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Regulation of PKC autophosphorylation by calponin in contractile vascular smooth muscle tissue.

Kim HR, Gallant C, Morgan KG - Biomed Res Int (2013)

Bottom Line: We found that calponin increases the level of in vitro PKCα phosphorylation at the PDK and hydrophobic sites, but not the turn motif site.Calponin knockdown inhibits autophosphorylation of cellular PKC in response to phenylephrine, confirming results with recombinant PKC.Thus these results show that autophosphorylation of PKC is regulated in dVSM and calponin is necessary for autophosphorylation of PKC in VSM.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Sciences, Boston University, 635 Commonwealth Avenue, Boston, MA 02215, USA ; Department of Pharmacology, College of Medicine, Dankook University, 119 Dandaero, Chungnam, Cheonan-si 330-714, Republic of Korea.

ABSTRACT
Protein kinase C (PKC) is a key enzyme involved in agonist-induced smooth muscle contraction. In some cases, regulatory phosphorylation of PKC is required for full activation of the enzyme. However, this issue has largely been ignored with respect to PKC-dependent regulation of contractile vascular smooth muscle (VSM) contractility. The first event in PKC regulation is a transphosphorylation by PDK at a conserved threonine in the activation loop of PKC, followed by the subsequent autophosphorylation at the turn motif and hydrophobic motif sites. In the present study, we determined whether phosphorylation of PKC is a regulated process in VSM and also investigated a potential role of calponin in the regulation of PKC. We found that calponin increases the level of in vitro PKCα phosphorylation at the PDK and hydrophobic sites, but not the turn motif site. In vascular tissues, phosphorylation of the PKC hydrophobic site, but not turn motif site, as well as phosphorylation of PDK at S241 increased in response to phenylephrine. Calponin knockdown inhibits autophosphorylation of cellular PKC in response to phenylephrine, confirming results with recombinant PKC. Thus these results show that autophosphorylation of PKC is regulated in dVSM and calponin is necessary for autophosphorylation of PKC in VSM.

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Calponin increases autophosphorylation on the PDK and hydrophobic sites but not the turn motif site of recombinant PKC. (a) Mean densitometric results of 6 experiments using a site-specific phosphoantibody to the PDK site (T497) as described in methods. (b) Mean densitometric results of 6 experiments, phosphorylation detected with a site-specific phosphor-antibody to the hydrophobic site (S657). (c) Mean densitometric results of 6 experiments, phosphorylation detected with a site-specific antibody for the turn motif site (T638). No ATP and BSA lanes represent negative controls. Protamine is a positive control. Data are mean ± SE. *P < 0.05 and **P < 0.01 versus No ATP.
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fig1: Calponin increases autophosphorylation on the PDK and hydrophobic sites but not the turn motif site of recombinant PKC. (a) Mean densitometric results of 6 experiments using a site-specific phosphoantibody to the PDK site (T497) as described in methods. (b) Mean densitometric results of 6 experiments, phosphorylation detected with a site-specific phosphor-antibody to the hydrophobic site (S657). (c) Mean densitometric results of 6 experiments, phosphorylation detected with a site-specific antibody for the turn motif site (T638). No ATP and BSA lanes represent negative controls. Protamine is a positive control. Data are mean ± SE. *P < 0.05 and **P < 0.01 versus No ATP.

Mentions: We have previously reported [21] and confirmed here that the addition of calponin to PKC invitro in the presence of ATP, but the absence of lipids, increases the amount of phosphorylation at the PKCα hydrophobic site (Figure 1(b)). We now report that the addition of calponin also increases phosphorylation at T497, the putative PDK transphosphorylation site, (detected with a site specific phosphoantibody) 2-3-fold over that in the absence of ATP (Figure 1(a)). In contrast, the addition of an equimolar amount of BSA as a negative control had no effect. The addition of an equimolar amount of protamine produced similar levels of activation to that caused by calponin. Protamine is used as a positive control since it is also known to be able to activate PKC in the absence of lipid cofactors [24, 25]. In contrast, as can be seen in Figure 1(c), the addition of calponin in vitro does not significantly change the level of phosphorylation of PKCα at the turn motif site (T638). Interestingly, protamine, a known direct activator of PKC [24, 25], also had no effect on the phosphorylation at this site.


Regulation of PKC autophosphorylation by calponin in contractile vascular smooth muscle tissue.

