Specificity of pSer1 Ab against the phosphorylated regulatory light chain of myosin II. (A) Unphosphorylated (0-p) and phosphorylated MLC₂₀ (1-p and 2-p) of myosin II either by MLCK or PKC were separated by alkali-urea/glycerol gel (left panel, Ponceau S staining), followed by immunoblotting with pSer1 Ab (right panel). Lane 1, unphosphorylated MLC₂₀; lane 2, phosphorylated MLC₂₀ by MLCK; and lane 3, phosphorylated MLC₂₀ by PKC. ELCs, essential light chains. (B) Immunoblot analysis with pSer1 Ab was carried out for wild-type MLC₂₀ (Wt) and S1A/S2A MLC₂₀ (AA). Both wild-type MLC₂₀ and S1A/S2A MLC₂₀ were incubated with PKC in the presence of Mg²⁺-ATP, and the sample was analyzed by immunoblotting with pSer1 Ab. (C) Immunoblot of the whole lysates of TPA stimulated cells with pSer1 Ab. After serum starvation, REF-2A fibroblasts, CHO-K1, NIH3T3, or NRK52E epithelial cells were treated with 200 nM TPA or 0.1% DMSO (control) for 15 min, and then the whole cell lysates were subjected to immunoblotting with pSer1 Ab.

Effect of the protein kinase inhibitors on the PDGF-mediated Ser1 phosphorylation. (A) Immunoblotting of PDGF-stimulated NIH3T3 cells in the presence of various kinase inhibitors. Serum-starved NIH3T3 cells were preincubated with the protein kinase inhibitors for 10 min (3 μM GF109203X, PKC inhibitor; 10 μM LY294002, PI3K inhibitor; 50 μM PD98059, MEK inhibitor; and SB203580, p38 MAP kinase inhibitor; respectively) before PDGF (20 ng/ml) stimulation. The reaction was stopped at 30 min after stimulation, and the whole cell lysates were subjected to immunoblot analysis with pSer1 Ab (top panel) and MLC₂₀ Ab (bottom panel), respectively. NC, no treatment with PDGF; control, 0.1% DMSO. (B) Immunoblotting of PDGF-stimulated NIH3T3 cells in the presence of PKC inhibitors. Serum-starved NIH3T3 cells were treated with 20 ng/ml PDGF for 30 min in the presence of 0.1% DMSO (control), 3 μM GF109203X, 1 μM or 100 nM Go6976, or 1 μM Rottlerin. Cells were preincubated with the PKC inhibitors for 10 min before cell stimulation. The whole cell lysates were subjected to Western blotting with pSer1 Ab (top panel) and MLC₂₀ Ab (bottom panel), respectively. NC, no treatment with PDGF. (C) Immunostaining of PDGF-stimulated NIH3T3 cells in the presence of PKC inhibitors. Serum-starved NIH3T3 cells were treated with 20 ng/ml PDGF for 30 min under the same condition as in B. The cells were stained with pSer1 Ab (a–d), Myosin II Ab (e–h) and Alexa Fluor546-phalloidin (i–l). Bar, 25 μm.

Effect of the phosphorylation of MLC₂₀ at the inhibitory sites on the disassembly of stress fibers in MEF/3T3 Tet-Off cells. MEF/3T3 Tet-Off cells were cultured in the absent of doxycycline (Dox) to induce the expression of myc-tagged wild-type or S1A/S2A MLC₂₀. After 3 d, the cells were serum-starved for 18 h and then treated with PDGF for 40 min, followed by immunostaining with myc Ab, paxillin Ab, and Alexa Fluor546-phalloidin. Bar, 30 μm. (A) Immunostainig of MEF/3T3 Tet-Off cells expressed with wild-type MLC₂₀. (B) Immunostaining of S1A/S2A MLC₂₀ stable cells in the absent or presence of Dox. myc Ab (green), Alexa Fluor546-phalloidin (red), and paxillin (blue). Bar, 25 μm. (C) Quantification of PDGF-induced the disassembly of stress fibers in MEF/3T3 Tet-Off cells. *p < 0.003 compared with controls (+Dox) and wild-type MLC₂₀s. (D) Quantitative comparisons of the focal adhesions in MEF/3T3 Tet-Off cells. ^#p < 0.02 compared with controls (+Dox) and wild-type MLC₂₀s. The values shown are means ± SD from three independent experiments (>300 cells; 100 cells/experiment). The focal adhesion area were defined as follows: paxillin foci: large focal adhesions, 3.22–16.05 μm² (6–30 pixels); and small focal adhesions, 0.535–2.675 μm² (1–5 pixels).

