cell Line, Cloning, Molecular, Cytoskeleton, Endoplasmic Reticulum, Humans, Isoenzymes, Kidney Tubules, Proximal, Microscopy, Fluorescence, PC12 Cells, Peptides, Phosphotransferases (Alcohol Group Acceptor), Protein Binding, Protein Structure, Tertiary, Rats, Journal Article, Research Support, Non-U.S. Gov't",
author = "Brehm, {Maria A} and Isabell Schreiber and Uwe Bertsch and Albrecht Wegner and Mayr, {Georg W}",
year = "2004",
month = aug,
day = "15",
doi = "10.1042/BJ20031751",
language = "English",
volume = "382",
pages = "353--362",
journal = "BIOCHEM J",
issn = "0264-6021",
publisher = "PORTLAND PRESS LTD",
number = "1",

}

RIS

TY - JOUR

T1 - Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B)

AU - Brehm, Maria A

AU - Schreiber, Isabell

AU - Bertsch, Uwe

AU - Wegner, Albrecht

AU - Mayr, Georg W

PY - 2004/8/15

Y1 - 2004/8/15

N2 - 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.

AB - 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.

KW - Actin Cytoskeleton

KW - Actins

KW - Animals

KW - Blotting, Western

KW - Cell Line

KW - Cloning, Molecular

KW - Cytoskeleton

KW - Endoplasmic Reticulum

KW - Humans

KW - Isoenzymes

KW - Kidney Tubules, Proximal

KW - Microscopy, Fluorescence

KW - PC12 Cells

KW - Peptides

KW - Phosphotransferases (Alcohol Group Acceptor)

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - Rats

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1042/BJ20031751

DO - 10.1042/BJ20031751

M3 - SCORING: Journal article

C2 - 15130091

VL - 382

SP - 353

EP - 362

JO - BIOCHEM J

JF - BIOCHEM J

SN - 0264-6021

IS - 1

M1 - 1

ER -