Localization and Activity of Myosin Light Chain Kinase Isoforms during the Cell Cycle

Abstract

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH2-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH2-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735–744) act cooperatively to regulate the level of Ser 19–phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.

DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation

“…Also, within a single cell, GFP-DAPk was not evenly distributed on the filaments, with some areas of individual stress fibers containing more DAPk than others (compare shades of yellow and orange of overlap in right panel of Figure 1b), and some fibers lacking visible GFP-DAPk. This contrasts with the previously described long myosin light chain kinase (MLCK L -GFP) fusion, 24 which localizes to well-organized, thick filaments (e.g. Figure 1a) that coincide completely and uniformly with actin stress fibers (Figure 1c), extending to the focal contacts (Figure 1e).…”

“…Figure 1a) that coincide completely and uniformly with actin stress fibers (Figure 1c), extending to the focal contacts (Figure 1e). In fact, expression of MLCK results in increased fiber formation in these same cells (see also Poperechnaya et al 24 and Blue et al 25 ). GFP alone, in contrast, exhibited a diffuse cytoplasmic localization with strong nuclear accumulation (Figure 1a), and showed no significant overlap with actin filaments (data not shown).…”

“…1,24 In-frame C-terminal fusions of GFP to full-length DAPk, or the catalytically inactive K42A mutant, were generated by excising the 4.7 kB EcoR1 fragment encoding HA-tagged DAPk from pcDNA3-DAPk or pcDNA3-DAPk K42A 1 and inserting it into pEGFP-C2 (Clontech), to produce pEGFP-DAPk or pEGFP-DAPk K42A, respectively. A construct in which the DAPk cassette was inserted in the reverse orientation was used as a control for a GFP-expressing plasmid of identical size that does not produce a fusion protein (pEGFP-DAPk(rev)).…”

“…293T human embryonic kidney cells and HeLa cells 24 were grown in DMEM (Biological Industries) supplemented with 10% fetal bovine serum (Sigma), L-glutamine (2 mM), penicillin (50 U/ml), and streptomycin (50 mg/ ml, Gibco BRL). Cells were transiently transfected by the Ca 2 þ -phosphate precipitation method.…”

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

“…It is hypothesized that binding to F-actin via DFRXXL and to myosin via telokin facilitates RLC phosphorylation by MLCK. When the 5DFRXXL region was deleted, the full length or N-terminus of long MLCK could still bind to stress fiber, implying the existence of a novel binding motif in the extension region [23][24][25]. Furthermore, Poperechnaya et al found that, in dividing cells, the extension region was required for MLCK localization to the cleavage furrow.…”