Tropomyosin-binding properties of the CHASM protein are dependent upon its calponin homology domain

Ulke-Lemée, Annegret; Ishida, Hiroaki; Borman, Meredith A.; Valderrama, Alexandra; Vogel, Hans J.; MacDonald, Justin A.

doi:10.1016/j.febslet.2010.07.012

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Volume 584, Issue 15 , Pages 3311-3316, 4 August 2010

Tropomyosin-binding properties of the CHASM protein are dependent upon its calponin homology domain

Edited by Dietmar J. Manstein

Annegret Ulke-Lemée
- Department of Biochemistry and Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, Alberta, Canada T2N 4Z6
Hiroaki Ishida
- Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
Meredith A. Borman
- Department of Biochemistry and Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, Alberta, Canada T2N 4Z6
Alexandra Valderrama
- Department of Biochemistry and Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, Alberta, Canada T2N 4Z6
Hans J. Vogel
- Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
Justin A. MacDonald
- Department of Biochemistry and Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, Alberta, Canada T2N 4Z6
- Corresponding author. Address: Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, Alberta, Canada T2N 4Z6. Fax: +1 403 270 2211.

Received 3 June 2010; received in revised form 6 July 2010; accepted 6 July 2010. published online 12 July 2010.

Abstract

The calponin homology-associated smooth muscle protein (CHASM) can modulate muscle contractility, and its biological action may involve an interaction with the contractile filament. In this study, we demonstrate an interaction between CHASM and tropomyosin. Deletion constructs of CHASM were generated, and pull-down assays revealed a minimal deletion construct that could bind tropomyosin. Removal of the calponin homology (CH) domain or expression of the CH domain alone did not enable binding. The interaction was characterized by microcalorimetry with a dissociation constant of 2.0×10⁻⁶M. Confocal fluorescence microscopy also showed green fluorescent protein (GFP)–CHASM localization to filamentous structures within smooth muscle cells, and this targeting was dependent upon the CH domain.

Structured summary

MINT-7966126: CHASM (uniprotkb:Q99LM3), Tropomyosin alpha (uniprotkb:P04268) and Tropomyosin beta (uniprotkb:P19352) physically interact (MI:0915) by isothermal titration calorimetry (MI:0065)

MINT-7966073: CHASM (uniprotkb:Q99LM3) physically interacts (MI:0914) with Tropomyosin beta (uniprotkb:P58776) and Tropomyosin alpha (uniprotkb:P58772) by pull down (MI:0096)

MINT-7966187: Tropomyosin alpha (uniprotkb:P04268) and Tropomyosin beta (uniprotkb:P19352) physically interact (MI:0915) with CHASM (uniprotkb:Q99LM3) by pull down (MI:0096)

MINT-7966090: CHASM (uniprotkb:Q99LM3) binds (MI:0407) to Tropomyosin alpha (uniprotkb:P04268) by pull down (MI:0096)

Abbreviations: CaM, calmodulin, CH, calponin homology, CHASM, calponin homology-associated smooth muscle protein, DMEM, Dulbecco’s Modified Eagle Medium, FBS, fetal bovine serum, ITC, isothermal titration calorimetry, K_d, dissociation constant, GFP, green fluorescent protein, GST, glutathione-S-transferase, MS/MS, tandem mass spectrometry, PBS, phosphate-buffered saline, TBD, tropomyosin-binding domain, Tn, troponin