Identification of Human CPI-17, an Inhibitory Phosphoprotein for Myosin Phosphatase

Yamawaki, Koji; M, Ito; Machida, Hirofumi; Moriki, Nobuyuki; Okamoto, Ryuji; Isaka, Naoki; Shimpo, Hideto; Kohda, Atsushi; Okumura, Katsuhiko; Hartshorne, David J.; Nakano, Takeshi

doi:10.1006/bbrc.2001.5290

Cited by 33 publications

(22 citation statements)

References 23 publications

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“…In addition, our data demonstrated the crucial role of CS1β and MYPT1 subunits of MLCP in the barrier regulation when barrier protective (ATP and its hydrolysis product, adenosine) or barrier disruptive (thrombin) agents were applied. The result showing the involvement of MYPT1 in the barrier protection confirms the previously reported result that the inhibition of adenosine induced barrier enhancement by depletion of MYPT1 (Umapathy et al, 2010) MYPT1 is considered as a housekeeping gene because it is ubiquitously expressed in many cell types (Yamawaki et al, 2001), particularly in smooth muscle cells . Several isoforms are generated by alternative splicing in chicken as well as in rat (Dirksen et al, 2000); and the existence of human MYPT1 variants have also been tested in HeLa and HEK293 cell lines (Xia et al, 2005).…”

Section: Discussionsupporting

confidence: 90%

Molecular Characterization Of Myosin Phosphatase In Endothelium

Kim

Csortos

Verin

2010

B60. Regulation of the Endothelium: Barrier Function and Endothelial Cell Integrity

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The phosphorylation status of myosin light chain (MLC) is regulated by both MLC kinases and type 1 Ser/Thr phosphatase (PPase 1), MLC phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (CS1β) towards myosin depends on its associated regulatory subunit, namely myosin PPase targeting subunit 1 (MYPT1). Our previously published data strongly suggested the involvement of MLCP in endothelial cell (EC) barrier regulation. In this paper, our new data demonstrates that inhibition of MLCP by either CS1β or MYPT1 siRNA-based depletion results in significant attenuation of purine nucleotide (ATP and adenosine)-induced EC barrier enhancement. Consistent with the data, thrombin-induced EC F-actin stress fiber formation and permeability increase were attenuated by the ectopic expression of constitutively active (C/A) MYPT1. The data demonstrated for the first time direct involvement of MLCP in EC barrier enhancement/protection. Cloning of MYPT1 in human pulmonary artery EC (HPAEC) revealed the presence of two MYPT1 isoforms, long and variant 2 (V2) lacking 56 amino acids from 553 to 609 of human MYPT1 long, which were previously identified in HeLa and HEK 293 cells. Our data demonstrated that in Cos-7 cells ectopically-expressed EC MYPT1 isoforms coimmunoprecipitated with intact CS1β suggesting the importance of PPase 1 activity for the formation of functional complex of MYPT1/CS1β. Interestingly, MYPT1 V2 shows decreased binding affinity compared to MYPT1 long for radixin (novel MLCP substrate and a member of ERM family proteins). These results suggest functional difference between EC MYPT1 isoforms in the regulation of MLCP activity and cytoskeleton.

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Section: Discussionsupporting

confidence: 90%

Molecular Characterization Of Myosin Phosphatase In Endothelium

Kim

Csortos

Verin

2010

B60. Regulation of the Endothelium: Barrier Function and Endothelial Cell Integrity

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“…Although Rho kinase is capable of activating CPI-17 in vitro, albeit to a lesser extent when compared with PKC, its predominant effect in vivo appears to be via phosphorylation of the large, 110-130 kDa regulatory myosin phosphatase targeting subunit (MYPT1), and inhibition of MLCP activity [27]. In both vascular and visceral smooth muscle, CPI-17 expression varies widely and is most abundant in tonic smooth muscle, consistent with a role for CPI-17 in sustained contraction [28,29]. CPI-17 expression correlated with the ability of phorbol esters to increase contraction and MLC 20 phosphorylation at submaximal Ca 2+ concentrations ('Ca 2+ sensitization').…”

Section: Phosphorylation Of Sermentioning

confidence: 91%

Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway

Murthy

Zhou

Grider

et al. 2003

Biochemical Journal

137

250

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Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gβγ i3 with activation of phospholipase C-β3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Gα q/11 with activation of phospholipase C-β1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44 + − 5 %). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr 38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28 + − 3 %). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC 20 ) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKCdependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC 20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities, it could not be used to ascertain the contribution of MYPT1 to inhibition of MLCP activity. m2-dependent phosphorylation and inactivation of MLCK precluded its involvement in sustained MLC 20 phosphorylation and contraction.

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“…Ca 2ϩ sensitization responses with MYPT1 and CPI-17 phosphorylation were measured in tissues from MLCK SMϪ/Ϫ mice. With the gradient gel system used for these analyses, the different isoforms for MYPT1 and CPI-17 were resolved (33,34). KCl treatment of bladder smooth muscles from MLCK SMϩ/ϩ mice showed modest increases in MYPT1 and CPI-17 phosphorylation (Fig.…”

Section: Distinct Effects On Developed Contractile Force Rlc Phosphomentioning

confidence: 99%