Effects of inhibitors of protein kinase C and calpain in experimental delayed cerebral vasospasm

✓ Vasospasm was produced in adult mongrel dogs by a two-hemorrhage method, and the spastic basilar arteries were exposed via the transclival route on Day 7. Tonic contraction was produced in the normal canine basilar arteries by a local application of KCl or serotonin after transclival exposure. The exposed spastic and tonic basilar arteries then received a topical application of the following: 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7), a potent inhibitor of protein kinase C acting at the catalytic domain; calphostin C, a specific inhibitor of protein kinase C acting at the regulatory domain; or calpeptin, a selective inhibitor of calpain. Both spastic and tonic basilar arteries were effectively dilated by H-7. Calphostin C caused only slight dilation of spastic basilar arteries but moderate dilation of tonic basilar arteries. Dilation in response to calpeptin was remarkable in the spastic basilar arteries but slight in the tonic basilar arteries. The doses of calphostin C and calpeptin required to obtain maximum effect were markedly lower in the tonic model than in the spastic model. The spastic and tonic models had a similar dose-dependent response to H-7 but quite a different response to calphostin C or calpeptin, suggesting a difference in the function of protein kinase C and calpain in the two models. Furthermore, the effect of calphostin C on the reversal of vasospasm was increased significantly after topical treatment with calpeptin. It is suggested that the majority of the catalytic domain of protein kinase C is dissociated from the regulatory domain, probably by a limited proteolysis with calpain, and is markedly activated in vasospasm.

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The regulatory domain of protein kinase C-ε restricts the catalytic-domain-specificity

Biochemical Journal ◽

10.1042/bj2760257 ◽

1991 ◽

Vol 276 (1) ◽

pp. 257-260 ◽

Cited By ~ 31

Author(s):

C Pears ◽

D Schaap ◽

P J Parker

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Catalytic Domain ◽

Structural Basis ◽

Regulatory Domain ◽

Substrate Selection ◽

Kinase C ◽

Pkc Epsilon ◽

Pkc Alpha

Protein kinase C (PKC) consists of a family of closely related enzymes that can be divided into two subfamilies (alpha, beta and gamma and delta, epsilon and zeta) on the basis of primary sequence. Functional differences have also been described; thus PKC-alpha, PKC-beta and PKC-gamma readily phosphorylate histone IIIS in vitro, whereas PKC-epsilon will not employ this substrate efficiently. We have previously demonstrated, however, that proteolytic cleavage of PKC-epsilon generates a constitutive kinase activity that is an efficient histone IIIS kinase [Schaap, Hsuan, Totty & Parker (1990) Eur. J. Biochem. 191, 431-435]. In order to investigate the structural basis for this switch in specificity, we have constructed a chimaeric protein containing the regulatory domain of PKC-epsilon fused to the catalytic domain of PKC-gamma. When this is expressed in COS1 cells the chimaeric kinase shows a substrate-specificity similar to that of PKC-epsilon rather than to that of PKC-gamma. This demonstrates a role for the regulatory domain in substrate selection of PKC-epsilon.

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Faculty Opinions recommendation of Protein kinase M zeta synthesis from a brain mRNA encoding an independent protein kinase C zeta catalytic domain. Implications for the molecular mechanism of memory.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1008709.793469674 ◽

2013 ◽

Author(s):

David Glanzman

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Molecular Mechanism ◽

Catalytic Domain ◽

Kinase C ◽

Brain Mrna ◽

Protein Kinase C Zeta ◽

Independent Protein

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The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium- and phosphatidylserine-dependent phorbol diester binding activity.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)66795-x ◽

1986 ◽

Vol 261 (32) ◽

pp. 14867-14870 ◽

Cited By ~ 18

Author(s):

M H Lee ◽

R M Bell

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Lipid Binding ◽

Binding Activity ◽

Regulatory Domain ◽

Kinase C

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Differential effects of the protein kinase C inhibitors H7 and calphostin C on the cell cycle of neuroblastoma cells

Brain Research ◽

10.1016/0006-8993(92)91543-n ◽

1992 ◽

Vol 596 (1-2) ◽

pp. 157-162 ◽

Cited By ~ 8

Author(s):

MaDolores Miñana ◽

Vicente Felipo ◽

Santiago Grisolía

Keyword(s):

Cell Cycle ◽

Protein Kinase C ◽

Protein Kinase ◽

Neuroblastoma Cells ◽

Differential Effects ◽

Protein Kinase C Inhibitors ◽

Calphostin C ◽

Kinase C

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Three-Dimensional Reconstruction of Protein Kinase C-delta and Its Regulatory Domain by Electron Microscopy of Two-Dimensional Crystals

Microscopy and Microanalysis ◽

10.1017/s1431927605502290 ◽

2005 ◽

Vol 11 (S02) ◽

Author(s):

A S Solodukhin ◽

R H Kretsinger ◽

J J Sando

Keyword(s):

Electron Microscopy ◽

Protein Kinase C ◽

Protein Kinase ◽

Three Dimensional ◽

Regulatory Domain ◽

Two Dimensional ◽

Three Dimensional Reconstruction ◽

Protein Kinase C Delta ◽

Kinase C ◽

Dimensional Reconstruction

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Stimulation of protein kinase C activity by tumor necrosis factor-α in bovine bronchial epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.5.l1007 ◽

1997 ◽

Vol 273 (5) ◽

pp. L1007-L1012 ◽

Cited By ~ 16

Author(s):

