Properties of long myosin light chain kinase binding to F-actin in vitro and in vivo

Lula Smith, Mojgan Parizi-Robinson, Min Sheng Zhu, Gang Zhi, Ryosuke Fukui, Kristine E. Kamm, James T. Stull

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Abstract

Short and long myosin light chain kinases (MLCKs) are Ca2+/calmodulin-dependent enzymes that phosphorylate the regulatory light chain of myosin II in thick filaments but bind with high affinity to actin thin filaments. Three repeats of a motif made up of the sequence DFRXXL at the N terminus of short MLCK are necessary for actin binding (Smith, L., Su, X., Lin, P., Zhi, G., and Stull, J. T. (1999) J. Biol. Chem. 274, 29433-29438). The long MLCK has two additional DFRXXL motifs and six Ig-like modules in an N-terminal extension, which may confer unique binding properties for cellular localization. Two peptides containing either five or three DFRXXL motifs bound to F-actin and smooth muscle myofilaments with maximal binding stoichiometries consistent with each motif binding to an actin monomer in the filaments. Both peptides cross-linked F-actin and bound to stress fibers in cells. Long MLCK with an internal deletion of the five DFRXXL motifs and the unique NH2-terminal fragment containing six Ig-like motifs showed weak binding. Cell fractionation and extractions with MgCl2 indicate that the long MLCK has a greater affinity for actin-containing filaments than short MLCK in vitro and in vivo. Whereas DFRXXL motifs are necessary and sufficient for short MLCK binding to actin-containing filaments, the DFRXXL motifs and the N-terminal extension of long MLCK confer high affinity binding to stress fibers in cells.

Original languageEnglish (US)
Pages (from-to)35597-35604
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number38
DOIs
StatePublished - Sep 20 2002

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Myosin-Light-Chain Kinase
Actins
Actin Cytoskeleton
Stress Fibers
Myosin Type II
Cell Fractionation
Peptides
Magnesium Chloride
In Vitro Techniques
Fibers
Myofibrils
Calmodulin
Fractionation
Stoichiometry
Smooth Muscle
Muscle
Monomers
Light

ASJC Scopus subject areas

  • Biochemistry

Cite this

Properties of long myosin light chain kinase binding to F-actin in vitro and in vivo. / Smith, Lula; Parizi-Robinson, Mojgan; Zhu, Min Sheng; Zhi, Gang; Fukui, Ryosuke; Kamm, Kristine E.; Stull, James T.

In: Journal of Biological Chemistry, Vol. 277, No. 38, 20.09.2002, p. 35597-35604.

Research output: Contribution to journalArticle

Smith, Lula ; Parizi-Robinson, Mojgan ; Zhu, Min Sheng ; Zhi, Gang ; Fukui, Ryosuke ; Kamm, Kristine E. ; Stull, James T. / Properties of long myosin light chain kinase binding to F-actin in vitro and in vivo. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 38. pp. 35597-35604.
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