scholarly journals The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts

2020 ◽  
Vol 21 (16) ◽  
pp. 5709
Author(s):  
Irene Lang ◽  
Guneet Virk ◽  
Dale C. Zheng ◽  
Jason Young ◽  
Michael J. Nguyen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Phosphatase ◽  
Smooth Muscle Contraction ◽  
Cellular Systems ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Myosin Phosphatase ◽  
Phosphatase Inhibitors ◽  
Regulatory Myosin Light Chain

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

Abstract 70: Protein Phosphatase 1 Contributes to Atrial Stunning in Atrial Fibrillation

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Srikanth Perike ◽  
Xander Wehrens ◽  
Dawood Darbar ◽  
Mark McCauley
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Stroke Risk ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Future Studies ◽  
Hek Cells

Background: Atrial fibrillation (AF) is the most common cardiac arrhythmia, and increases a patient’s stroke risk five-fold. Reduced atrial contractility (stunning) is observed in AF and contributes to stroke risk; however, the mechanisms responsible for atrial stunning in AF are unknown. Recent data from our laboratory indicate that protein phosphatase 1 (PP1) dephosphorylation of myosin light chain 2a (MLC2a) may contribute to atrial stunning in AF. Objective: To determine how the PP1 regulatory subunit 12C (PPP1R12C) and catalytic (PPP1c) subunits modify atrial sarcomere phosphorylation in AF. Methods: We evaluated the protein expression, binding and phosphorylation among PPP1R12C, PPP1c, and MLC2a in transfected HL-1 cells, murine atrial tissue (Pitx2null +/– mice, with a genetic predisposition AF), and in HEK cells. An inhibitor of PPP1R12C phosphorylation, BDP5290, was used to enhance the PPP1R12C-PPP1C interaction. Results: In Pitx2 null +/– mice, PPP1R12C was increased by 2-fold ( P <0.01) and associated with a 40% reduction in S-19-MLC2a phosphorylation versus WT mice ( P <0.058). BDP5290 increased PPP1R12C-PPP1C binding by >3-fold in HL-1 cells ( P <0.01). BDP5290 reduced MLC2a phosphorylation by 40% through an enhanced interaction with PPP1R12C by >3-fold in HEK cells ( P <0.01). Conclusion: In Pitx2 null+/- mice, increased expression of PPP1R12C is associated with PP1 holoenzyme targeting to sarcomeric MLC2a, and is associated with reduced S19-MLC2a phosphorylation. Additionally, BDP5290 enhances the PPP1R12C-PPP1C interaction and models PP1 activity in AF. Future studies will examine the effects of both AF and BDP5290 upon atrial contractility in vitro.

scholarly journals Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors

1999 ◽  
Vol 112 (2) ◽  
pp. 157-168 ◽  
Author(s):  
L. Trinkle-Mulcahy ◽  
P. Ajuh ◽  
A. Prescott ◽  
F. Claverie-Martin ◽  
S. Cohen ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mrna Splicing ◽  
Dominant Negative ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Dominant Negative Mutant ◽  
Splicing Factors ◽  
Nuclear Extracts ◽  
Nuclear Organisation

Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that ‘nuclear inhibitor of PP1’ (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a ‘dominant negative’ mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.

scholarly journals Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function

1992 ◽  
Vol 286 (1) ◽  
pp. 197-203 ◽  
Author(s):  
S J Winder ◽  
M D Pato ◽  
M P Walsh
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Smooth Muscle Contraction ◽  
Inhibitory Effect ◽  
Chicken Gizzard ◽  
Kinase C

