Expression, purification, and characterization of coiled coil and leucine zipper domains of C-terminal myosin binding subunit of myosin phosphatase for solution NMR studies

Abstract Vascular smooth muscle contractile state is tightly coupled to myosin light chain phosphorylation. Myosin light chain phosphorylation is regulated by the counter regulatory activities of myosin light chain kinase and myosin phosphatase. Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase. This in turn functions to dephosphorylate myosin light chains. Mechanistically this involves a protein-protein interaction mediated by a leucine zipper (LZ) domain that is localized within the N-terminus of cGK1α and the C-terminus of myosin-binding subunit (MBS). To better understand the importance of this interaction in regulating smooth muscle vascular tone we have determined the three-dimensional structure of the LZ domain of cGK1α, (cGKIα1–59) using triple resonance, multidimensional nuclear magnetic resonance (NMR) spectroscopy. We have expressed the LZ domain (cGKIα1–59) in E. coli under conditions facilitating the uniform incorporation of 13C/15N and 2H isotopic labels and purified this expressed protein. Circular dichroism data supported that cGK1α is comprised of an α-helix, while our NMR data identified that this helix commences at residue Ala9 and extends through to residue Leu47 of the LZ domain. 15N backbone longitudinal and transverse relaxation rates are best fit to a rotational diffusion anisotropy model that is consistent with a coiled-coil dimmer structure, not a monomeric helix. This suggests that cGKIα1–59 is comprised of a coiled-coil conformation over this same region. A time course investigation carried out with the LZ domain and a homobifunctional amine reactive crosslinker [dithio-bis(succinimidyl propionate)] (DSP) supported the presence of a homodimer. Furthermore, 15N heteronuclear single quantum correlation (HSQC) chemical shift mapping data has been used to identify residues that are critical for interacting with the C-terminal region of the myosin binding subunit (MBS). These data have allowed us to identify those residues necessary for cGK1α dimerization, while simultaneously permitting a better understanding of the critical determinants of cGK1α and MBS that are involved in regulating the contractile state of smooth muscles within blood vessels. The spatial and temporal contractile state of these blood vessels is important in the physiologic control of blood vessel tone and plays a role in the pathogenesis of hypertension and atherosclerosis a leading cause of death in the US and worldwide.

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Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43828-8 ◽

1994 ◽

Vol 269 (48) ◽

pp. 30407-30411

Author(s):

H Shimizu ◽

M Ito ◽

M Miyahara ◽

K Ichikawa ◽

S Okubo ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Myosin ◽

Myosin Phosphatase ◽

Myosin Binding ◽

Muscle Myosin ◽

Binding Subunit

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Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies

Protein Expression and Purification ◽

10.1016/s1046-5928(03)00127-x ◽

2003 ◽

Vol 30 (2) ◽

pp. 253-261 ◽

Cited By ~ 69

Author(s):

Bethany Buck ◽

Jamillah Zamoon ◽

Tara L Kirby ◽

Tara M DeSilva ◽

Christine Karim ◽

...

Keyword(s):

Solid State ◽

High Resolution ◽

Solid State Nmr ◽

Purification And Characterization ◽

Nmr Studies

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Association of the Myosin-binding Subunit of Myosin Phosphatase and Moesin: Dual Regulation of Moesin Phosphorylation by Rho-associated Kinase and Myosin Phosphatase

The Journal of Cell Biology ◽

10.1083/jcb.141.2.409 ◽

1998 ◽

Vol 141 (2) ◽

pp. 409-418 ◽

Cited By ~ 163

Author(s):

Yuko Fukata ◽

Kazushi Kimura ◽

Noriko Oshiro ◽

Hideyuki Saya ◽

Yoshiharu Matsuura ◽

...

Keyword(s):

Plasma Membrane ◽

Phosphatase Activity ◽

Mdck Cells ◽

Rho Kinase ◽

Small Gtpase ◽

Myosin Phosphatase ◽

Membrane Ruffling ◽

Amino Terminal ◽

Myosin Binding ◽

Binding Subunit

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho- kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. We found that in MDCK epithelial cells, MBS accumulated at the tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling area, where moesin, a member of the ERM (ezrin, radixin, and moesin) family, was localized. Neither membrane ruffling nor an accumulation of moesin and MBS at the free-end plasma membrane was induced when MDCK cells were stimulated with TPA after the microinjection of C3, which ADP-ribosylates and inactivates Rho. MBS was colocalized with moesin at the cell–cell contact sites in MDCK cells. We also found that moesin was coimmunoprecipitated with MBS from MDCK cells. Recombinant MBS interacted with the amino-terminal domains of moesin and ezrin. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward moesin, which was phosphorylated by Rho-kinase. The phosphatase activity was inhibited when MBS was phosphorylated by Rho-kinase. These results suggest that MBS is recruited with moesin to the plasma membrane and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of moesin downstream of Rho.

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