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

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 aminoacids in mice (33 in humans). The 41 amino acids are inserted into loop 2 ofthe NM II-C heavy chain within the actin binding region. Unlike mostvertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressedmouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatorymyosin light chain (MLC₂₀) phosphorylation for maximumactin-activated MgATPase activity or maximum in vitro motility asmeasured by the sliding actin filament assay. In contrast, noninserted HMMII-C0 and another alternatively spliced isoform HMM II-C1, which contains 8amino acids inserted into loop 1, are dependent on MLC₂₀phosphorylation for both actin-activated MgATPase activity and invitro 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, whichcontains both the C1 and C2 inserts, does not require MLC₂₀phosphorylation for full activity similar to HMM II-C2. These constitutivelyactive 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 ubiquitouslyexpressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes tofilaments in interphase cells and to the cytokinetic ring in dividingcells.Mammalian non-muscle myosin IIs (NMIIs)² belong to theconventional Class II myosins and are hexameric proteins composed of two heavychains and two pairs of light chains, referred as the 20-kDa regulatory myosinlight chain (MLC₂₀) and the 17-kDa essential myosin light chain(MLC₁₇). These myosins self-associate through their tail regions toform bipolar filaments that pull on actin filaments to produce force to driveimportant cellular functions such as cytokinesis, cell polarity, and cellmigration(1-4).Three isoforms of the non-muscle myosin heavy chain (NMHC), II-A, II-B, andII-C, have been identified in vertebrates. They are products of threedifferent genes, MYH9(5,6), MYH10(6), and MYH14(7,8), respectively, in humans. Itis well established that the enzymatic activity of these myosins is regulatedby phosphorylation of MLC₂₀, which is catalyzed by a number ofenzymes, including myosin light chain kinase (MLCK), and Rho kinase(9-14).Alternative splicing of pre-mRNA of NMHC II genes generates multiple mRNAsto enhance protein diversity in the NM II family. Work from this laboratoryand others (8,15-18)has established that both NMHC II-B and II-C undergo alternative splicing togenerate several isoforms. In the case of NMHC II-B, 10 amino acids areincorporated into loop 1 at amino acid 212 (NMHC II-B1), and 21 amino acidsare inserted into loop 2 at amino acid 622 (NMHC II-B2; see Ref.15). These isoforms have beenexpressed as proteins, and their biochemical and functional importance hasbeen studied extensively(19-22).Recently, it has been reported that baculovirus-expressed heavy meromyosin(HMM) II-B2 lacks actin-activated MgATPase activity and cannot propel actinfilaments in an in vitro motility assay following MLC₂₀phosphorylation (22) eventhough HMM II-B0 and II-B1 show normal phosphorylation-dependent activities(21). These two insertedisoforms (NM II-B1 and NM II-B2) are only expressed in neuronal tissues, andthe results of ablating each of them and NM II-B in mice have been reported(23-25).For NMHC II-C, an alternative exon encoding 8 amino acids is incorporatedinto loop 1 at amino acid 227 (NMHC II-C1) at a location homologous to that ofthe B1 insert. Unlike NMHC II-B1, which is only expressed in neuronal tissue,NMHC II-C1 is found in a variety of tissues such as liver, kidney, testes,brain, and lung (8). Thepresence of the C1 insert in baculovirus-expressed HMM II-C1 increases boththe actin-activated MgATPase activity and in vitro motility of HMMII-C1 compared with HMM II-C0, the noninserted form. The activity of both HMMII-C0 and HMM II-C1 is dependent on MLC₂₀ phosphorylation(26). NM II-C1 has been shownto be expressed in a number of tumor cell lines, and decreasing its expressionusing small interfering RNA delays a late step in cytokinesis in the lungtumor cell line A549 (27).In this study, we report that an exon encoding 41 amino acids can beincorporated into loop 2 near the actin binding region at amino acid 636 ofNMHC II-C in mice. Expression of NM II-C2 is limited to neural tissue in mice.We used the baculovirus system to express all four isoforms of HMM II-C andfound that inclusion of the 41 amino acids in loop 2 results in an HMM with anactin-activated MgATPase activity and in vitro motility that areindependent of MLC₂₀ phosphorylation.