Heart-specific small subunit of myosin light chain phosphatase activates rho-associated kinase and regulates phosphorylation of myosin phosphatase target subunit 1

Abstract

Phosphorylation of myosin regulatory light chain (MLC) plays a regulatory role in muscle contraction, and the level of MLC phosphorylation is balanced by MLC kinase and MLC phosphatase (MLCP). MLCP consists of a catalytic subunit, a large subunit (MYPT1 or MYPT2), and a small subunit. MLCP activity is regulated by phosphorylation of MYPTs, whereas the role of small subunit in the regulation remains unknown. We previously characterized a human heart-specific small subunit (hHS-M(21)) that increased the sensitivity to Ca(2+) in muscle contraction. In this study, we investigated the role of hHS-M(21) in the regulation of MLCP phosphorylation. Two isoforms of hHS-M(21), hHS-M(21)A and hHS-M(21)B, preferentially bound the C-terminal one-third region of MYPT1 and MYPT2, respectively. Amino acid substitutions at a phosphorylation site of MYPT1, Ser-852, impaired the binding of MYPT1 and hHS-M(21). The hHS-M(21) increased the phosphorylation level of MYPT1 at Thr-696, which was attenuated by Rho-associated kinase (ROCK) inhibitors and small interfering RNAs for ROCK. In addition, hHS-M(21) bound ROCK and enhanced the ROCK activity. These findings suggest that hHS-M(21) is a heart-specific effector of ROCK and plays a regulatory role in the MYPT1 phosphorylation at Thr-696 by ROCK.

FIGURE 1.

Schematic representation of constructs used in M2H and pulldown assays. Full-length and deletion mutants for large and small regulatory subunits of MLCP are schematically indicated along with their covering residues. A, constructs for myosin phosphatase target subunits (MYPT1 and MYPT2). Diagonal hatched boxes in the N terminus of MYPTs represent ankyrin repeats. Shaded and open boxes in the C terminus of MYPTs indicate LZ motifs of MYPT1 and MYPT2, respectively. B, constructs for MLCP small subunits (hHS-M₂₁ and sm-M20). Open and dotted boxes in the C terminus of hHS-M₂₁ indicate LZ motifs in hHS-M₂₁A and hHS-M₂₁B, respectively. C terminus of MYPT2 has identical sequences to that of hHS-M₂₁A and sm-M20. N-terminal 34 residues of sm-M20 (vertical hatched box of sm-M20) are different from that of hHS-M₂₁.

FIGURE 2.

M2H assay in evaluating the binding of hHS-M₂₁/sm-M20 with MYPTs. Luciferase activities obtained in the M2H assay. A, binding pairs were deletion mutants of MYPT1 or MYPT2 with hHS-M₂₁A, hHS-M₂₁B, or sm-M20. pACT and pBIND indicate transfection with pACT or pBIND vectors, respectively, as controls (no VP16- and GAL4-tagged proteins). B, binding pairs were deletion mutants of hHS-M₂₁A or hHS-M₂₁B with MYPT1-P or MYPT2-P. Data for MYPT1-P with hHS-M₂₁A were arbitrarily defined as 1.00 AU. Data are represented as means ± S.E. (n = 3 for control or n = 4 for each case). ***, p < 0.001 versus controls.

FIGURE 3.

Pulldown assay for binding of hHS-M₂₁/sm-M20 with C terminus of MYPT1 or MYPT2. Binding of hHS-M₂₁A, hHS-M₂₁B, or sm-M20 with MYPT1 (A) or MYPT2 (B) was analyzed. VP16-tagged MYPT1-P or -MYPT2-P was pulled down (PD) with His₆-tagged hHS-M₂₁A, -hHS-M₂₁B, or -sm-M20 and detected by using anti-VP16 Ab. Expression levels of VP16-tagged MYPTs and His₆-tagged small subunits were evaluated by immunoblotting of whole-cell lysates. Densitometric data obtained in the pulldown assays are shown in lower panel. Bars indicate the amounts of pulldown products normalized to the amount of input VP16-MYPTs. Data for VP16-MYPT1-P with His₆-hHS-M₂₁A (A) or VP16-MYPT2-P with His₆-hHS-M₂₁B (B) were arbitrarily defined as 1.00 AU. Data are represented as means ± S.E. (n = 5 for each case). C, VP16-tagged MYPT1s pulled down with His₆ protein alone or VP16 protein alone pulled down with His₆-tagged small subunits were not detected.

