Functional Characterization of Vertebrate Nonmuscle Myosin IIB Isoforms Using Dictyostelium Chimeric Myosin II

Takahashi, Masayuki; Takahashi, Keita; Hiratsuka, Yuichi; Uchida, Keiji; Yamagishi, Akihiko; Uyeda, Taro Q.P.; Yazawa, Michio

doi:10.1074/jbc.m005370200

Cited by 8 publications

(20 citation statements)

References 61 publications

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“…As Table 2 shows, in the absence of MLC 20 phosphorylation, there was no significant movement (Ͻ0.01 m/s) of actin filaments bound to HMM II-C0-and II-C1-coated surfaces. Following MLC 20 phosphorylation, as expected, the actin filaments were propelled by HMM II-C0 and II-C1 with an average velocity of 0.04 and 0.09 m/s, respectively, similar to a previous report (26). In contrast, HMM II-C2 and II-C1C2 can translocate actin filaments with a velocity of 0.06 m/s whether or not MLC 20 is phosphorylated.…”

Section: Actin-activated Mgatpase Activity Of Hmm Ii-c Isoforms-supporting

confidence: 64%

“…vertebrate NM IIs and smooth muscle myosin II (SM II) is regulated by phosphorylation of MLC 20 . These myosins have a very low actin-activated MgATPase activity when their light chain is unphosphorylated, and this activity increases upon phosphorylation of MLC 20 on Ser-19/Thr-18.…”

Section: Actin-activated Mgatpase Activity Of Hmm Ii-c Isoforms-mentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Jana

Kim

Mao

et al. 2009

Journal of Biological Chemistry

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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 ...

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Section: Actin-activated Mgatpase Activity Of Hmm Ii-c Isoforms-supporting

confidence: 64%

Section: Actin-activated Mgatpase Activity Of Hmm Ii-c Isoforms-mentioning

confidence: 99%

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

Jana

Kim

Mao

et al. 2009

Journal of Biological Chemistry

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“…This suggests that the effect of the B2 insert depends on the specificity of myosin heavy chain. In an experiment more directly relevant to the present report, Takahashi et al [21] introduced loop 2 from II-B0, and separately loop 2 from II-B2, into the Dictyostelium myosin II backbone replacing the endogenous loop 2. They found that introducing the II-B0 loop 2 lowered the actin-activated MgATPase activity by one-half compared to the wild-type activity and that introducing the II-B2 loop 2 reduced it by twothirds.…”

Section: Discussionmentioning

confidence: 99%

“…A further detailed study by Murphy and Spudich [20] showed that the V max of the actin-activated MgATPase activity and the affinity of Dictyostelium myosin for actin are both affected by substitutions of the loop 2 sequence. Takahashi et al [21] demonstrated that the insertion of the human II-B2 sequence into the Dictyostelium MHC reduced the motor activity of Dictyostelium myosin, with reduction of both the maximal actin-activated MgATPase activity and a decrease in the affinity for actin. In vivo studies with genetically ablated NMHC II-B2 mice demonstrated impaired motor coordination and misplaced Purkinje cells in the cerebellar molecular layer [13].…”

Section: Introductionmentioning

confidence: 99%

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

Kim

Kawamoto

Bao

et al. 2008

Biochemical and Biophysical Research Communications

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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 non-spliced 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 (Ma et al., 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 . 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 acids 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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A brain specific alternatively spliced isoform of nonmuscle myosin IIA lacks its mechanoenzymatic activities

Das¹,

Mallick²,

Sarkar³

et al. 2023

Journal of Biological Chemistry

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Functional Characterization of Vertebrate Nonmuscle Myosin IIB Isoforms Using Dictyostelium Chimeric Myosin II

Cited by 8 publications

References 61 publications

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

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

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

A brain specific alternatively spliced isoform of nonmuscle myosin IIA lacks its mechanoenzymatic activities

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