Toward the identification of selective modulators of protein kinase C (PKC) isozymes

Shindo, Mayumi; Irie, Kazuhiro; Nakahara, Akifumi; Ohigashi, Hajime; Konishi, Hiroaki; Kikkawa, Ushio; Fukuda, Hiroyuki; Wender, Paul A.

doi:10.1016/s0968-0896(01)00100-6

Cited by 74 publications

(60 citation statements)

References 26 publications

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“…PKC␥ is quite sensitive to small changes in the natural activator, DAG, because of the presence of two tandem zincbinding motifs in the C1 domain which bind DAG at very low DAG levels (1-6 nM) (33). Because LEDGF can increase DAG levels, does the elevation of DAG in the presence of LEDGF increase PKC␥ enzyme activity as well?…”

Section: Resultsmentioning

confidence: 99%

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

Nguyen¹,

Takemoto

Takemoto³

2004

Journal of Biological Chemistry

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Gap junction activity in lens epithelial cells is regulated by PKC␥-mediated phosphorylation of Cx43. PKC␥ activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKC␥-C1B domain that are responsible for its interaction with 14-3-3⑀. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKC␥. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3⑀, resulting in the release of endogenous cellular PKC␥ from 14-3-3⑀, in vivo or in vitro, in activation of PKC␥ enzyme activity, phosphorylation of PKC␥, in the subsequent translocation of PKC␥ to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 M of C1B1 or C1B5 peptides also caused a 10-or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKC␥ are regulated by its binding to 14-3-3⑀ at the C1B domain of PKC␥. Synthetic peptides corresponding to these regions of PKC␥ successfully competed for the binding of 14-3-3⑀ to endogenous PKC␥, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.

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Section: Resultsmentioning

confidence: 99%

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

Nguyen¹,

Takemoto

Takemoto³

2004

Journal of Biological Chemistry

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“…2) were synthesized in a stepwise fashion on 0.1 mmol of preloaded Fmoc-Gly-PEG-PS resin (Applied Biosystems) by Pioneer TM using the Fmoc method as reported previously (23,24). The coupling reaction was carried out using each Fmoc amino acid (0.4 (29), and N,N-diisopropylethylamine (0.8 mmol) in N,Ndimethylformamide for 30 min (flow rate, 30 ml/min).…”

Section: Methodsmentioning

confidence: 99%

“…After completion of the chain assembly, each peptide resin was cleaved, and the resultant crude peptide was precipitated by diethyl ether. The crude peptide was purified by gel filtration, followed by HPLC as reported previously (23,24). Lyophilization gave a corresponding pure C1 peptide, the purity of which was confirmed by HPLC (Ͼ98%).…”

Section: Methodsmentioning

confidence: 99%

“…[ 3 H]PDBu Binding Assay of DGK Isozyme C1 Peptides and FLAG-DGK␥-The PDBu binding assay was carried out using the procedure of Sharkey and Blumberg (30) with slight modification (23,24). The standard assay mixture (250 l) in 1.5-ml Eppendorf tubes contained 50 mM Tris maleate, pH 7.4, 50 g/ml 1,2-di-(cis-9-octadecenoyl)-sn-glycero-3-phospho-L-serine (phosphatidylserine dioleoyl), 3 mg/ml bovine ␥-globulin, [ 3 H]PDBu (17.0 Ci/mmol), and each DGK␥ C1 peptide or FLAG-DGK␥.…”

Section: Methodsmentioning

confidence: 99%

“…Each PKC C1 domain has six conserved cysteines and two histidines in the typical core structure HX 12 CX 2 CX 13-14 CX 2 CX 4 HX 2 CX 7 C (where X is any amino acid) that coordinates two atoms of zinc in a tetrahedral geometry (21,22). We previously synthesized the 50-mer core structure of the C1 domains of all PKC isozymes and showed that most of the C1 domains of conventional and novel PKC isozymes strongly bind phorbol 12,13-dibutyrate (PDBu) with dissociation constants (K d ) in the nanomolar range (23,24).…”

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Synthesis and Phorbol Ester Binding of the Cysteine-rich Domains of Diacylglycerol Kinase (DGK) Isozymes

Shindo¹,

Irie

Masuda

et al. 2003

Journal of Biological Chemistry

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Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGK␥-C1A and DGK␤-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGK␥-C1A, human-DGK␥-C1A, and human-DGK␤-C1A gave K d values of 3.6, 2.8, and 14.6 nM, respectively, suggesting that DGK␥ and DGK␤ are new targets of phorbol esters. An A12T mutation of human-DGK␤-C1A enhanced the affinity to bind PDBu, indicating that the ␤-hydroxyl group of Thr-12 significantly contributes to the binding. The K d value for PDBu of FLAG-tagged whole rat-DGK␥ (4.4 nM) was nearly equal to that of rat-DGK␥-C1A (3.6 nM). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGK␥ and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGK␥ to cause its translocation.Diacylglycerol kinase (DGK) 1 and protein kinase C (PKC) both interact with the second messenger diacylglycerol (DG) (1, 2). DGK phosphorylates DG to produce phosphatidic acid, whereas PKC is allosterically activated by DG in the presence of phosphatidylserine. Therefore, DGK may inhibit the activation of PKC by attenuating DG levels, contributing to the regulation of intracellular signal transduction.To date, nine subtypes of mammalian DGKs have been cloned (3-15). All DGK isozymes consist of a conserved catalytic domain and two or three cysteine-rich C1 domains designated as C1A, C1B, and C1C (16). These isozymes are classified into five classes according to the other functional domains (Fig. 1). The class I isozymes (DGK␣, -␤, and -␥) have calcium binding domains (EF-hands). The class II isozymes (DGK␦ and -) have a pleckstrin homology domain at the N terminus, and their catalytic region is split into two domains unlike the other DGK isozymes. DGK⑀ has a simple structure and is classified as a class III isozyme. The class IV isozymes DGK and have a myristoylated alanine-rich C kinase substrate homology domain and four ankyrin repeats. DGK , which has three C1 domains unlike other DGK and PKC isozymes, is the only isozyme in class V.The similarity between DGK and PKC isozymes in structure is in the cysteine-rich C1 domains. Recent investigations using NMR spectroscopy and x-ray crystallography have revealed the three-dimensional structure of C1B domains of PKC␣, PKC␥,. Each PKC C1 domain has six conserved cysteines and two histidines in the typical core structure HX 12 CX 2 CX 13-14 CX 2 CX 4 HX 2 CX 7 C (where X is any amino acid) that coordinates two atoms of zinc in...

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Importance of Interaction between C1 Domain and Lipids in Protein Kinase Cα Activation: Hydrophobic Side Chain Direction in Isobenzofuranone Ligands Controls Enzyme Activation Level

et al. 2007

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Toward the identification of selective modulators of protein kinase C (PKC) isozymes

Cited by 74 publications

References 26 publications

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

Synthesis and Phorbol Ester Binding of the Cysteine-rich Domains of Diacylglycerol Kinase (DGK) Isozymes

Importance of Interaction between C1 Domain and Lipids in Protein Kinase Cα Activation: Hydrophobic Side Chain Direction in Isobenzofuranone Ligands Controls Enzyme Activation Level

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