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An increase or a decrease in myosin II phosphorylation inhibits macrophage motility
A K Wilson1, G Gorgas, W D Claypool
1Department of Physiology, College of Medicine, University of Illinois, Chicago 60680.
Abstract:
Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin-independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells.
Insights
Myosin light chain kinase (MLCK) regulates cell motility by controlling MLC20 phosphorylation. Inhibiting or overactivating MLCK impairs macrophage migration, indicating phosphorylation must be tightly controlled.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Myosin II is crucial for cell motility.
- Myosin light chain kinase (MLCK) phosphorylates the myosin light chain 20 (MLC20) subunit.
- The role of MLC20 phosphorylation in regulating cell motility is not fully understood.
Purpose of the Study:
- To investigate the importance of MLC20 phosphorylation in regulating cell motility.
- To determine the effects of modulating MLCK activity on macrophage chemotaxis.
Main Methods:
- Macrophages were introduced with antibodies to MLCK (MK-Ab) or a constitutively active form of MLCK (MK-).
- Quantitative chemotaxis assays were used to measure cell motility.
- Intracellular MLC20 phosphorylation levels and myosin association with the cytoskeleton were analyzed.
Main Results:
- Macrophage chemotaxis was significantly diminished with MK-Ab, showing an inverse relationship between MK-Ab levels and cell migration.
- Constitutively active MK- also inversely correlated with cell migration, despite increasing MLC20 phosphorylation.
- MK-Ab decreased, while MK- increased, intracellular MLC20 phosphorylation and myosin-cytoskeleton association.
Conclusions:
- MLCK regulation is critical for mammalian cell motility.
- MLC20 phosphorylation must be maintained within narrow limits for effective cell migration.
- These findings highlight the precise control required for myosin-driven cellular processes.