Calponin, a thin-filament protein of smooth muscle, has been implicated in the regulation of smooth-muscle contraction, since in vitro the isolated protein inhibits the actin-activated myosin MgATPase. This inhibitory effect, and the ability of calponin to bind to actin, is lost after its phosphorylation by protein kinase C or Ca2+/calmodulin-dependent protein kinase II [Winder & Walsh (1990) J. Biol. Chem. 265, 10148-10155]. If this phosphorylation reaction is of physiological significance, there must be a protein phosphatase in smooth muscle capable of dephosphorylating calponin and restoring its inhibitory effect on the actomyosin MgATPase. We demonstrate here the presence, in chicken gizzard smooth muscle, of a single major phosphatase activity directed towards calponin. This phosphatase was purified from the soluble fraction of chicken gizzard by (NH4)2SO4 fractionation and sequential chromatography on Sephacryl S-300, DEAE-Sephacel, omega-amino-octyl-agarose and thiophosphorylated myosin 20 kDa light-chain-Sepharose columns. The purified phosphatase contained three polypeptide chains of 60, 55 and 38 kDa which were shown to be identical with the subunits of SMP-I, a smooth-muscle phosphatase capable of dephosphorylating the isolated 20 kDa light chain of myosin but not intact myosin [Pato & Adelstein (1983) J. Biol. Chem. 258, 7047-7054]. Consistent with its identity with SMP-I, calponin phosphatase was classified as a type-2A protein phosphatase. Of several potential phosphoprotein substrates examined, calponin proved to be kinetically the best, suggesting that calponin may be a physiological substrate for this phosphatase. Finally, dephosphorylation of calponin which had been phosphorylated by protein kinase C restored completely its ability to inhibit the actin-activated MgATPase of smooth-muscle myosin. These observations support the hypothesis that calponin plays a role in regulating the contractile state of smooth muscle and that this function in turn is controlled by phosphorylation-dephosphorylation.

NIMA-related kinase 2 (Nek2), a cell-cycle-regulated protein kinase localized to centrosomes, is complexed to protein phosphatase 1

2000 ◽  
Vol 349 (2) ◽  
pp. 509-518 ◽  
Author(s):  
Nicholas R. HELPS ◽  
Xinmei LUO ◽  
Hazel M. BARKER ◽  
Patricia T. W. COHEN
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Protein Phosphatase ◽  
Cell Cycle Progression ◽  
Protein Phosphatase 1 ◽  
Binding Motif ◽  
Phosphatase Inhibitors ◽  
Cell Extracts ◽  
Co Precipitation

The cell cycle-regulated protein serine/threonine NIMA-related kinase 2 (Nek2), which shows a predominant localization at centrosomes, is identified as a protein which interacts with protein phosphatase 1 (PP1) using the yeast two-hybrid system. Complex formation between Nek2 and PP1 is supported by co-precipitation of the two proteins using transfected expression constructs of Nek2 and the endogenous Nek2/PP1 proteins. The sequence KVHF in the C-terminal region of Nek2, which conforms to the consensus PP1-binding motif, is shown to be essential for the interaction of Nek2 with PP1. Nek2 activity increases with autophosphorylation and addition of phosphatase inhibitors and decreases in the presence of PP1. PP1 is a substrate for Nek2 and phosphorylation of PP1γ1 on two C-terminal sites reduces its phosphatase activity. The presence of a ternary complex containing centrosomal Nek2-associated protein (C-Nap1), Nek2 and PP1 has also been demonstrated, and C-Nap1 is shown to be a substrate for both Nek2 and PP1 in vitro and in cell extracts. The implications of kinase-phosphatase complex formation involving Nek2 and PP1 are discussed in terms of the coordination of centrosome separation with cell cycle progression.

scholarly journals Role of p47phox in regulating Cdc42GAP, vimentin, and contraction in smooth muscle cells

2009 ◽  
Vol 297 (6) ◽  
pp. C1424-C1433 ◽  
Author(s):  
Qing-Fen Li ◽  
Dale D. Tang
Keyword(s):  
Hydrogen Peroxide ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Contraction ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Regulatory Subunit ◽  
Ros Generation ◽  
Vitro Assay

