Phosphorylation on threonine-18 of the regulatory light chain dissociates the ATPase and motor properties of smooth muscle myosin II

Anne R. Bresnick, Vicki L. Wolff-Long, Otto Baumann, Thomas D. Pollard

Research output: Contribution to journalArticle

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Abstract

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 Tl8A 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.

Original languageEnglish (US)
Pages (from-to)12576-12583
Number of pages8
JournalBiochemistry
Volume34
Issue number39
StatePublished - 1995
Externally publishedYes

Fingerprint

Smooth Muscle Myosins
Myosin Type II
Phosphorylation
Threonine
Myosins
Adenosine Triphosphatases
Light
Xenopus
Myosin-Light-Chain Kinase
Myosin Light Chains
Assays
Avian Gizzard
Trifluoperazine
Actin Cytoskeleton
Alanine
Protein Kinase C
Oocytes
Actins
Chickens
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation on threonine-18 of the regulatory light chain dissociates the ATPase and motor properties of smooth muscle myosin II. / Bresnick, Anne R.; Wolff-Long, Vicki L.; Baumann, Otto; Pollard, Thomas D.

In: Biochemistry, Vol. 34, No. 39, 1995, p. 12576-12583.

Research output: Contribution to journalArticle

Bresnick, Anne R. ; Wolff-Long, Vicki L. ; Baumann, Otto ; Pollard, Thomas D. / Phosphorylation on threonine-18 of the regulatory light chain dissociates the ATPase and motor properties of smooth muscle myosin II. In: Biochemistry. 1995 ; Vol. 34, No. 39. pp. 12576-12583.
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