Properties of Long Myosin Light Chain Kinase Binding to F-Actin in Vitro and in Vivo

• Published in: The Journal of biological chemistry 2002-09, Vol.277 (38), p.35597-35604
• Main Authors: Smith, Lula, Parizi-Robinson, Mojgan, Zhu, Min-Sheng, Zhi, Gang, Fukui, Ryosuke, Kamm, Kristine E, Stull, James T
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Actins - metabolism; Animals; Base Sequence; DNA Primers; In Vitro Techniques; Mice; Myosin-Light-Chain Kinase - metabolism; Protein Binding; Rabbits
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M206483200

Short and long myosin light chain kinases (MLCKs) are Ca 2+ /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 DFR XX L 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 DFR XX L 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 DFR XX L 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 DFR XX L motifs and the unique NH 2 -terminal fragment containing six Ig-like motifs showed weak binding. Cell fractionation and extractions with MgCl 2 indicate that the long MLCK has a greater affinity for actin-containing filaments than short MLCK in vitro and in vivo . Whereas DFR XX L motifs are necessary and sufficient for short MLCK binding to actin-containing filaments, the DFR XX L motifs and the N-terminal extension of long MLCK confer high affinity binding to stress fibers in cells.