Binding of Myosin Light Chain Kinase to Cellular Actin-Myosin Filaments

Lin, Po‐Chun; Luby-Phelps, Katherine; Stull, James T.

doi:10.1074/jbc.272.11.7412

Cited by 47 publications

(59 citation statements)

References 41 publications

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“…4). Additional experiments showed that short MLCK containing no DFRXXL motifs did not bind to stress fibers (data not shown), confirming previously reported observations (1,18,26). These results suggest that there are at least two components in long MLCK involved in stress fiber binding: the five DFRXXL motifs in addition to the N-terminal extension.…”

Section: In Vivo Localization Of 3dfr-mlck and 5dfr-mlck-tosupporting

confidence: 89%

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

Smith¹,

Parizi-Robinson²,

Zhu³

et al. 2002

Journal of Biological Chemistry

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Section: In Vivo Localization Of 3dfr-mlck and 5dfr-mlck-tosupporting

confidence: 89%

“…Cell Imaging-Fluorescence imaging was performed as described previously (26). Twelve-bit images were obtained with a CCD camera (Quantix Photometrics, Tucson, AZ) and Oncor-image software (Oncor, Gaithersburg, MD).…”

Section: Methodsmentioning

confidence: 99%

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

Smith¹,

Parizi-Robinson²,

Zhu³

et al. 2002

Journal of Biological Chemistry

Self Cite

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“…Evidence also suggests that myosin light chain kinase binds to cellular actin filaments and is localized to actinomyosin-containing stress fibers in nonmuscle cells. In a very elegant study on smooth muscle cells that used GFP-labeled myosin light chain kinase the filament-bound kinase was shown to be sufficient for Ca 2ϩ -dependent phosphorylation of myosin regulatory light chain and contraction of stress fiber (43). One of the recently identified PAK1 substrates is indeed myosin light chain kinase, and its phosphorylation by PAK1 in vitro and in vivo was shown to decrease myosin light chain activity, leading to a decrease in cell spreading and thus less actin-myosin contraction (44).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Microfilament Reorganization and Invasiveness of Breast Cancer Cells by Kinase Dead p21-activated Kinase-1

Adam¹,

Vadlamudi²,

Mandal³

et al. 2000

Journal of Biological Chemistry

161

117

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Stimulation of growth factor signaling has been implicated in the development of invasive phenotype and p21-activated kinase (PAK1) activation in human breast epithelial cancer cells. To further explore the roles of PAK1 in the invasive behavior of breast cancer cells, in the present study we investigated the influence of inhibition of PAK1 activity on the reorganization of cytoskeleton components that control motility and invasiveness of cells, using a highly invasive breast cancer MDA-MB435 as a model system. Our results demonstrate that overexpression of a kinase dead K299R PAK1 mutant leads to suppression of motile phenotypes as well as invasiveness of cells both in the absence or presence of exogenous heregulin-␤1. In addition, these phenotypic changes were accompanied by a blockade of disassembly of focal adhesion points, stabilization of stress fibers, and enhanced cell spreading and were dependent on the presence of the kinase dead domain but independent of the presence of the Rac/cdc42 intact (Cdc42/Rac interactive binding) domain of PAK1. We also demonstrated that in K299R PAK1-expressing cells, F-actin filaments were stabilized by persistent co-localization with the actin-binding proteins tropomyosin and caldesmon. Extension of these studies to invasive breast cancer MDA-MB231 cells illustrated that conditional expression of kinase-defective K299R PAK1 was also accompanied by persistent cell spreading, multiple focal adhesion points, and reduced invasiveness. Furthermore, inhibition of PAK1 activity in breast cancer cells was associated with a reduction in c-Jun N-terminal kinase activity, inhibition of DNA binding activity of transcription factor AP-1, and suppression of in vivo transcription driven by AP-1 promoter (known to be involved in breast cancer invasion). These findings suggest that PAK1 downstream pathways have a role in the development and maintenance of invasive phenotypes in breast cancer cells.Breast cancer is one of the most common malignancies in the United States, affecting one in nine women. Localized breast cancer prior to metastasis can be cured by surgery. The high mortality rate associated with breast cancer, however, is related to its ability to metastasize beyond the mammary gland and invade distant sites while the primary tumor is small and undetected. Thus, tumor cell migration/invasion is an important factor in formation of solid tumors and is necessary for their spread to distinct organs. The process of malignancy requires, among other steps, changes in growth factor pathways and increased migration. The exposure of cells to growth factors has been shown to cause cytoskeleton reorganization, formation of lamellipodia, membrane ruffling, and altered cell morphology, and accordingly, such exposure is implicated in stimulating cell migration and invasion (1). Most eukaryotic cells possess the capacity to move over or through a substrate, and cell migration plays a key role in both normal physiology and in a disease setting, including invasion and metastasis (2). In many tiss...

