Baculovirus expression of chicken nonmuscle heavy meromyosin II-B - Characterization of alternatively spliced isoforms

We have expressed two truncated isoforms of chicken nonmuscle myosin II-B using the baculovirus expression system, One of the expressed heavy meromyosins (HMM(exp)) consists of two 150-kDa myosin heavy chains (MHCs), comprising amino acids 1-1231 as well as two pairs of 20-kDa and 17-kDa myosin light chains (MLCs) in a 1:1:1 molar ratio, The second HMM(exp) was identical except that it contained an insert of 10 amino acids (PESPKPVKHQ) at the 25-50-kDa domain boundary in the subfragment-1 region of the MHC. These 10 amino acids include a consensus sequence (SPK) for proline directed kinases, Expressed HMMs were soluble at low ionic strength and bound to rabbit skeletal muscle actin in an ATP-dependent manner, These properties afforded a rapid purification of milligram quantities of expressed protein, Both isoforms were capable of moving actin filaments in an in vitro motility assay and manifested a greater than 20-fold activation of actin-activated MgATPase activity following phosphorylation of the 20-kDa MLC. HMM(exp) with the 10-amino acid insert was phosphorylated by Cdc2, Cdk5, and mitogen-activated protein kinase in vitro to 0.3-0.4 mol of PO4/mol of MHC The site phosphorylated in the MHC was identified as the serine residue present in the 10-amino acid insert and its presence was confirmed in bovine brain MHCs. Characterization of the baculovirus expressed noninserted and inserted MHC isoforms with respect to actin-activated MgATPase activity and ability to translocate actin filaments in an in vitro motility assay produced the following average values following MLC phosphorylation: noninserted HMM(exp), V-max 0.28 s(-1), K-m = 12.7 mu M; translocation rate = 0.077 mu m/s; inserted HMM(exp) V-max = 0.37 s(-1), K-m = 15.1 mu M; translocation rate = 0.092 mu m/s.