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© The Rockefeller University Press, 0021-9525/1999//149 $5.00
The Journal of Cell Biology, Volume 146, Number 1, , 1999 149-164

Original Article

Myosin Light Chain Kinase Functions Downstream of Ras/ERK to Promote Migration of Urokinase-Type Plasminogen Activator-Stimulated Cells in an Integrin-Selective Manner



Diem H.D. Nguyena, Andrew D. Catlingb, Donna J. Webbc, Mauricio Sankovicc, Lori A. Walkerd, Avril V. Somlyoc,d, Michael J. Weberb, and Steven L. Goniasa,c

a Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
b Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
c Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
d Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
Department of Pathology, Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Box 214, Charlottesville, VA 22908.(804) 982-0283(804) 924-9192

SLG2T{at}VIRGINIA.EDU

Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a β1-integrin (probably {alpha}Vβ1) and {alpha}Vβ5. When MCF-7 cells were transfected to express {alpha}Vβ3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of {alpha}Vβ3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

Key Words: urokinase-type plasminogen activator • myosin light chain kinase • integrins • vitronectin • cellular migration

© 1999 The Rockefeller University Press

1.used in this paper: ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GPI, glycosyl-phosphatidylinositol; HA, hemagglutinin; MAP kinase, mitogen activated protein kinase; MEK, MAP kinase kinase; MLCK, myosin light chain kinase; PAI, plasminogen activator inhibitor; RLC, myosin II regulatory light chain; sc, single chain; tc, two chain; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor


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