Properties of long myosin light chain kinase binding to F-actin in vitro and in vivo.
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 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 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 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.
Smith
Lula
L
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
Parizi-Robinson
Mojgan
M
Zhu
Min-Sheng
MS
Zhi
Gang
G
Fukui
Ryosuke
R
Kamm
Kristine E
KE
Stull
James T
JT
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D066298:In Vitro Techniques; D051379:Mice; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D017931:DNA Primers; D009219:Myosin-Light-Chain Kinase