MLCK-independent phosphorylation of MLC20 and its regulation by MAP kinase pathway in human bladder smooth muscle cells

Abstract

Myosins are a superfamily of actin-based molecular motor proteins, which hydrolyze ATP and generate various forms of eukaryotic motility and muscle contraction. Myosin light chain 20 (MLC20) is small ring around the neck region of heavy chain of myosins. Phosphorylation of MLC20 is thought to play a key role in regulation of smooth muscle contraction. Calcium- and calmodulin-dependent myosin light chain kinase (MLCK) is considered the primary regulator of MLC20 phosphorylation. However, several observations in smooth muscle contraction cannot be explained by the mode of phosphorylation. By performing a series of experiments in vitro and in vivo, we report here MLCK-independent MLC20 phosphorylation. Gene expression study reveals that expression of MLCK in smooth muscles is inconsistent with MLC20 phosphorylation at Ser19. None of inactivating calmodulin/MLCK, depriving of calcium and silencing MLCK expression by siRNA blocks effectively the phosphorylation of MLC20 at Ser19. In addition, by overexpressing active human MAP (mitogen-activated protein)-ERK kinase kinase-1 (MEKK1) and blocking its downstream messengers, we have demonstrated a new regulatory system of MLC phosphorylation via MEKK1, which downregulates Ser19 phosphorylation of MLC20 through its downstream molecules, p38, JNK, and ERK in human bladder smooth muscle cells.

Fig. 1. Inconsistence between expression of MLCK and phosphorylation of MLC20 in mouse smooth muscles

Western blotting shows inconsistency between MLCK level and phosphorylation of MLC20 in smooth muscles from one female and two male mice. *Large and small intestines; †MLCK; ‡phosphor-MLC20.

Fig. 2. None of MLCK, inhibitory peptides of calmodulin, or MLCK and EGTA affects MLC20 phosphorylation

(A) Immunoprecipitated MLCK from 100 μg tissue was added to tissue extract 1 containing low −p-MLC20 and MLCK (Line 2). The immunocomplex beads containing MLCK from 50 μg tissue was loaded onto line 4. Reaction was stopped after 1-h incubation at 30°C by loading buffer and reaction mixture was loaded onto SDS-PAGE gel. Antibodies against p-MLC20 and actin were used to detect membranes. Western blotting shows that immunoprecipitated MLCK did not trigger phosphorylation of MLC20 in tissue extract 1 containing low phosphor-MLC20 (p-MLC20) and low MLCK (Line 2). However, 4 μg extract 2 containing high p-MLC20 and no detectable MLCK phosphorylated MLC20 (line 3). (B) Permeabilized hBSM cells were treated with calmodulin and MLCK inhibitory peptides (1000 nM each) for 3 h. EGTA (20 mM) was incubated with cells for 2 h. Cells were harvested for Western blotting and mixture of antibodies for p-MLC20 and actin was used to detected membranes. No significant inhibition on MLC20 phosphorylation was observed. (C) CaM and MLCK inhibitors did not inhibit MLC20 phosphorylation when they were incubated with smooth muscle extracts. (D) Small amount of high p-MLC20 without detectable MLCK tissue catalyzed MLC20 phosphorylation in low p-MLC20 and MLCK tissue extract. *CaM or MLCK inhibitor.

Fig. 3. siRNA-mediated knockdown of MLCK does not affect MLC20 phosphorylation but adenovirus-mediated MEKK1 over-expression activates downstream messengers and thereby inhibits MLC20 phosphorylation

(A) siRNA against MLCK was introduced to hBSM cells by transfection. Cells were harvested for Western blotting. MLCK, actin and phosphorylated MLC20 were detected with antibodies. (B) Overexpression of HA-tagged MEKK1 in hBSM cells was detected by HA antibody. (C–E) Overexpressed MEKK1 activated its downstream molecules, JNK1/2, ERK1, and p38. (F) MEKK1 inhibited MLC20 phosphorylation in a dose-dependent manner in hBSM cells. *Plaque-forming units. Here, it stands for pfu per cell. † and ‡ phosphor-ERK1/2 and phosphor–p38.

Fig. 4. MAP kinase inhibitors counteract the inhibition of MEKK1 on MLC20 phosphorylation

(A) Three days after adenovirus infection (90 pfu/cell), MEKK1-adenovirus-infected cells were incubated with SB (30 μM), SP (30 μM), and ERK inhibitor (60 μM) for 40 min. Cells were harvested for Western blotting. Membranes were detected by antibodies against p-MLC20 and actin. (B) Permeabilized cells were treated with 1000 nM of calmodulin and MLCK inhibitors for 3 h and then exposed to MAP kinase inhibitors for 40 min. Cell lysates were analyzed by Western blotting. Neither calmodulin or MLCK inhibitors affected the inducement of MAP kinase inhibitors on MLC20 phosphorylation.SB, SB 203580, a p38 inhibitor; SP, SP600125, a JNK inhibitor; ERK in, ERK inhibitory peptide.

Fig. 5. MAP kinase inhibitors increase MLC20 phosphorylation in hBSM cells

(A) Cells were incubated with 30 μM of SB and SP, and 60 μM ERK inhibitor for 40 min. Same volume of DMSO was used as vehicle control. (B–D) MAP inhibitors increased phosphorylation of MLC20 in dose-dependent manner. *ERK inhibitor.

Fig. 6. Migration of cells expressing MEKK1

Two days after cells grown on 24-well plate were infected with MEKK- and GFP-containing adenovirus (30 pfu/cell), a scratch was made with a pipette tip in the middle of the well in a 24-well plate. Pictures were taken at 14 h after wounding. The figure shows that fluorescent cells, which express MEKK1 that inhibits MLC20 phosphorylation, were migrating across the wound.

Fig. 7. Effects of Rho A, PKC, and MAPK on BSM cell migration

Two days after infected by adenovirus (30 pfu/cell), a wound was made with a pipette tip in the middle of each well. The cells were kept in inhibitor-containing media for 24 h. Cell migration was photographed at 0, 6, and 20 h. Rho A, infected by Rho A-containing adenovirus (30 pfu/cell); Y27632, a specific inhibitor of Rho kinase (20 μM); G1918, PKC inhibitor (150 nM); PD, PD 98059, a MEK1 inhibitor (50 μM).

Fig. 8. Effects of PKC and Rho A on MLC20 phosphorylation

(A) Rho A-containing adenovirus was tested by Western blotting. Two days after infected with control and Rho A-containing adenoviruses (20 pfu/cell), cells were harvested for Western blotting. Rho A and actin were detected by corresponding antibodies separately. (B) G1918, a PKC inhibitor (150 nM) resulted in decrease of MLC20 phosphorylation in cultured BSM cells. (C) Rho kinase inhibitor Y27632 (20 μM) inhibited MLC20 phosphorylation at Ser19.