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.
Highlights
Phosphorylation of myosin regulatory light chain (MLC)2 plays pivotal roles in activation of actomyosin, regulation of cell shape, cell motility, and cytokinesis in eukaryotic cells (1– 4)
Expression analysis of smwere as follows: mouse anti-c-Myc monoclonal antibody (mAb) (1:100), rabbit anti- M20 in various human tissues by RT-PCR showed a high level phospho-MYPT1 threonine 696 (Thr-696) polyclonal antibody (pAb) (1:500), rabbit anti-MYPT1 expression in the tissues abundant with smooth muscle, includpAb (1:100), rabbit anti-ROK␣/ROCK2 pAb (1:500), and ing uterus, small intestine, and colon, whereas a weak expresmouse anti-GAPDH mAb (1:100, Santa Cruz Biotechnology). sion and no expression were observed in skeletal muscle and
When the luciferase activity in the transfectants with hHS-M21A plus MYPT1-P was arbitrarily defined as 1.00 Arbitrary units (AU), that of hHS-M21A plus MYPT2-P showed a significant binding (0.40 Ϯ 0.01 AU, p Ͻ 0.001, as compared with negative controls containing either myosin phosphatase target subunit (MYPT) or action between the C-terminal one-third of MYPT1 (MYPT1-P) and MLC phosphatase (MLCP) small subunits, Western blotting (WB) analysis of pulldown products from the mixture of cell lysates prepared from transfectants of VP16MYPT1-P in combination with His6-hHS-M21A, -hHS-M21B, or -sm-M20 revealed that the C-terminal one-third of MYPT1
Summary
Mammalian Two-hybrid (M2H) Assay—The cDNA fragments for MYPT1 corresponding to amino acids (aa) 1–344 (N-terminal one-third of MYPT1; MYPT1-A), aa 345– (middle one-third of MYPT1; MYPT1-M), and aa –1030 (C-terminal one-third of MYPT1; MYPT1-P) and for MYPT2 corresponding to aa 1–328 (N-terminal one-third of MYPT2; MYPT2-A), aa 329 – 656 (middle one-third of MYPT2; MYPT2-M), and aa 657–982 (C-terminal one-third of MYPT2; MYPT2-P) were obtained by PCR from total heart cDNA. Each mMYPT1 cDNA fragment was cloned into ogy), rabbit anti-phospho-MYPT1-Thr-696 pAb (1:500), rabbit pACT to obtain VP16-tagged protein in transfection experi- anti-phospho-MYPT1-Thr-853 pAb (1:350, Millipore), rabbit ments. COS-7 cells (1 ϫ 106) were seeded onto 100-mm dishes anti-MYPT1 pAb (1:100, Santa Cruz Biotechnology), rabbit antiand co-transfected with a combination of pcDNA4/HisMax- ROK␣/ROCK2 pAb (1:500, Millipore), rabbit anti-ezrin/radixin/. The cDNA frag- Hygro full-length MYPT1 (2 g) plus pcDNA3.1/Zeo-ROCK2-act ments of full-length MYPT1 (major isoform in human heart; (2 g), or pcDNA3.1/Hygro full-length MYPT1 (2 g) plus spliced out of exon 13) and constitutively active form of ROCK2 pCMV-Tag3-hHS-M21 (2 g) were transfected using COSFectin (ROCK2-act, aa 1–557) were amplified by RT-PCR from human lipid reagent. COS-7 cells (1 ϫ 106) were seeded onto 100-mm dishes, and either pCMV-Tag3-hHS-M21 (4 g) or pCMV-Tag2-full-length MYPT1 (4 g) alone or both were transfected using COSFectin lipid reagent. Statistical analyses were performed using the R statistical computing environment version 2.6.1. p values less than 0.05 were considered to be statistically significant
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