Todd A. Wyatt ◽

Harumasa Ito ◽

Thomas J. Veys ◽

John R. Spurzem

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tumor Necrosis Factor ◽

Epithelial Cells ◽

Tumor Necrosis ◽

Bronchial Epithelial ◽

Calphostin C ◽

Kinase C ◽

Tnf Α ◽

Necrosis Factor

Bronchial epithelial cell migration, attachment, and proliferation are important processes in response to airway injury. We have shown that tumor necrosis factor (TNF)-α stimulates the migration of bovine bronchial epithelial cells (BBEC) in vitro. We hypothesized that protein kinase C (PKC) may be one of the intracellular signaling mediators of TNF-α in BBEC. In this study, we have identified multiple PKC isoforms in BBEC and measured total cellular PKC activity. Polyclonal antibodies to the PKC-α, -β2, -δ, and -ε isoforms recognized protein bands around 80–90 kDa. BBEC primary cultures treated with either 500 U/ml TNF-α for 2–4 h or 100 ng/ml 12- O-tetradecanoylphorbol 13-acetate for 15 min resulted in three- to fivefold increases in PKC activity in the particulate fractions of crude cell lysates. This activity was inhibited by 1 μM calphostin C or 10 μM H-7. Similarly, TNF-α-stimulated BBEC migration was reduced at least twofold in the presence of H-7 or calphostin C. These studies suggest that the activation of PKC is necessary for TNF-α-stimulated BBEC migration.

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Differential effects of putative protein kinase C inhibitors on contraction of rat aortic smooth muscle

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.264.4.h1300 ◽

1993 ◽

Vol 264 (4) ◽

pp. H1300-H1306 ◽

Cited By ~ 10

Author(s):

Y. Shimamoto ◽

H. Shimamoto ◽

C. Y. Kwan ◽

E. E. Daniel

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinases ◽

Induced Response ◽

Dependent Manner ◽

Contractile Responses ◽

Putative Protein ◽

Calphostin C ◽

Kinase C ◽

Putative Protein Kinase

We investigated effects of three kinds of putative protein kinase C (PKC) inhibitors, calphostin C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and stauro-sporine, on aortic muscle contractions induced by KCl, phenylephrine, 12-O-tetradecanoylphorbol-13-acetate (TPA), and phorbol 12, 13-dibutyrate (PDBu). Calphostin C noncompetitively inhibited TPA-induced contractions in a concentration-dependent manner. At 10(-6) M, calphostin C completely abolished responses to TPA and also effectively inhibited PDBu-induced contractions. Such a concentration of calphostin C had no effect on KCl-induced contractions but decreased the maximal tension of phenylephrine-induced response curve by 35.3 +/- 6.6% H-7 (10(-5) M had little effect on TPA-induced contraction but significantly inhibited contractile responses to phenylephrine and KCl. Staurosporine (10(-8) M, 3 x 10(-8) M) inhibited contractile responses to KCl, phenylephrine, and TPA. We suggest that staurosporine and H-7, which are known to act on the catalytic domain of PKC carrying high degree of sequence homology with other protein kinases, are relatively nonselective for PKC. On the other hand, calphostin C acting on the regulatory domain of PKC, which is distinct from other protein kinases, may serve as a relatively more selective PKC inhibitor.

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The regulatory domain of protein kinase C delta positively regulates insulin receptor signaling

Journal of Molecular Endocrinology ◽

10.1677/jme-09-0119 ◽

2009 ◽

Vol 44 (3) ◽

pp. 155-169 ◽

Cited By ~ 9

Author(s):

Avraham I Jacob ◽

Miriam Horovitz-Fried ◽

Shlomit Aga-Mizrachi ◽

Tamar Brutman-Barazani ◽

Hana Okhrimenko ◽

...

Keyword(s):

Skeletal Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Insulin Receptor ◽

Glycogen Synthase ◽

Regulatory Domain ◽

Protein Kinase C Delta ◽

Kinase C ◽

Regulatory Effects ◽

In Cells

Protein kinase C delta (PKCδ) is induced by insulin to rapidly associate with insulin receptor (IR) and upregulates insulin signaling. We utilized specific JM and CT receptor domains and chimeras of PKCα and PKCδ regulatory and catalytic domains to elucidate which components of PKCδ are responsible for positive regulatory effects of PKCδ on IR signaling. Studies were performed on L6 and L8 skeletal muscle myoblasts and myotubes. PKCδ was preferentially bound to the JM domain of IR, and insulin stimulation increased this binding. Both PKCδ/α and PKCα/δ chimeras (regulatory/catalytic) were bound preferentially to the JM but not to the CT domain of IR. Although IR–PKCδ binding was higher in cells expressing either the PKCδ/α or PKCα/δ chimera than in control cells, upregulation of IR signaling was observed only in PKCδ/α cells. Thus, in response to insulin increases in tyrosine phosphorylation of IR and insulin receptor substrate-1, downstream signaling to protein kinase B and glycogen synthase kinase 3 (GSK3) and glucose uptake were greater in cells overexpressing PKCδ/α and the PKCδ/δ domains than in cells expressing the PKCα/δ domains. Basal binding of Src to PKCδ was higher in both PKCδ/α- and PKCα/δ-expressing cells compared to control. Binding of Src to IR was decreased in PKCα/δ cells but remained elevated in the PKCδ/α cells in response to insulin. Finally, insulin increased Src activity in PKCδ/α-expressing cells but decreased it in PKCα/δ-expressing cells. Thus, the regulatory domain of PKCδ via interaction with Src appears to determine the role of PKCδ as a positive regulator of IR signaling in skeletal muscle.

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