Journal of Cell Science, Vol 108, Issue 10 3253-3265, Copyright © 1995 by Company of Biologists

JOURNAL ARTICLES

Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon

MF Pittenger, A Kistler and DM Helfman
Cold Spring Harbor Laboratory, NY 11724, USA.

The rat beta-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle beta-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. Skeletal muscle beta-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal beta gene products, fibroblast TM-1 and beta skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 microM for beta Sk TM, 8.5 for TM-6/10, 25 microM for TM-1, and 30 microM for TM-7/11 at an F-actin concentration of 42 microM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the beta skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the alpha gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed beta gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other beta gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The beta tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				G. Schevzov, T. Fath, B. Vrhovski, N. Vlahovich, S. Rajan, J. Hook, J. E. Joya, F. Lemckert, F. Puttur, J. J.-C. Lin, et al. Divergent Regulation of the Sarcomere and the Cytoskeleton J. Biol. Chem., January 4, 2008; 283(1): 275 - 283. [Abstract] [Full Text] [PDF]

				P. Gunning, G. O'neill, and E. Hardeman Tropomyosin-Based Regulation of the Actin Cytoskeleton in Time and Space Physiol Rev, January 1, 2008; 88(1): 1 - 35. [Abstract] [Full Text] [PDF]

				F. Houle, A. Poirier, J. Dumaresq, and J. Huot DAP kinase mediates the phosphorylation of tropomyosin-1 downstream of the ERK pathway, which regulates the formation of stress fibers in response to oxidative stress J. Cell Sci., October 15, 2007; 120(20): 3666 - 3677. [Abstract] [Full Text] [PDF]

				A. J. Kee, G. Schevzov, V. Nair-Shalliker, C. S. Robinson, B. Vrhovski, M. Ghoddusi, M. R. Qiu, J. J.-C. Lin, R. Weinberger, P. W. Gunning, et al. Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy J. Cell Biol., August 30, 2004; 166(5): 685 - 696. [Abstract] [Full Text] [PDF]

				R. Maytum, F. Bathe, M. Konrad, and M. A. Geeves Tropomyosin Exon 6b Is Troponin-specific and Required for Correct Acto-myosin Regulation J. Biol. Chem., April 30, 2004; 279(18): 18203 - 18209. [Abstract] [Full Text] [PDF]

				S. Bharadwaj, S. Hitchcock-DeGregori, A. Thorburn, and G. L. Prasad N Terminus Is Essential for Tropomyosin Functions: N-TERMINAL MODIFICATION DISRUPTS STRESS FIBER ORGANIZATION AND ABOLISHES ANTI-ONCOGENIC EFFECTS OF TROPOMYOSIN-1 J. Biol. Chem., April 2, 2004; 279(14): 14039 - 14048. [Abstract] [Full Text] [PDF]

				N. S. Bryce, G. Schevzov, V. Ferguson, J. M. Percival, J. J.-C. Lin, F. Matsumura, J. R. Bamburg, P. L. Jeffrey, E. C. Hardeman, P. Gunning, et al. Specification of Actin Filament Function and Molecular Composition by Tropomyosin Isoforms Mol. Biol. Cell, March 1, 2003; 14(3): 1002 - 1016. [Abstract] [Full Text] [PDF]

				D. M. Helfman, E. T. Levy, C. Berthier, M. Shtutman, D. Riveline, I. Grosheva, A. Lachish-Zalait, M. Elbaum, and A. D. Bershadsky Caldesmon Inhibits Nonmuscle Cell Contractility and Interferes with the Formation of Focal Adhesions Mol. Biol. Cell, October 1, 1999; 10(10): 3097 - 3112. [Abstract] [Full Text]

				C. Dufour, R. P. Weinberger, G. Schevzov, P. L. Jeffrey, and P. Gunning Splicing of Two Internal and Four Carboxyl-terminal Alternative Exons in Nonmuscle Tropomyosin 5 Pre-mRNA Is Independently Regulated during Development J. Biol. Chem., July 17, 1998; 273(29): 18547 - 18555. [Abstract] [Full Text] [PDF]

				R. L. Hammell and S. E. Hitchcock-DeGregori The Sequence of the Alternatively Spliced Sixth Exon of alpha -Tropomyosin Is Critical for Cooperative Actin Binding but Not for Interaction with Troponin J. Biol. Chem., September 5, 1997; 272(36): 22409 - 22416. [Abstract] [Full Text] [PDF]

				M Gimona, Z Lando, Y Dolginov, J Vandekerckhove, R Kobayashi, A Sobieszek, and D. Helfman Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins J. Cell Sci., January 3, 1997; 110(5): 611 - 621. [Abstract] [PDF]

				R. L. Hammell and S. E. Hitchcock-DeGregori Mapping the Functional Domains within the Carboxyl Terminus of alpha-Tropomyosin Encoded by the Alternatively Spliced Ninth Exon J. Biol. Chem., February 23, 1996; 271(8): 4236 - 4242. [Abstract] [Full Text] [PDF]

				S. E. Hitchcock-DeGregori and Y. An Integral Repeats and a Continuous Coiled Coil Are Required for Binding of Striated Muscle Tropomyosin to the Regulated Actin Filament J. Biol. Chem., February 16, 1996; 271(7): 3600 - 3603. [Abstract] [Full Text] [PDF]