Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts

Abstract

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions. We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.

Figure 1

M130Ab inhibits myosin phosphatase activity in vitro. (a) Specificity of M130Ab. Immunoblot showing that M130Ab raised against the NH₂-terminal region (1–296) of chick MBS specifically recognizes 130-kD MBS in a variety of nonmuscle cells. Total cell lysates were separated by SDS-PAGE followed by immunoblotting using M130Ab. Lane 1, REF 2A cells; lane 2, Balb/c 3T3 cells; lane 3, NRK cells. (b) Inhibition of myosin phosphatase activity by M130Ab. Myosin phosphatase was first incubated with M130Ab, then myosin phosphatase activity was determined using ³²P-labeled MLC as a substrate. The values shown are means ± SEM from three independent experiments.

Figure 2

Microinjection of M130Ab induces stress fiber formation and increases MLC phosphorylation in serum-starved 3T3 cells. M130Ab (5 mg/ml) was microinjected into serum-starved 3T3 cells. FITC-dextran was coinjected to identify injected cells (b, d, and f). 2 h after injection, cells were fixed and stained with rhodamine-phalloidin (a–d), or anti–S19-phosphorylated MLC antibody (e and f). Asterisks indicated uninjected cells. Bars, 10 μm.

Figure 3

Microinjection of M130Ab induces focal adhesion formation in serum-starved 3T3 cells. a–f: M130Ab was microinjected into serum-starved 3T3 cells as in Fig. 2. Cells were fixed and stained with anti-vinculin antibody (a and b), anti-paxillin antibody (c and d), or anti-FAK antibody (e and f). FITC-dextran was coinjected to identify injected cells (b, d, and f). (g–j) M130Ab-injected cells were double stained with rhodamine-phalloidin (h, red) and anti-vinculin antibody (i, green). Injected cells were detected by anti-rabbit IgG secondary antibody (g). A merged image of rhodamine-phalloidin staining (red) and vinculin localization (green) is shown in j. Asterisks indicated uninjected cells. Bars, 10 μm.

Figure 4

MBS₂₉₆ inhibits MLC phosphorylation, stress fiber assembly, and focal adhesion formation of serum-stimulated 3T3 cells. MBS₂₉₆ (1 mg/ml) was microinjected into serum-starved 3T3 cells 30 min before serum stimulation. Because subconfluent serum-starved cells were difficult to examine for focal adhesion assembly due to their overlaps, individually separated cells were prepared by replating as described in Materials and Methods. Cells were stimulated with serum for 10 min, then fixed and stained with rhodamine-phalloidin (a and b), anti–S19-phophorylated MLC antibody (c and d), or anti–vinculin antibody (e and f). Note that stress fibers of the uninjected cell at the top of panel a are not obvious because of out of focus. As a control, MBS_278–415 was microinjected into serum-starved 3T3 cells, and cells were stimulated with serum in the same way. The injection of MBS_278–415 showed no effect when examined by rhodamine-phalloidin staining (g and h) or by anti–S19-phophorylated MLC antibody (i and j). It is noticed that MBS₂₉₆-injected cells show prominent vesicles distributed circumferentially around the cells. The identity of these vesicles is currently unknown. Injected cells were detected by coinjection of FITC-dextran (b, d, f, h, and j). Asterisks indicate uninjected cells. Bars, 10 μm.

Figure 5

Effects of ROCK or MLCK inhibitors on M130Ab-induced stress fiber and focal adhesion assembly. Serum-starved 3T3 cells were first treated for 30 min with 10 μM Y-27632 alone (a–f), 10 μM ML-9 alone (g and h) or both 10 μM Y-27632 and 10 μM ML-9 (i and j). M130Ab (5 mg/ml) was then microinjected together with FITC-dextran. 2 h after injection, cells were fixed and stained with rhodamine-phalloidin (a, b, and g–j), anti S19-phosphorylated MLC antibody (c and d), or anti-vinculin antibody (e and f). In k–n, 3T3 cells grown with 10% serum were pretreated with 10 μM Y-27632 (k and l) or 10 μM ML-9 (m and n) for 30 min, and microinjected with M130Ab. 2 h after injection, cells were fixed and stained with rhodamine-phalloidin. Injected cells were detected by coinjected FITC-dextran (b, d, f, h, j, l and n). Uninjected cells are indicated by asterisks. Bars, 10 μm.

Figure 6

Localization of MLCK in cortical actin bundles induced by M130Ab injection in the presence of Y-27632. Serum-starved 3T3 cells (a–c) or 3T3 cells grown with 10% serum (d–f) were first treated for 30 min with 10 μM Y-27632, and M130Ab (5 mg/ml) was then microinjected. 2 h after injection, cells were fixed and double stained with rhodamine-phalloidin (b and e) or anti-MLCK antibody (c and f). Injected cells were detected by anti-rabbit IgG secondary antibody (a and d). Arrowheads indicate cortical actin bundles. Asterisks indicate uninjected cells. Bars, 10 μm.

Figure 7

Model of spatially differentiated regulation of MLC phosphorylation by ROCK and MLCK. (a) in the center of cells; (b) at the cell periphery. See the text for detail.