Phosphorylation on threonine-18 of the regulatory light chain dissociates the ATPase and motor properties of smooth muscle myosin II.
We cloned the full-length cDNA for the cytoplasmic myosin II regulatory light chain (RLC) from a stage 1-2 Xenopus oocyte library. The Xenopus RLC is 94% identical to the chicken smooth muscle myosin RLC. All of the protein kinase C and myosin light chain kinase phosphorylation sites are conserved. Using trifluoperazine [Trybus, K. M., Waller, G. S., & Chatman, T. A. (1994) J. Cell Biol. 124, 963-969], we removed the RLC of smooth muscle myosin and replaced it with recombinant Xenopus RLCs. The wild-type Xenopus RLC substitutes for the gizzard RLC in actin-activated ATPase and in vitro motility assays. We made alanine substitutions of the two residues phosphorylated by myosin light chain kinase, Ser-19 and Thr-18. All of the myosin hybrids, regardless of their mutations or phosphorylation, have similar K+EDTA ATPase activities. As expected, the T18A, S19A hybrid had no actin-activated ATPase, whereas the T18A hybrid phosphorylated on Ser-19 had an actin-activated ATPase similar to that of wild-type hybrids phosphorylated only on Ser-19. The actin-activated ATPase of myosin phosphorylated only on Thr-18 is approximately 15-fold lower than that of myosin phosphorylated on Ser-19. Phosphorylation of either Ser-19 or Thr-18 permits the formation of filaments. Remarkably, in the gliding filament assay, myosin phosphorylated only on Thr-18 moves actin filaments at velocities similar to myosin phosphorylated on Ser-19 or both Thr-18 and Ser-19.
Bresnick
A R
AR
Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Wolff-Long
V L
VL
Baumann
O
O
Pollard
T D
TD
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D004247:DNA; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D000199:Actins; D013912:Threonine; D004247:DNA; D009219:Myosin-Light-Chain Kinase; D000251:Adenosine Triphosphatases; D009218:Myosins; D000409:Alanine