Model for the role of myosin II phosphorylation in a negative and positive manner. (A) The generation of force by the complex of actin and active form of myosin II stabilizes the actomyosin filaments and focal adhesions. (B) The effects of stimulation of the cells with PDGF. PDGF transiently activates PKCα and Rho GTPase family (RhoE, Rac, and Cdc42) resulting in the down-regulation of myosin II and RhoA activities, which contributes to the reorganization of actin cytoskeleton.

PDGF mediates the phosphorylation of MLC₂₀ at Ser1 in cells. (A) NIH3T3 cells were treated with PDGF (20 ng/ml) as described in Materials and Methods for the indicated times. Top and bottom panels show the confocal microscopic images of cells stained with pSer1 Ab (a–d), Myosin II Ab (e–h), and Alexa Fluor546-phalloidin (i–l). The focal plane is close to the bottom of the cell. Bar, 25 μm. (B) Immunoblot of PDGF-stimulated cell lysates with pSer1 Ab, pSer19 Ab, pTS Ab, and MLC₂₀ Ab. The whole cell lysates of PDGF-stimulated cells were subjected to SDS-PAGE followed by immunoblotting with pSer1 Ab, pSer19 Ab, pTS Ab, and MLC₂₀ Ab. Right, the amount of phosphorylated MLC₂₀ was determined by scanning densitometry (NIH image program). (C) Amount of phosphorylated MLC₂₀ at the inhibitory sites and the activation sites. NIH3T3 cells were treated with 20 ng/ml PDGF for 30 min (lane 2) and then subjected to alkali-urea/glycerol gel electrophoresis, followed by immunoblotting with anti-MLC₂₀ Ab (lane 1, control; untreated cells). Right, the fraction of phosphorylated MLC₂₀ was determined by scanning densitometry (NIH image program). The values shown are means ± SD from three independent experiments.

Inducible expression of myc-tagged wild-type or S1A/S2A MLC₂₀ in the stable transfectants. MEF/3T3 Tet-Off cells were cultured with (+) or without (−) doxycycline (Dox) to suppress or induce the expression of myc-tagged wild-type or S1A/S2A MLC₂₀. Myosin II having endogenous and/or expressed MLC₂₀s in cell lysates were coprecipitated with F-actin (see Materials and Methods for details). After releasing myosin II from F-actin by ATP, the supernatants were subjected to immunoblot, and the signals were detected with Myosin II Ab, MLC₂₀ Ab, and myc Ab.

Deletion of C-terminal heavy-chain phosphorylation sites in the nonhelical tailpiece. (A) NIH3T3 cells were transfected with either YFP-tagged wild-type or ΔC-Myosin IIB. Twelve hours after transfection, the cells were serum-starved for 18 h and then the starved transfected cells were treated with 20 ng/ml PDGF for 30 min, followed by staining with Alexa Fluor546-phalloidin for visualizing F-actin. Bar, 25 μm. (B) The bar graphs show quantification of PDGF-induced disassembly of stress fibers in NIH3T3 cells transfected with YFP-wild-type Myosin IIB or ΔC-Myosin IIB. Transfected cells (n = 40–65) were analyzed in three independent experiments (wild type, n = 165; ΔC, n = 137; p > 0.98, t tests). The values shown are means ± SD from three independent experiments.