An Alternatively Spliced Isoform of Non-muscle Myosin II-C Is Not Regulated by Myosin Light Chain Phosphorylation

Siddhartha S Jana,Kye-Young Kim,Jian Mao,Sachiyo Kawamoto,James R Sellers,Robert S Adelstein

doi:10.1074/jbc.m806574200

Abstract

We report a novel isoform of non-muscle myosin II-C (NM II-C), NM II-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC(20)) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1, which contains 8 amino acids inserted into loop 1, are dependent on MLC(20) phosphorylation for both actin-activated MgATPase activity and in vitro motility ( Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-22775 ). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC(20) phosphorylation for full activity similar to HMM II-C2. These constitutively active C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells.

Highlights

We report a novel isoform of non-muscle myosin II-C (NM II-C), non-muscle myosin IIs (NM IIs)-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans)
Unlike most vertebrate NM II and smooth muscle myosin II (SM II) isoforms characterized to date, heavy meromyosin (HMM) II-C2 and II-C1C2 do not require phosphorylation of their regulatory light chain at Ser-19/Thr-18 for activity, as measured by actin-activated MgATPase activity and by the sliding actin in vitro motility assay
Because of its low activity we speculated that NM II-B2 plays a role in cellular functions that depend more on the structural properties, rather than the motor activity of myosin II

Summary

EXPERIMENTAL PROCEDURES

Identification of the C2 Insert in Mouse and Human NMHC II-C—Total RNA from various mouse tissues was isolated using the RNeasy minikit (Qiagen, Valencia, CA). The slower migrating bands (694 bp for mouse and 650 bp for human) from cerebellum were extracted from the gel and subcloned into the pCR2.1-TOPO vector for sequencing using the TOPO TA cloning kit (Invitrogen), which identified the inserted isoform of both mouse and human NMHC II-C. ATPase Assay—Actin-activated MgATPase activities were measured by an NADH-linked assay as described by Wang et al [29] in a buffer containing 10 mM MOPS (pH 7.0), 2 mM MgCl2, 0.1 mM EGTA, 1 mM ATP, 0.2 mM CaCl2, 1 ␮M calmodulin, with and without 10 ␮g/ml MLCK, 40 units/ml lactate dehydrogenase, 200 units/ml pyruvate kinase, 1 mM phosphoenolpyruvate, and 0.2 mM NADH, various concentrations of actin (1–30 ␮M), and HMM II-C isoforms (0.2– 0.8 ␮M) at 25 °C. 150 mM NaCl, 10 mM MgCl2, 5 mM ATP, 4 mM EDTA, 1 mM dithiothreitol, 1% Nonidet P-40, and protease inhibitors

RESULTS

In vitro motility

Findings

DISCUSSION