Kim HR, Gallant C, Morgan KG - Biomed Res Int (2013)

Calponin increases autophosphorylation on the PDK and hydrophobic sites but not the turn motif site of recombinant PKC. (a) Mean densitometric results of 6 experiments using a site-specific phosphoantibody to the PDK site (T497) as described in methods. (b) Mean densitometric results of 6 experiments, phosphorylation detected with a site-specific phosphor-antibody to the hydrophobic site (S657). (c) Mean densitometric results of 6 experiments, phosphorylation detected with a site-specific antibody for the turn motif site (T638). No ATP and BSA lanes represent negative controls. Protamine is a positive control. Data are mean ± SE. *P < 0.05 and **P < 0.01 versus No ATP.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3852320&req=5

fig1: Calponin increases autophosphorylation on the PDK and hydrophobic sites but not the turn motif site of recombinant PKC. (a) Mean densitometric results of 6 experiments using a site-specific phosphoantibody to the PDK site (T497) as described in methods. (b) Mean densitometric results of 6 experiments, phosphorylation detected with a site-specific phosphor-antibody to the hydrophobic site (S657). (c) Mean densitometric results of 6 experiments, phosphorylation detected with a site-specific antibody for the turn motif site (T638). No ATP and BSA lanes represent negative controls. Protamine is a positive control. Data are mean ± SE. *P < 0.05 and **P < 0.01 versus No ATP.
Mentions: We have previously reported [21] and confirmed here that the addition of calponin to PKC invitro in the presence of ATP, but the absence of lipids, increases the amount of phosphorylation at the PKCα hydrophobic site (Figure 1(b)). We now report that the addition of calponin also increases phosphorylation at T497, the putative PDK transphosphorylation site, (detected with a site specific phosphoantibody) 2-3-fold over that in the absence of ATP (Figure 1(a)). In contrast, the addition of an equimolar amount of BSA as a negative control had no effect. The addition of an equimolar amount of protamine produced similar levels of activation to that caused by calponin. Protamine is used as a positive control since it is also known to be able to activate PKC in the absence of lipid cofactors [24, 25]. In contrast, as can be seen in Figure 1(c), the addition of calponin in vitro does not significantly change the level of phosphorylation of PKCα at the turn motif site (T638). Interestingly, protamine, a known direct activator of PKC [24, 25], also had no effect on the phosphorylation at this site.

Bottom Line: We found that calponin increases the level of in vitro PKCα phosphorylation at the PDK and hydrophobic sites, but not the turn motif site.Calponin knockdown inhibits autophosphorylation of cellular PKC in response to phenylephrine, confirming results with recombinant PKC.Thus these results show that autophosphorylation of PKC is regulated in dVSM and calponin is necessary for autophosphorylation of PKC in VSM.

View Article: PubMed Central - PubMed

Affiliation: Department of Health Sciences, Boston University, 635 Commonwealth Avenue, Boston, MA 02215, USA ; Department of Pharmacology, College of Medicine, Dankook University, 119 Dandaero, Chungnam, Cheonan-si 330-714, Republic of Korea.

ABSTRACT
Protein kinase C (PKC) is a key enzyme involved in agonist-induced smooth muscle contraction. In some cases, regulatory phosphorylation of PKC is required for full activation of the enzyme. However, this issue has largely been ignored with respect to PKC-dependent regulation of contractile vascular smooth muscle (VSM) contractility. The first event in PKC regulation is a transphosphorylation by PDK at a conserved threonine in the activation loop of PKC, followed by the subsequent autophosphorylation at the turn motif and hydrophobic motif sites. In the present study, we determined whether phosphorylation of PKC is a regulated process in VSM and also investigated a potential role of calponin in the regulation of PKC. We found that calponin increases the level of in vitro PKCα phosphorylation at the PDK and hydrophobic sites, but not the turn motif site. In vascular tissues, phosphorylation of the PKC hydrophobic site, but not turn motif site, as well as phosphorylation of PDK at S241 increased in response to phenylephrine. Calponin knockdown inhibits autophosphorylation of cellular PKC in response to phenylephrine, confirming results with recombinant PKC. Thus these results show that autophosphorylation of PKC is regulated in dVSM and calponin is necessary for autophosphorylation of PKC in VSM.

Show MeSH
Related in: MedlinePlus