Man-Sik Kang

Increase in the level of m-calpain correlates with the elevated cleavage of filamin during myogenic differentiation of embryonic muscle cells.

The activity of Ca(2+)-activated proteinase requiring millimolar Ca2+ (m-calpain) was found to increase dramatically in cultured chick embryonic myoblasts during the early period of myogenic differentiation. Furthermore, the protein level of m-calpain also markedly increased in parallel with the rise in its activity, and both remained elevated thereafter. On the other hand, the activity level of calpastatin, an endogenous inhibitor of the proteinase, remained similar during the entire period of the culture. In addition, the activity of Ca(2+)-activated proteinase requiring micromolar Ca2+ (mu-calpain) was not detected in either proliferating or differentiated myoblasts. Thus, the overall capacity of Ca(2+)-dependent proteolysis is likely to increase in differentiating myoblasts and should be contributed by m-calpain. Filamin (250 kDa), that is known to facilitate actin microfilament assembly and interfere with actin-myosin filament formation, was found to be cleaved in cultured myoblasts to 240 kDa products. This filamin-cleavage occurred in a manner similar to the in vitro cleavage of the cytoskeletal protein by the purified m-calpain. Moreover, the filamin-cleavage was most evident at the period of the cell fusion. Thus, it seems likely that the in vivo cleavage of filamin is mediated by m-calpain. These results suggest that m-calpain may play an important role in cytoskeletal reorganization that is requisite for myoblast fusion.

Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease

HslVU in E. coli is a new type of ATP‐dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain β‐type subunit of the 20S proteasomes. To gain an insight into the catalytic mechanism of HslV, site‐directed mutagenesis was performed to replace each of the Thr residues with Ser or Val and to delete the first or both Thr. Also each of the five internal Ser residues in HslV were replaced with Ala. The results obtained by the mutational analysis revealed that the N‐terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation.

Inhibitors of the proteasome block the myogenic differentiation of rat L6 myoblasts

Myogenesis is characterized by membrane fusion and accumulation of muscle specific proteins. We have previously shown that nitric oxide acts as a messenger for membrane fusion. Here we show that inhibitors of the proteasome, such as lactacystin, reversibly block both the fusion of L6 myoblasts and the accumulation of muscle specific proteins, such as myosin heavy chain (MHC). The inhibitors also reversibly prevented the induction of the NF‐κB activity, which is required for the expression of nitric oxide synthase (NOS). Moreover, the inhibition of the NF‐κB activity occurred in parallel with that of the NOS activity upon treatment with increasing concentrations of lactacystin. While pyrrolidine dithiocarbamate, an inhibitor of NF‐κB, blocked both membrane fusion and accumulation of MHC, N G‐monomethyl‐l‐arginine, a specific inhibitor of NOS, inhibited only the fusion. These results suggest that the proteasome plays an essential role in the regulation of myogenic differentiation through the activation of NF‐κB and that the target of NF‐κB for the expression of muscle specific proteins is distinct from that for myoblast fusion.

Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli

HslU is the ATPase component of the ATP‐dependent HslVU protease in Escherichia coli. To gain an insight into the structure and function of HslU, site‐directed mutagenesis was performed to generate a mutation in the ATP‐binding site of the ATPase (i.e., to replace the Lys63 with Thr). Unlike the wild‐type HslU, the mutant form (referred to as HslU/K63T) could not hydrolyze ATP or support the ATP‐dependent hydrolysis of N‐carbobenzoxy‐Gly‐Gly‐Leu‐7‐amido‐4‐methyl coumarin by HslV. The wild‐type HslU (a mixture of monomer and dimer) formed a multimer containing 6–8 subunits in the presence of either ATP or ADP, indicating that ATP‐binding, but not its hydrolysis, is required for oligomerization of HslU. However, HslU/K63T remained as a monomer whether or not the adenine nucleotides were present. Furthermore, ATP or ADP could protect HslU, but not HslU/K63T, from degradation by trypsin. These results suggest that the mutation in the ATP‐binding site results in prevention of the binding of the adenine nucleotides to HslU and hence in impairment of both oligomerization and ATPase function of HslU.

Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein in chick embryonic muscle cells in culture☆

The pattern of protein phosphorylation was found to change in differentiating chick embryonic myoblasts in culture. The extent of phosphorylation of 42-, 50-, and 100-kDa proteins increased while that of a 63-kDa protein declined in extracts of myoblasts that had been cultured for increasing periods. Of these, the increase in phosphorylation of the 100-kDa protein occurred most dramatically in extracts of myoblasts in an early stage of differentiation and was specifically inhibited by trifluoperazine (TFP) and other calmodulin (CaM) antagonists including chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). Treatment of increasing concentrations of TFP to culture medium also decreased the phosphorylation state of the 100-kDa protein and the degree of myoblast fusion in parallel. In addition, levels of both the kinase activity and the 100-kDa protein but not of CaM appeared to rise in the cells cultured for longer periods. These results suggest that (1) a Ca2+/CaM-dependent protein kinase is responsible for phosphorylation of the 100-kDa protein, (2) the TFP-mediated myoblast fusion block may be associated with the inhibitory effect of the drug against the kinase activity, and (3) the increase in phosphorylation state of the 100-kDa protein during myogenic differentiation is due to the rise in levels of the kinase and its substrate.

Okadaic acid blocks membrane fusion of chick embryonic myoblasts in culture

Okadaic acid was found to block membrane fusion of chick embryonic myoblasts in culture. It also induced morphological change of the cells from bipolar to spherical shape. These effects were dose-dependent, and could be reversed upon removal of the drug from the culture medium. It showed, however, no effect on the induction of muscle specific proteins including tropomyosin and creatine kinase. When okadaic acid was treated to the cell lysates, the phosphorylation state of many proteins significantly increased. These results suggest that the inhibition of myoblast fusion by okadaic acid may be mediated by the increase in the phosphorylation of certain, unknown protein(s) that regulate the fusion process.

Purification and characterization of a poly-l-lysine-activated serine endoprotease from Lumbricus rubellus

An endoprotease in earthworm (Lumbricus rubellus) is purified to apparent homogeneity using 125I-lactalbumin as a substrate. The protease has a molecular mass of 27 kDa and is markedly activated by poly-L-lysine or poly-L-arginine. It is a chymotrypsin-like serine protease. Its activity is distributed to coelomic fluid but relatively little to coelomocytes.

Mutational analysis for the role of C‐terminal region of ecotin, a dimeric inhibitor of pancreatic serine proteases

Ecotin is a dimeric molecule that is capable of inhibiting a variety of serine proteases. To clarify the role of the C‐terminal region, mutagenesis was performed to delete the C‐terminal residues from 130 to 142. The mutant inhibitor behaved as a monomer upon cross‐linking analysis followed by gel filtration. The mutation also resulted in a significant increase of the Ki of ecotin on trypsin, chymotrypsin, and elastase (i.e., by 1 to 2 order of magnitude). The mutant ecotin was slightly more sensitive to heating at 100°C than the wild‐type ecotin, but became much more sensitive to the heat treatment upon reduction of the intra‐chain disulfide bond in its subunits. In addition, treatment with 4 M urea resulted in complete loss of the activity of the mutant ecotin but not that of the wild‐type inhibitor. Thus, the C‐terminal region of ecotin seems to be required not only for dimerization of the subunits but also for optimal interaction with target proteases and for maintenance in its structural stability, particularly under reducing or denaturing conditions.

NF‐κB activation by disruption of microtubule array during myogenesis of L6 cells

We have previously reported that NF‐κB is involved in the regulation of nitric oxide synthase gene expression during differentiation of chick embryonic myoblasts. However, how NF‐κB is timely activated during myogenesis remains elusive. One of the most prominent events in myogenesis is myoblast membrane fusion, which is accompanied with massive cytoskeletal reorganization. Here we show that the activity of NF‐κB markedly increases in L6 rat myogenic cells that have just initiated morphological changes by treating nocodazole, a microtubule‐disrupting agent. Furthermore, the induction of NF‐κB activation was closely correlated with the myoblast fusion. In addition, a variety of agents that disrupt microtubules stimulated the myoblast fusion as well as the induction of NF‐κB activation. In contrast, taxol, a microtubule‐stabilizing agent, suppressed the induction of NF‐κB activation and inhibited spontaneous differentiation of L6 cells as well. In addition, we found that the NF‐κB in the cells consists of p5...