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 (HMMexp) 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 HMMexp 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. HMMexp 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 HMMexp, Vmax = 0.28 s-1, Km = 12.7 microM; translocation rate = 0.077 micron/s; inserted HMMexp, Vmax = 0.37 s-1, Km = 15.1 microM; translocation rate = 0.092 micron/s.
Pato
M D
MD
Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1762, USA.
Sellers
J R
JR
Preston
Y A
YA
Harvey
E V
EV
Adelstein
R S
RS
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002417:Cattle; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D018411:Spodoptera
D015879:Myosin Subfragments