scholarly journals Smooth-muscle caldesmon phosphatase is SMP-I, a type 2A protein phosphatase

1993 ◽  
Vol 293 (1) ◽  
pp. 35-41 ◽  
Author(s):  
M D Pato ◽  
C Sutherland ◽  
S J Winder ◽  
M P Walsh
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Gel Filtration ◽  
Cytosolic Fraction ◽  
Chicken Gizzard ◽  
Kinase C

Caldesmon phosphatase was identified in chicken gizzard smooth muscle by using as substrates caldesmon phosphorylated at different sites by protein kinase C, Ca2+/calmodulin-dependent protein kinase II and cdc2 kinase. Most (approximately 90%) of the phosphatase activity was recovered in the cytosolic fraction. Gel filtration after (NH4)2SO4 fractionation of the cytosolic fraction revealed a single major peak of phosphatase activity which coeluted with calponin phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203] and myosin LC20 phosphatase. Further purification of caldesmon phosphatase was achieved by sequential chromatography on columns of DEAE-Sephacel, omega-amino-octyl-agarose, aminopropyl-agarose and thiophosphorylated myosin LC20-Sepharose. A single peak of caldesmon phosphatase activity was detected at each step of the purification. The purified phosphatase was identified as SMP-I [Pato and Adelstein (1980) J. Biol. Chem. 255, 6535-6538] by subunit composition (three subunits, of 60, 55 and 38 kDa) and Western blotting using antibodies against the holoenzyme which recognize all three subunits and antibodies specific for the 38 kDa catalytic subunit. SMP-I is a type 2A protein phosphatase [Pato, Adelstein, Crouch, Safer, Ingebritsen and Cohen (1983) Eur. J. Biochem. 132, 283-287; Winder et al. (1992), cited above]. Consistent with the conclusion that SMP-I is the major caldesmon phosphatase of smooth muscle, purified SMP-I from turkey gizzard dephosphorylated all three phosphorylated forms of caldesmon, whereas SMP-II, -III and -IV were relatively ineffective. Kinetic analysis of dephosphorylation by chicken gizzard SMP-I of the three phosphorylated caldesmon species and calponin phosphorylated by protein kinase C indicates that calponin is a significantly better substrate of SMP-I than are any of the three phosphorylated forms of caldesmon. We therefore suggest that caldesmon phosphorylation in vivo can be maintained after kinase inactivation due to slow dephosphorylation by SMP-I, whereas calponin and myosin are rapidly dephosphorylated by SMP-I and SMP-III/SMP-IV respectively. This may have important functional consequences in terms of the contractile properties of smooth muscle.

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.

Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKCζ

1996 ◽  
Vol 74 (1) ◽  
pp. 51-65 ◽  
Author(s):  
Odile Clément-Chomienne ◽  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Specific Activity ◽  
Regulation Of Gene Expression ◽  
Smooth Muscle Contraction ◽  
Particulate Fraction ◽  
Partial Purification ◽  
Chicken Gizzard ◽  
Kinase C

The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: α, δ, ε, η, and ζ isoenzymes were detected. PKCα associated with the particulate fraction in the presence of Ca2+ and was extracted by divalent cation chelators. PKCδ required detergent treatment for extraction from the EDTA – EGTA-washed particulate fraction. PKCε, η, and ζ were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKCζ, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKCζ-immunoreactive protein (Mr 76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKCζ with peptide ε (a synthetic peptide based on the pseudosubstrate domain of PKCε) as substrate was 2.1 μmol Pi∙min−1∙(mg PKCζ)−1 and, with peptide ζ as substrate, was 1.2 μmol Pi min−1∙(mg PKCζ)−1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKCζ (specific activity, 0.1–0.3 μmol Pi∙min−1∙mg−1). Two proteins, calponin and caldesmon, which have been implicated in the regulation of smooth muscle contraction and are phosphorylated by cPKC (a mixture of α, β, and γ isoenzymes), were also poor substrates of PKCζ (specific activities, 0.04 and 0.02 μmol Pi∙min−1∙mg−1, respectively). Chicken gizzard PKCζ was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKCζ are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.Key words: protein kinase C, isoenzymes, smooth muscle.

Protein kinase C from chicken gizzard: characterization and detection of an inhibitor and endogenous substrates

1989 ◽  
Vol 67 (6) ◽  
pp. 260-270 ◽  
Author(s):  
Gwyneth DeVries ◽  
Elaine D. Fraser ◽  
Michael P. Walsh
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Chicken Gizzard ◽  
Substrate Protein ◽  
Kinase C ◽  
Endogenous Substrates

Protein kinase C was purified from the cytosolic fraction of chicken gizzard by Ca2+-dependent hydrophobic interaction chromatography, anion-exchange chromatography, and hydrophobic chromatography. The molecular weight was estimated as 61 500 by gel filtration and 80 000 by denaturing gel electrophoresis, indicating that the native enzyme is a monomer. Using the mixed micellar assay, with histone III-S as the substrate, protein kinase C required Ca2+, phospholipid, and diacylglycerol for activity, with half-maximal activation at ~5 × 10−7 M Ca2+ in the presence of L-α-phosphatidyl-L-serine and 1,2-diolein. No activation by Ca2+ was observed in the absence of diacylglycerol. Protein kinase C requires free Mg2+, in addition to the MgATP2− substrate, for activity. The Km for ATP was determined to be 20 μM. Activity was sensitive to ionic strength, with half-maximal inhibition at 70 mM NaCl. Using the liposomal assay, phosphorylation of platelet P47 protein and smooth muscle vinculin was more strongly dependent on Ca2+ and lipids than was histone phosphorylation. Partial digestion of protein kinase C with trypsin yielded a constitutively active fragment. A heat-stable inhibitor and three major endogenous protein substrates of protein kinase C were also detected in chicken gizzard smooth muscle.Key words: protein kinase C, gizzard, inhibitor, endogenous substrates.