FIGURE 4.

Binding of hHS-M₂₁ and MYPT1 in mimicking phosphorylation or dephosphorylation status at Ser-852/Thr-853. A, schematic structures of wild-type (WT) and mutant MYPT1 (mMYPT1) truncated the LZ motif from MYPT1-P. Nonphosphorylatable (Ala) and/or phosphorylation-mimicking (Asp) substitutions at Ser-852 and Thr-853 within mMYPT1 are indicated. B, binding of hHS-M₂₁A and MYPT1 with or without the substitutions at Ser-852/Thr-853. VP16-mMYPT1s pulled down (PD) with His₆-hHS-M₂₁A were detected by using anti-VP16 Ab. Expression levels of VP16-MYPTs and His₆-hHS-M₂₁A were evaluated by immunoblotting of whole-cell lysates. Densitometric data obtained in the pulldown assay are shown in the lower panel. Bars indicate the amounts of pulldown products normalized to the amounts of VP16-mMYPT1s. Data for VP16-mMYPT1-WT with His₆-hHS-M₂₁A were arbitrarily defined as 1.00 AU. Data are represented as means ± S.E. (n = 5 for each case). **, p < 0.01; ***, p < 0.001 versus WT.

FIGURE 5.

MYPT1 phosphorylation at Thr-696 and Thr-853 in the presence of hHS-M₂₁. A, amounts of MYPT1 phosphorylation level in COS-7 cells transfected with nontagged MYPT1 alone (2 μg) or in the combination of nontagged MYPT1 (2 μg) and Myc-tagged hHS-M₂₁ (2 μg) (referred to in Fig. 1B) were measured by using anti-phospho-MYPT1-Thr-696 (pThr696) or -Thr-853 (pThr853) Abs. Expression levels of MYPT1 and Myc-hHS-M₂₁ were evaluated by immunoblotting of whole-cell lysates. B, densitometric analysis of MYPT1-Thr(P)-696 blotting data in A. Bars indicate the Thr(P)-696 phosphorylation levels normalized to the amounts of total MYPT1. Data for MYPT1 without Myc-hHS-M₂₁ was arbitrarily defined as 1.00 AU. Data are represented as means ± S.E. (n = 5 for each case). ***, p < 0.001 versus MYPT1 without Myc-hHS-M₂₁. C, MYPT1 phosphorylation at Thr-696 in the cells transfected with nontagged MYPT1 (2 μg) in combination of Myc-tagged hHS-M₂₁A (0, 0.1, 0.5, 1, and 2 μg). D, densitometric analysis of MYPT1-Thr(P)-696 blotting data in C. Bars indicate the Thr-696 phosphorylation levels normalized to the amounts of total MYPT1. Data for MYPT1 without Myc-hHS-M₂₁ were arbitrarily defined as 1.00 AU. Data are represented as means ± S.E. (n = 5 for each case). *, p < 0.05; ***, p < 0.001 versus MYPT1 without Myc-hHS-M₂₁.

FIGURE 6.

Effect of ROCK-specific inhibitors on MYPT1 phosphorylation in the presence of hHS-M₂₁. A, COS-7 cells co-transfected with nontagged MYPT1 alone (left panel) or nontagged MYPT1 plus Myc-tagged hHS-M₂₁A (right panel) were treated with ROCK-specific inhibitor, Y-27632 (0, 1, 10, and 20 μm; upper panel) or fasudil (0, 1, 10, and 20 μm; lower panel). MYPT1 phosphorylation level was measured by using anti-phospho-MYPT1-Thr-696 (pThr696) or -Thr-853 (pThr853) Abs. Expression levels of MYPT1, Myc-hHS-M₂₁A, and endogenous ROCK were evaluated by immunoblotting of whole-cell lysates. B, densitometric analysis of MYPT1-Thr(P)-696 blotting data in A. Bars indicate the Thr-696 phosphorylation levels normalized to the amounts of total MYPT1. Data for without Y-27632 or fasudil were arbitrarily defined as 1.00 AU. Data are represented as means ± S.E. (n = 5 for each case). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus no treatment with Y-27632 or fasudil.