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“…MLCK has been reported to directly interact with actin filaments (34). To examine whether the formation of the cortical actin cytoskeleton is required for peripheral accumulation of MLCK, FAK Ϫ/Ϫ cells were treated with 2.5 M cytochalasin D for 1 h to disrupt actin cytoskeleton and then co-stained for actin and MLCK (Fig.…”

Section: And Fakmentioning

confidence: 99%

Roles of Rho-associated Kinase and Myosin Light Chain Kinase in Morphological and Migratory Defects of Focal Adhesion Kinase-null Cells

Chen

Tzen

Bresnick

et al. 2002

Journal of Biological Chemistry

140

121

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Rho GTPases including Rho, Rac, and Cdc42 are key modulators of the actin cytoskeleton (1-3). They are critical for the cell shape changes and adhesion dynamics that drive cell migration (4 -7). Among the Rho GTPase family, Rho induces the formation of focal adhesions and stress fibers (7,8). Interestingly, although a basal level of Rho activity is needed for fibroblast migration, too much Rho activity impedes migration (4, 9, 10). It has been shown that the concerted action of two of the immediate Rho targets, Rho-associated kinase (Rho-kinase) 1 /ROCK and the formin homology protein mDia1, mediate the effect of Rho on matrix adhesion and the actin cytoskeleton (11). In particular, Rho-kinase was shown to stimulate myosindriven contractility in smooth muscle and nonmuscle cells by phosphorylating, thereby inactivating myosin light chain (MLC) phosphatase (12, 13), and possibly by direct phosphorylation of MLC (14 -16). In addition to Rho-kinase, MLC kinase (MLCK) is another kinase that phosphorylates the MLC in both smooth muscle and nonmuscle cells (17, 18). The phosphorylation of MLC on Ser-19 and to a lesser extent on Thr-18 by MLCK promotes the assembly of myosin II into filaments and activates its ATPase activity, which stabilizes the actin-myosin interaction and promotes cell contractility (19 -22). Recently, Rho-kinase and MLCK were suggested to play distinct roles in spatial regulation of MLC phosphorylation. Rho-kinase appears to be important for MLC phosphorylation in the center of cells, and MLCK is responsible for phosphorylating MLC at the cell periphery (16). Focal adhesion kinase (FAK), a 125-kDa cytoplasmic tyrosine kinase localized in focal contacts, has been known to play an important role in integrin-mediated cell migration (23). Fibroblasts derived from FAK-null mouse embryos are more rounded and poorly spread than their wild-type counterparts (23). They show an overabundance of focal adhesions, enriched cortical actin filaments at the cell periphery, and a decreased migration rate (23,24). It has been suggested that the increase in peripheral adhesions results from an inhibition of turnover in FAK Ϫ/Ϫ cells (23), which may result from constitutive activation of Rho (24). Because of the known involvement of Rhokinase and MLCK in cell contractility, a major factor controlling cell migration, we hypothesize that abnormal regulation of Rho-kinase and MLCK may underlie the migratory defect of FAK Ϫ/Ϫ cells. EXPERIMENTAL PROCEDURESMaterials-Fetal bovine serum, non-essential amino acids, sodium pyruvate, and 2-mercaptoethanol were purchased from Invitrogen. Y27632, a specific inhibitor of Rho-kinase, was purchased from Calbiochem. The monoclonal anti-MLCK, monoclonal anti-MLC, monoclonal anti-␤-tubulin, bovine MLC, myelin basic protein (MBP), cytochalasin D, and 2,3-butanedione monoxime (BDM) were purchased from SigmaAldrich. The polyclonal anti-Rho-kinase and monoclonal anti-paxillin were purchased from Transduction Laboratories (Lexington, KY). The plasmid pEGFP-N1-MLCK and polyclonal anti-MLCK ...

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Binding of Myosin Light Chain Kinase to Cellular Actin-Myosin Filaments

Cited by 47 publications

References 41 publications

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

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

Regulation of Microfilament Reorganization and Invasiveness of Breast Cancer Cells by Kinase Dead p21-activated Kinase-1

Roles of Rho-associated Kinase and Myosin Light Chain Kinase in Morphological and Migratory Defects of Focal Adhesion Kinase-null Cells

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