Protein phosphatase activity against protein kinase C-phosphorylated substrates in human placenta

Placenta ◽  
1994 ◽  
Vol 15 (7) ◽  
pp. 721-732 ◽  
Author(s):  
K.M. Eyster ◽  
M.S. Waller ◽  
T.L. Miller ◽  
C.J. Miller ◽  
D.M. Olson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatase Activity ◽  
Human Placenta ◽  
Protein Phosphatase ◽  
Kinase C

scholarly journals α-Tocopherol specifically inactivates cellular protein kinase C α by changing its phosphorylation state

1998 ◽  
Vol 334 (1) ◽  
pp. 243-249 ◽  
Author(s):  
Roberta RICCIARELLI ◽  
Andrea TASINATO ◽  
Sophie CLÉMENT ◽  
Nesrin K. ÖZER ◽  
Daniel BOSCOBOINIK ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Protein Phosphatase ◽  
Antioxidant Properties ◽  
Muscle Cells ◽  
Cellular Protein ◽  
Kinase C

The mechanism of protein kinase C (PKC) regulation by α-tocopherol has been investigated in smooth-muscle cells. Treatment of rat aortic A7r5 smooth-muscle cells with α-tocopherol resulted in a time- and dose-dependent inhibition of PKC. The inhibition was not related to a direct interaction of α-tocopherol with the enzyme nor with a diminution of its expression. Western analysis demonstrated the presence of PKCα, β, δ, ε, ζ and µ isoforms in these cells. Autophosphorylation and kinase activities of the different isoforms have shown that only PKCα was inhibited by α-tocopherol. The inhibitory effects were not mimicked by β-tocopherol, an analogue of α-tocopherol with similar antioxidant properties. The inhibition of PKCα by α-tocopherol has been found to be associated with its dephosphorylation. Moreover the finding of an activation of protein phosphatase type 2A in vitro by α-tocopherol suggests that this enzyme might be responsible for the observed dephosphorylation and subsequent deactivation of PKCα. It is therefore proposed that PKCα inhibition by α-tocopherol is linked to the activation of a protein phosphatase, which in turn dephosphorylates PKCα and inhibits its activity.

Chronic treatment by the calcium channel blocker ± PN 200-110 in the rat counteracts the stimulations of pituitary weight, prolactin release and pituitary C-kinase activity induced by a chronic estradiol treatment, in vivo

1990 ◽  
Vol 122 (3) ◽  
pp. 403-408
Author(s):  
Ph. Touraine ◽  
P. Birman ◽  
F. Bai-Grenier ◽  
C. Dubray ◽  
F. Peillon ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Channel ◽  
Calcium Channel Blocker ◽  
Channel Blocker ◽  
Plasma Prolactin ◽  
Estradiol Treatment ◽  
Kinase C ◽  
Protein Kinase C Activity

Abstract In order to investigate whether a calcium channel blocker could modulate the protein kinase C activity in normal and estradiol pretreated rat pituitary, female Wistar rats were treated or not (controls) with ± PN 200-110 (3 mg · kg−1 · day−1, sc) for 8 days or with estradiol cervical implants for 8 or 15 days, alone or in combination with PN 200-110 the last 8 days. Estradiol treatment induced a significant increase in plasma prolactin levels and pituitary weight. PN 200-110 administered to normal rats did not modify these parameters, whereas it reduced the effects of the 15 days estradiol treatment on prolactin levels (53.1 ± 4.9 vs 95.0 ±9.1 μg/l, p<0.0001) and pituitary weight (19.9 ± 0.4 vs 23.0 ± 0.6 mg, p <0.001), to values statistically comparable to those measured after 8 days of estradiol treatment. PN 200-110 alone did not induce any change in protein kinase C activity as compared with controls. In contrast, PN 200-110 treatment significantly counteracted the large increase in soluble activity and the decrease in the particulate one induced by estradiol between day 8 and day 15. We conclude that PN 200-110 opposed the stimulatory effects of chronic in vivo estradiol treatment on plasma prolactin levels and pituitary weight and that this regulation was related to a concomitant modulation of the protein kinase C activity.

Regulation of Scavenger Receptor Expression in Smooth Muscle Cells by Protein Kinase C

1997 ◽  
Vol 17 (5) ◽  
pp. 969-978 ◽  
Author(s):  
Michele Mietus-Snyder ◽  
Annabelle Friera ◽  
Christopher K. Glass ◽  
Robert E. Pitas
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Scavenger Receptor ◽  
Muscle Cells ◽  
Receptor Expression ◽  
Kinase C
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