Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase

Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery

“…Consistent with a kinase-independent mechanism, L-MLCK-specific N-terminal sequences (Fig. 11A), which lack the myosin-binding and kinase domains (Stull et al, 1998) but bundle actin filaments (Smith et al, 2002;Yang et al, 2006), slowed the rate of cell spreading by 30-53% (Fig. 11B).…”

“…Myosin II and actin filaments have long been known to assemble at the membrane surface (DeBiasio et al, 1988;McKenna et al, 1989;Rhee et al, 1994) and to interact with both L-MLCK and S-MLCK (Blue et al, 2002;Dulyaninova et al, 2004;Poperechnaya et al, 2000;Stull et al, 1998). The additional F-actin-binding and -bundling sequences in the unique L-MLCK N-terminus apparently increase the affinity of L-MLCK, relative to S-MLCK, for actin filaments (Smith et al, 2002;Yang et al, 2006). SV also binds to both myosin and actin filaments, with the physical separation of the F-actin-and myosin II-binding sites within SV consistent with its suggested potential for regulated recruitment of actin and myosin filaments during myofibril and stress fiber assembly at the membrane (Oh et al, 2003).…”

“…Murine L-MLCK contains the entire S-MLCK coding sequence. In addition, L-MLCK contains an extra N-terminal sequence of 954 amino acids with multiple actin-binding sites within its immunoglobulin (Ig) domains and two DXR repeats (repeats of a motif made up of the sequence DXRXXL) (Kudryashov et al, 2004;Poperechnaya et al, 2000;Smith et al, 2002;Stull et al, 1998;Yang et al, 2006). The shared S-MLCK C-terminus contains another three DXR repeats, the kinase domain and a myosin II-binding site (Fig.…”

Myosin Light Chain Kinase (MLCK) Regulates Cell Migration in a Myosin Regulatory Light Chain Phosphorylation-independent Mechanism

“…Adv-MLCK and Adv-2Ig were prepared by releasing L-MLCK and 2Ig fragments from pEGFP-MLCK210 (described in Yang et al and Zhang et al) (29,30) and introducing them into the pShuttle-IRES-hrGFP-1 vector followed by virus preparation according to the AdEasy TM Adenoviral Vector System kit (Stratagene). For the nucleofection rescue experiments, we subcloned kinase-dead MLCK (with a deletion of the ATPbinding domain (1460 -1482 aa, GSGKFGQVFRLVEKKTG-KVWAGK) in chicken L-MLCK) (KD-MLCK), and a five-DFRXXL-motif (5DFRXXL)-containing fragment (29) into pShuttle-IRES-hrGFP-1 using a ClonExpress TM II One Step Cloning Kit (Vazyme).…”

Myosin Light Chain Kinase Is Central to Smooth Muscle Contraction and Required for Gastrointestinal Motility in Mice

“…Smooth muscle MLCK containing a kinase catalytic core with substrate binding sites and several structural motifs is ubiquitously expressed in different cells in the body. [8][9][10][11] Conventional deletion of MLCK expression leads to embryonic or perinatal lethality 12 making it impossible to determine the functional importance of MLCK in contraction of mature smooth muscle. Thus, we crossed mice containing floxed MLCK alleles with SM-CreER T2 (ki) mice expressing a tamoxifen-activated Cre recombinase driven by the SM-22 promoter to delete MLCK expression specifically in smooth muscles of adult mice.…”