SITE-DIRECTED MUTAGENESIS OF NONMUSCLE MYOSIN HEAVY CHAINS

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Cloning of the CDNA encoding a chicken brain nonmuscle myosin heavy chain (MHC-B) revealed the presence of cassettes of amino acids at the 25-50 and 50-20 Kda junctions, located in the head region of the MHC (Takahashi et al., J. Biol. Chem. 267: 17,864, 1992). These inserts are located near to the ATP-binding and actin-binding sites in the nonmuscle myosin heavy chain, respectively. The isoforms that expressed the inserted cassettes were confined to myosin expressed in the nervous system and were present along with the noninserted isoform. The latter is also expressed in numerous other tissues in addition to the brain and spinal cord. To study the differences in biological activity between the inserted and noninserted isoforms, we initiated studies to express CDNA encoding the amino-terminal 1231 amino acids of the MHC-B noninserted isoform along with the 20 Kd and 17 Kd myosin light chains. HMM-exp (containing the 150 Kd, 20 Kd and 17 Kd polypeptides in the proper stoichiometry) was soluble at low ionic strength and bound to rabbit skeletal muscle actin in an ATP-dependent manner. It has the ability to propel actin filaments in an in vitro motility assay system and to be activated by actin to hydrolyze MgATP. Both of these biological properties required phosphorylation of the 20 Kda myosin light chain by myosin light chain kinase. Tryptic peptide maps of the expressed 20 Kd myosin light chain, previously phosphorylated by myosin light chain kinase and protein kinase C, resembled those generated from the native light chain. Since protein kinase C phosphorylates an acetylated serine residue (serine-1), this similarity in peptide maps suggests that insect cell posttranslationally processes the expressed light chain in a similar manner. Recently, we have succeeded in expressing the MHC that contains an inserted sequence of 10 amino acids near to the ATP-binding site (i.e., at the 25-50 Kda junction). This insert also contains a putative site for phosphorylation by cyclin-p34-cdc2 kinase and/or MAP II kinase. We are presently characterizing this expressed isoform.