The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility

doi:10.1016/j.bbrc.2007.11.108

Biochemical and Biophysical Research Communications

Volume 369, Issue 1, 25 April 2008, Pages 124-134

https://doi.org/10.1016/j.bbrc.2007.11.108 Get rights and content

Abstract

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the nonspliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells [X. Ma, S. Kawamoto, J. Uribe, R.S. Adelstein, Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development, Mol. Biol. Cell 15 (2006) 2138–2149]. In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20 kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acid II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific.

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Materials and methods

Construction of baculovirus expression vectors. Recombinant HMM-like proteins of human NMHC II-B and mouse NMHC II-C were expressed in the baculovirus/Sf9 system. Briefly, the cDNA (nucleotides 1–3135) for human NMHC II-B (Accession No. M69181 with a change in amino acid Cys 800 to Tyr, which is the more commonly occurring amino acid) and the cDNA (nt 1–4071) for mouse NMHC II-C (Accession No. AY205605) were truncated at amino acid 1045 and 1357, respectively, to create an HMM fragment.

Enzymatic and motile activity of HMM II-B0 and HMM II-B2

HMM II-B2 has a 21-aa insert in loop 2 starting after residue 621 in human NMHC II-B (see Fig. 1). HMM II-B0 and II-B2 proteins were purified in mg quantities from 1 × 10⁹ cells following baculovirus expression and each HMM heavy chain bound the co-expressed MLC-20 and MLC-17 in an approximate 1:1:1 ratio. Fig. 2A is a Coomassie Blue-stained SDS–polyacrylamide gel showing each of the purified HMMs following concentration using a Q Sepharose column. The enzymatic activity was initially assessed by

Discussion

In this report, we describe the biochemical and cellular characterization of an alternatively spliced isoform of NM II-B. This spliced isoform results from the introduction of 21-aa into the heavy chain of NM II-B in loop 2, at a location that is close to the actin-binding region. A homologous location in NMHC II-C is also used for a larger insert of 33-aa in humans and 41-aa in mice. Of note is that the loop 2 flanking sequence is fairly well conserved between NMHC II-B and II-C (see Fig. 1).

Acknowledgments

We acknowledge the expert technical assistance of Estelle V. Harvey and Antoine Smith. We also acknowledge the expert advice of Mihaly Kovacs. Toshihiko Takenaka generated the original human HMM II-B0 construct. We also thank Catherine S. Magruder for expert editorial assistance and Mary Anne Conti, Mary D. Pato, and Siddhartha S. Jana for reading and improving this manuscript.

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The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility

Abstract

Section snippets

Materials and methods

Enzymatic and motile activity of HMM II-B0 and HMM II-B2

Discussion

Acknowledgments

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