Cdc42GAP (GTPase activating protein) has been shown to regulate smooth muscle contraction as well as cell motility, adhesion, proliferation, and apoptosis. We have recently shown that Cdc42GAP activity is suppressed in smooth muscle cells during contractile activation, which is reversed by inhibitors of reactive oxygen species (ROS). Because p47phox, a regulatory subunit of NAD(P)H oxidase, has been implicated in smooth muscle signaling, we determined whether this subunit modulates Cdc42GAP activity in response to contractile stimulation. Transfection of smooth muscle cells with plasmids encoding short hairpin RNA (shRNA) against p47phox, but not plasmids for luciferase shRNA, inhibited the expression of p47phox. ROS production and the suppression of Cdc42GAP activity in response to stimulation with 5-hydroxytryptamine (5-HT) were attenuated in cells producing p47phox shRNA compared with cells producing luciferase shRNA. In contrast, the addition of hydrogen peroxide to p47phox-deficient cells suppressed the activity of Cdc42GAP. Furthermore, exposure to hydrogen peroxide led to a decrease in Cdc42GAP activity in an in vitro assay. Cdc42 activation, p21-activated kinase 1 (PAK1) phosphorylation at Thr-423 (an indication of PAK activation), and vimentin phosphorylation at Ser-56 in response to 5-HT activation were also attenuated in smooth muscle cells producing shRNA against p47phox. The knockdown of p47phox inhibited smooth muscle contraction during stimulation with 5-HT but not hydrogen peroxide. These results suggest that the p47phox subunit of NAD(P)H oxidase may mediate the agonist-induced GAP suppression by controlling ROS generation in smooth muscle cells during agonist stimulation. p47phox-regulated GAP affects smooth muscle contraction likely through the Cdc42/PAK1/vimentin pathway.

Kinase-related protein/telokin inhibits Ca2+-independent contraction in Triton-skinned guinea pig taenia coli

2010 ◽  
Vol 429 (2) ◽  
pp. 291-302 ◽  
Author(s):  
Olga V. Shcherbakova ◽  
Daria V. Serebryanaya ◽  
Alexander B. Postnikov ◽  
Mechthild M. Schroeter ◽  
Stefan Zittrich ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phosphorylation Site ◽  
Smooth Muscle Contraction ◽  
Taenia Coli ◽  
Related Protein ◽  
Smooth Muscle Contractility ◽  
Regulatory Myosin Light Chain

KRP (kinase-related protein), also known as telokin, has been proposed to inhibit smooth muscle contractility by inhibiting the phosphorylation of the rMLC (regulatory myosin light chain) by the Ca2+-activated MLCK (myosin light chain kinase). Using the phosphatase inhibitor microcystin, we show in the present study that KRP also inhibits Ca2+-independent rMLC phosphorylation and smooth muscle contraction mediated by novel Ca2+-independent rMLC kinases. Incubating KRP-depleted Triton-skinned taenia coli with microcystin at pCa>8 induced a slow contraction reaching 90% of maximal force (Fmax) at pCa 4.5 after ~25 min. Loading the fibres with KRP significantly slowed down the force development, i.e. the time to reach 50% of Fmax was increased from 8 min to 35 min. KRP similarly inhibited rMLC phosphorylation of HMM (heavy meromyosin) in vitro by MLCK or by the constitutively active MLCK fragment (61K-MLCK) lacking the myosin-docking KRP domain. A C-terminally truncated KRP defective in myosin binding inhibited neither force nor HMM phosphorylation. Phosphorylated KRP inhibited the rMLC phosphorylation of HMM in vitro and Ca2+-insensitive contractions in fibres similar to unphosphorylated KRP, whereby the phosphorylation state of KRP was not altered in the fibres. We conclude that (i) KRP inhibits not only MLCK-induced contractions, but also those elicited by Ca2+-independent rMLC kinases; (ii) phosphorylation of KRP does not modulate this effect; (iii) binding of KRP to myosin is essential for this inhibition; and (iv) KRP inhibition of rMLC phosphorylation is most probably due to the shielding of the phosphorylation site on the rMLC.

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