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LPA(1) -induced migration requires nonmuscle myosin II light chain phosphorylation in breast cancer cells.

ABSTRACT: The enhanced migration found in tumor cells is often caused by external stimuli and the sequential participation of cytoskeleton-related signaling molecules. However, until now, the molecular connection between the lysophosphatidic acid (LPA) receptor and nonmuscle myosin II (NM II) has not been analyzed in detail for LPA-induced migration. Here, we demonstrate that LPA induces migration by activating the LPA(1) receptor which promotes phosphorylation of the 20?kDa NM II light chain through activation of Rho kinase (ROCK). We show that LPA-induced migration is insensitive to pertussis toxin (PTX) but does require the LPA(1) receptor as determined by siRNA and receptor antagonists. LPA activates ROCK and also increases GTP-bound RhoA activity, concomitant with the enhanced membrane recruitment of RhoA. LPA-induced migration and invasion are attenuated by specific inhibitors including C3 cell-permeable transferase and Y-27632. We demonstrate that NM II plays an important role in LPA-induced migration and invasion by inhibiting its cellular function with blebbistatin and shRNA lentivirus directed against NM II-A or II-B. Inhibition or loss of either NM II-A or NM II-B in 4T1 cells results in a decrease in migration and invasion. Restoration of the expression of NM II-A or NM II-B also rescued LPA-induced migration. Taken together, these results suggest defined pathways for signaling through the LPA(1) receptor to promote LPA-mediated NM II activation and subsequent cell migration in 4T1 breast cancer cells.

SUBMITTER: Kim JH

PROVIDER: S-EPMC3115449 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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LPA(1) -induced migration requires nonmuscle myosin II light chain phosphorylation in breast cancer cells.

Kim Jong Hyun JH Adelstein Robert S RS

Journal of cellular physiology 20111101 11

The enhanced migration found in tumor cells is often caused by external stimuli and the sequential participation of cytoskeleton-related signaling molecules. However, until now, the molecular connection between the lysophosphatidic acid (LPA) receptor and nonmuscle myosin II (NM II) has not been analyzed in detail for LPA-induced migration. Here, we demonstrate that LPA induces migration by activating the LPA(1) receptor which promotes phosphorylation of the 20 kDa NM II light chain through acti ...[more]

PMID: 21302283

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Project description:Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.Significance of the studyPhosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

Project description:The objective of the study was to understand how estrogen modulates the rigidity of the cytoskeleton in epithelial cells. Estrogen depletion decreased, and treatment with 17beta-estradiol increased deformability of cervical-vaginal epithelial cells. Estrogen also induced redistribution of nonmuscle myosin II-B (NMM-II-B); lesser interaction of NMM-II-B with actin; increased phosphorylation of NMM-II-B-heavy chains at threonine and serine residues; and decreased filamentation of NMM-II-B in vitro. The effects of 17beta-estradiol were time and dose related and could be mimicked by diethylstilbestrol. The effects of estrogen were blocked by cotreatment with antisense oligonucleotide for the estrogen receptor-alpha and inhibited by ICI-182,780 and tamoxifen; omission of epithelial growth factor (EGF) from the culture medium; and cotreatments with the EGF receptor inhibitor AG1478, the ERK-MAPK inhibitor PD98059, the casein kinase-II (CK2) inhibitor 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole, the Rho-associated kinase inhibitor Y-27632, and the nonspecific phosphatase inhibitor okadaic acid. Coadministration of 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole plus okadaic acid blocked the 17beta-estradiol effect. H-89 or LY294002 did not significantly affect estrogen effects. Treatment with estrogen increased activation of ERK1/2 and CK2 activity. These data suggest a novel pathway of estrogen regulation of the cytoskeleton in epithelial cells. The effect is mediated by estrogen receptor-alpha and involves in part the EGF-EGF receptor and ERK-MAPK cascades as proximal signaling networks and the CK2 and Rho-associated kinase-regulated myosin heavy chain phosphatase as terminal effectors. Augmented phosphorylation of NMM-II-B can block filamentation and induce disassociation of the myosin from the cortical actin, and disruption of the actomyosin ring can increase cell deformability. This mechanism can explain estrogen regulation of paracellular permeability in cervical-vaginal epithelia in vivo.

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LPA(1) -induced migration requires nonmuscle myosin II light chain phosphorylation in breast cancer cells.

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LPA(1) -induced migration requires nonmuscle myosin II light chain phosphorylation in breast cancer cells.

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