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

Abstract

We report a novel isoform of non-muscle myosin II-C (NM II- Mammalian non-muscle myosin IIs (NM IIs)2 belong to the conventional Class II myosins and are hexameric proteins composed of two heavy chains and two pairs of light chains, referred as the 20-kDa regulatory myosin light chain (MLC 20 ) and the 17-kDa essential myosin light chain (MLC 17 ). These myosins self-associate through their tail regions to form bipolar filaments that pull on actin filaments to produce force to drive important cellular functions such as cytokinesis, cell polarity, and cell migration (1-4). Three isoforms of the non-muscle myosin heavy chain (NMHC), II-A, II-B, and II-C, have been identified in vertebrates. They are products of three different genes, MYH9 (5, 6), MYH10 (6), and MYH14 (7, 8), respectively, in humans. It is well established that the enzymatic activity of these myosins is regulated by phosphorylation of MLC 20 , which is catalyzed by a number of enzymes, including myosin light chain kinase (MLCK), and Rho kinase (9 -14).Alternative splicing of pre-mRNA of NMHC II genes generates multiple mRNAs to enhance protein diversity in the NM II family. Work from this laboratory and others (8,(15)(16)(17)(18) has established that both NMHC II-B and II-C undergo alternative splicing to generate several isoforms. In the case of NMHC II-B, 10 amino acids are incorporated into loop 1 at amino acid 212 (NMHC II-B1), and 21 amino acids are inserted into loop 2 at amino acid 622 (NMHC II-B2; see Ref. 15). These isoforms have been expressed as proteins, and their biochemical and functional importance has been 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 actin filaments in an in vitro motility assay following MLC 20 phosphorylation (22) even though HMM II-B0 and II-B1 show normal phosphorylation-dependent activities (21). These two inserted isoforms (NM II-B1 and NM II-B2) are only expressed in neuronal tissues, and the results of ablating each of them and NM II-B in mice have been reported (23)(24)(25).For NMHC II-C, an alternative exon encoding 8 amino acids is incorporated into loop 1 at amino acid 227 (NMHC II-C1) at a location homologous to that of the 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). The presence of the C1 insert in baculovirusexpressed HMM II-C1 increases both the actin-activated MgATPase activity and in vitro motility of HMM II-C1 compared with HMM II-C0, the noninserted form. The activity of both HMM II-C0 and HMM II-C1 is dependent on MLC 20 phosphorylation (26). NM II-C1 has been shown to be expressed in a number of tumor cell lines, and decreasing its expression using small interfering RNA delays a late step in cytokinesis in the lung tumor cell line A549 (27).In this study, we report that an exon encoding 41 amino acids can be incorporated into loop 2 near the ...

The Effect of Including the C2 Insert of Nonmuscle Myosin II-C on Neuritogenesis

“…Possibly, other isoforms of NM II-C compensate for the loss of NM II-C1C2, or the cells are exposed to external cues that bypass the requirement for NM II-C1C2. All isoforms of NM II, including possible alternatively spliced isoforms of NM II-B and NM II-C, are found in neuronal cells (31,33,43), giving rise to complexity as to whether these isoforms have different or redundant functions in neuronal cells. Previously, NM II-C was shown to be involved in multitasking in neuronal cells (33).…”

“…We investigated the expression of another insert, C1, in NMHC II-C mRNA, which was shown to be expressed in neuronal tissue (31). We found that the C1 insert was expressed in both undifferentiated and differentiated Neuro-2a cells (panels 1 and 2 of Fig.…”

“…The differences were considered to be significant if p was Ͻ0.05. (31) reported that exon C2, encoding 41 amino acids in the mouse, was inserted into the loop 2 region of NMHC II-C due to alternative splicing of NMHC II-C pre-mRNA. This C2 insert was shown to be expressed only in mouse and human brain tissue.…”

“…Electrophoresis, Immunoblotting, and Immunoprecipitation-Extracts of Neuro-2a cells were prepared for SDS-PAGE as described previously (31). Briefly, cells on tissue culture plates were washed twice with cold phosphate-buffered saline and directly lysed with Laemmli sample buffer.…”

Conventional myosins – unconventional functions

“…The RLCs of all three myosin isoforms can be phosphorylated on Ser19 (numbering as for smooth muscle myosin), increasing actin-activated MgATPase activity and filament stability-hence referred to as an "activation site" (Komatsu and Ikebe 2007). A recent exception has been found, however: although the C2-inserted isoform of M2C can undergo RLC phosphorylation, the unphosphorylated molecule is constitutionally active (Jana et al 2009). Exactly what benefits this confers on the action of the C2 isoform is unclear, but whatever these may be, they are restricted to neuronal cells, the only cells that express this particular isoform.…”