FIGURE 7.

Effect of ROCK silencing on MYPT1 phosphorylation in the presence of hHS-M₂₁. A, COS-7 cells co-transfected with nontagged MYPT1 and Myc-hHS-M₂₁A (right panel) were transfected with siRNA against endogenous ROCK. Immunoblotting showed specific silencing of endogenous ROCK by double-stranded RNA oligonucleotides in COS-7 cells co-transfected with nontagged MYPT1 and Myc-tagged hHS-M₂₁A. GAPDH is shown as a loading control. B, MYPT1 phosphorylation level was measured by using anti-phospho-MYPT1 Thr-696 (pThr696) Ab. Expression levels of MYPT1 and Myc-hHS-M₂₁A were evaluated by immunoblotting of whole-cell lysates (upper panel). Results of densitometric analysis for MYPT1-Thr(P)-696 blotting data are shown (lower panel). Bars indicate the Thr-696 phosphorylation levels normalized to the amounts of total MYPT1. Data for treatment with control siRNA were arbitrarily defined as 1.00 AU. Data are represented as means ± S.E. (n = 6 for each case). ***, p < 0.001 versus treatment with control siRNA.

FIGURE 8.

Binding of ROCK with hHS-M₂₁ and MYPT1 in co-IP assay. Binding of endogenous ROCK with Myc-tagged hHS-M₂₁ and FLAG-tagged MYPT1 was analyzed by co-IP assay by using anti-FLAG and -Myc Abs. Endogenous ROCK co-immunoprecipitated with Myc-hHS-M₂₁ and FLAG-MYPT1 was detected by using anti-ROCK Ab. Expression levels of FLAG-MYPT1, Myc-hHS-M₂₁A, and endogenous ROCK were evaluated by immunoblotting of whole-cell lysates.

FIGURE 9.

Effect of hHS-M₂₁ on phosphorylation of MYPT1 in the presence of ROCK. A, amounts of MYPT1 phosphorylation level in COS-7 cells co-transfected with nontagged MYPT1 alone (2 μg), nontagged MYPT1 (2 μg) plus nontagged ROCK2-act (2 μg), or nontagged MYPT1 (2 μg) plus nontagged ROCK2-act (2 μg) plus Myc-tagged hHS-M₂₁ (2 μg) was measured by using anti-phospho-MYPT1-Thr-696 (pThr696) or -Thr-853 (pThr853) Abs. Expression levels of MYPT1, ROCK2-act, and Myc-hHS-M₂₁ were evaluated by immunoblotting of whole-cell lysates. B, densitometric analysis of MYPT1-Thr(P)-696 blotting data in A. C, densitometric analysis of MYPT1-Thr(P)-853 blotting data in A. Bars in B and C indicate the Thr-696 and Thr-853 phosphorylation levels, respectively, normalized to the amounts of total MYPT1. Data for MYPT1 alone were arbitrarily defined as 1.00 AU. Data are represented as means ± S.E. (n = 8 for each case). *, p < 0.05; ***, p < 0.001 versus MYPT1 plus ROCK2-act.

FIGURE 10.

Endogenous ERM phosphorylation in the presence of hHS-M₂₁. Top panel, immunoblotting showed amounts of endogenous ERM phosphorylation level in COS-7 cells transfected with Myc-tagged hHS-M₂₁ (2 μg) or nontagged ROCK2-act (2 μg) by using anti-phospho-ezrin(Thr-567)/radixin(Thr-564)/moesin(Thr-558) pAb. The cells were treated with a ROCK-specific inhibitor, fasudil (20 μm), before the transfection, when it is needed. Expression levels of Myc-hHS-M₂₁A, nontagged ROCK2-act, and endogenous ERM were evaluated by immunoblotting of whole-cell lysates. GAPDH is shown as a loading control. Densitometric analysis of ERM blotting data is shown in the lower panel. Bars indicate the endogenous phospho-ERM levels normalized to the amounts of total ERM. Data for whole-cell lysates only without transfection were arbitrarily defined as 1.00 AU. Data are represented as means ± S.E. (n = 9 for each case). *, p < 0.05; **, p < 0.01 versus whole-cell lysates lysate without transfection.