Protein Kinase C δ (PKCδ) Inhibits the Expression of Glutamine Synthetase in Glial Cells via the PKCδ Regulatory Domain and Its Tyrosine Phosphorylation

Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAKβ or CADTK) has been identified as a member of the focal adhesion kinase (FAK) family of protein-tyrosine kinases and it has been suggested that the mode of Pyk2 activation is distinct from that of FAK. In the present study we investigated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosphorylated, in a manner mostly dependent on αIIbβ3 integrin-mediated aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment with BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxymethyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C (PKC) inhibitors at concentrations that inhibited platelet aggregation. In contrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for αIIbβ3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. The activation of the von Willebrand factor (vWF)--glycoprotein Ib pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytoskeleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S-transferase fusion proteins containing Shc-SH2, Grb2-SH2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N-terminal SH3 domain of Grb2 and that the phosphotyrosine residue of Shc binds to the SH2 domain of Grb2. Although Pyk2 and FAK have been reported to be differentially regulated in many cell types, our results suggest that, in human platelets, the mode of Pyk2 activation is mostly similar to that of FAK, in terms of αIIbβ3 integrin-dependent and PKC-dependent tyrosine phosphorylation. Furthermore, Pyk2, as well as FAK, might have one or more important roles in post-aggregation tyrosine phosphorylation events, in association with the cytoskeleton and through interaction with adapter proteins including Grb2 and Shc.

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Protein kinase C activity in soluble fractions from glial cells in primary culture and subcultures

Neuroscience Letters ◽

10.1016/0304-3940(88)90361-8 ◽

1988 ◽

Vol 85 (2) ◽

pp. 255-260 ◽

Cited By ~ 11

Author(s):

John A. Murphy ◽

Jill A. Chapman ◽

Anthony J. Suckling ◽

Martin G. Rumsby

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Primary Culture ◽

Glial Cells ◽

Kinase C ◽

Protein Kinase C Activity ◽

Soluble Fractions

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Changes in Tight Junctional Resistance of the Cervical Epithelium Are Associated with Modulation of Content and Phosphorylation of Occludin 65-Kilodalton and 50-Kilodalton Forms

Endocrinology ◽

10.1210/en.2005-0916 ◽

2006 ◽

Vol 147 (2) ◽

pp. 977-989 ◽

Cited By ~ 17

Author(s):

Ling Zhu ◽

Xin Li ◽

Robin Zeng ◽

George I. Gorodeski

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tight Junction ◽

Tyrosine Phosphorylation ◽

Tight Junctions ◽

Early Stage ◽

Cervical Epithelium ◽

Kinase C ◽

Low Levels ◽

Junctional Resistance

Treatment of human cervical epithelial CaSki cells with ATP or with the diacylglyceride sn-1,2-dioctanoyl diglyceride (diC8) induced a staurosporine-sensitive transient increase, followed by a late decrease, in tight-junctional resistance (RTJ). CaSki cells express two immunoreactive forms of occludin, 65 and 50 kDa. Treatments with ATP and diC8 decreased the density of the 65-kDa form and increased the density of the 50-kDa form. ATP also decreased threonine phosphorylation of the 65-kDa form and increased threonine phosphorylation of the 50-kDa form and tyrosine phosphorylation of the 65- and 50-kDa forms. Staurosporine decreased acutely threonine and tyrosine phosphorylation of the two isoforms and in cells pretreated with staurosporine ATP increased acutely the density of the 65-kDa form and threonine phosphorylation of the 65-kDa form. Treatment with N-acetyl-leucinyl-leucinyl-norleucinal increased the densities of the 65- and 50-kDa forms. Pretreatment with N-acetyl-leucinyl-leucinyl-norleucinal attenuated the late decreases in RTJ induced by ATP and diC8 and the decrease in the 65-kDa and increase in the 50-kDa forms induced by ATP. Correlation analyses showed that high levels of RTJ correlated with the 65-kDa form, whereas low levels of RTJ correlated negatively with the 65-kDa form and positively with the 50-kDa form. The results suggest that in CaSki cells 1) occludin determines gating of the tight junctions, 2) changes in occludin phosphorylation status and composition regulate the RTJ, 3) protein kinase-C-mediated, threonine dephosphorylation of the 65-kDa occludin form increases the resistance of assembled tight junctions, 4) the early stage of tight junction disassembly involves calpain-mediated breakdown of occludin 65-kDa form to the 50-kDa form, and 5) increased levels of the 50-kDa form interfere with occludin gating of the tight junctions.

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

Journal of Neurosurgery ◽

10.3171/jns.1992.76.1.0111 ◽

1992 ◽

Vol 76 (1) ◽

pp. 111-118 ◽

Cited By ~ 48

Author(s):

Nobutaka Minami ◽

Eiichi Tani ◽

Yukio Maeda ◽

Ikuya Yamaura ◽

Masahiro Fukami

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Catalytic Domain ◽

Limited Proteolysis ◽

Maximum Effect ◽

Regulatory Domain ◽

Calphostin C ◽

Delayed Cerebral Vasospasm ◽

Kinase C ◽

Basilar Arteries

✓ 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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Cholecystokinin-stimulated Protein Kinase C-δ Kinase Activation, Tyrosine Phosphorylation, and Translocation Are Mediated by Src Tyrosine Kinases in Pancreatic Acinar Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m303119200 ◽

2003 ◽

Vol 278 (37) ◽

pp. 35220-35230 ◽

Cited By ~ 49

Author(s):

Jose A. Tapia ◽

Luis J. García-Marin ◽

Robert T. Jensen

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Kinase Activation ◽

Kinase C ◽

Src Tyrosine Kinases

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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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