Protein kinase C-catalyzed calponin phosphorylation in swine carotid arterial homogenate

Rokolya, Anikó; Walsh, Michael P.; Singer, Harold A.; Moreland, Robert S.

doi:10.1002/(sici)1097-4652(199809)176:3<545::aid-jcp11>3.0.co;2-z

“…Calponin is also a well-established in vitro substrate for PKC as well as a possible in vivo substrate for PKC (56). Rokolya et al (42) demonstrated that the physiological kinase for calponin phosphorylation is PKC. Studies (32) from other laboratories have indicated that PKC activity is related to its subcellular localization.…”

Section: Discussionmentioning

confidence: 99%

Direct association and translocation of PKC-α with calponin

Patil

¹

,

Pawar

²

,

Bitar

³

2004

American Journal of Physiology-Gastrointestinal and Liver Physi

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Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-α and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-α. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-α in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-α, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351–972 corresponding to amino acids 92–229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX -KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-α in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-α, indicating a direct association of GST-calponin with PKC-α. These data suggest that there is a direct association of translocated calponin and PKC-α in the membrane and a role for the complex calponin-PKC-α-HSP27, in contraction of colonic smooth muscle cells.

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“…We have previously reported the use of the protein kinase C substrate peptide, MBP 4-14 , in the determination of protein kinase C activity in a homogenate of the swine carotid artery (Rokolya et al, 1998). In our present study, we used MBP 4-14 to determine if protein kinase C is activated during Ca 2ϩ stimulation of the Triton X-100 detergent-skinned carotid arterial strip.…”

Section: Ca 2؉ -Dependent Activation Of Protein Kinase Cmentioning

confidence: 96%

Ca2‐ and protein kinase C‐dependent stimulation of mitogen‐activated protein kinase in detergent‐skinned vascular smooth muscle

Katoch¹,

Su²,

Moreland³

1999

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Protein kinase C and mitogen-activated protein (MAP) kinase are expressed in all smooth muscle cells and believed to be important in several physiologically relevant properties of this muscle. Our goal was to determine if protein kinase C and MAP kinase are activated by a simple increase in cellular Ca(2+) and to determine if protein kinase C is an upstream activator of MAP kinase. These studies were performed in the Triton X-100 detergent-skinned preparation of the swine carotid artery, which allows control of the intracellular environment without influence from membrane or receptor-mediated modulation. The p42 and p44 isoforms of MAP kinase were activated in a concentration-dependent fashion by an increase in Ca2+. This was shown by in-the-gel kinase assay and direct measurement of MAP kinase phosphotransferase activity. Protein kinase C was also activated by an increase in Ca2+, as shown by a novel assay that measures total active protein kinase C in the tissue. Inhibition of protein kinase C activity completely abolished MAP kinase activity. Additionally, inhibition of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) also abolished MAP kinase activity. Using intact swine carotid arteries, we showed p42 and p44 MAP kinase to be activated by both histamine and phorbol dibutyrate, but only the p42 isoform was calcium-sensitive. Our results suggest that a Ca(2+)-dependent isoform of protein kinase C and CaM kinase II are upstream activators of MAP kinase in the swine carotid artery.

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Ca ²⁺ ‐ and protein kinase C‐dependent stimulation of mitogen‐activated protein kinase in detergent‐skinned vascular smooth muscle

Katoch

¹

,

Su

²

,

Moreland

³

1999

Journal Cellular Physiology

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Protein kinase C and mitogen-activated protein (MAP) kinase are expressed in all smooth muscle cells and believed to be important in several physiologically relevant properties of this muscle. Our goal was to determine if protein kinase C and MAP kinase are activated by a simple increase in cellular Ca(2+) and to determine if protein kinase C is an upstream activator of MAP kinase. These studies were performed in the Triton X-100 detergent-skinned preparation of the swine carotid artery, which allows control of the intracellular environment without influence from membrane or receptor-mediated modulation. The p42 and p44 isoforms of MAP kinase were activated in a concentration-dependent fashion by an increase in Ca2+. This was shown by in-the-gel kinase assay and direct measurement of MAP kinase phosphotransferase activity. Protein kinase C was also activated by an increase in Ca2+, as shown by a novel assay that measures total active protein kinase C in the tissue. Inhibition of protein kinase C activity completely abolished MAP kinase activity. Additionally, inhibition of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) also abolished MAP kinase activity. Using intact swine carotid arteries, we showed p42 and p44 MAP kinase to be activated by both histamine and phorbol dibutyrate, but only the p42 isoform was calcium-sensitive. Our results suggest that a Ca(2+)-dependent isoform of protein kinase C and CaM kinase II are upstream activators of MAP kinase in the swine carotid artery.

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Protein kinase C-catalyzed calponin phosphorylation in swine carotid arterial homogenate

Cited by 5 publications

References 37 publications

Direct association and translocation of PKC-α with calponin

Direct association and translocation of PKC-α with calponin

Ca2‐ and protein kinase C‐dependent stimulation of mitogen‐activated protein kinase in detergent‐skinned vascular smooth muscle

Ca ²⁺ ‐ and protein kinase C‐dependent stimulation of mitogen‐activated protein kinase in detergent‐skinned vascular smooth muscle

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Protein kinase C-catalyzed calponin phosphorylation in swine carotid arterial homogenate

Cited by 5 publications

References 37 publications

Direct association and translocation of PKC-α with calponin

Direct association and translocation of PKC-α with calponin

Ca2‐ and protein kinase C‐dependent stimulation of mitogen‐activated protein kinase in detergent‐skinned vascular smooth muscle

Ca 2+ ‐ and protein kinase C‐dependent stimulation of mitogen‐activated protein kinase in detergent‐skinned vascular smooth muscle

Contact Info

Product

Resources

About

Ca ²⁺ ‐ and protein kinase C‐dependent stimulation of mitogen‐activated protein kinase in detergent‐skinned vascular smooth muscle