NMR Structure of a Protein Kinase C-γ Phorbol-Binding Domain and Study of Protein−Lipid Micelle Interactions

Xu, Renfeng; Pawełczyk, Tadeusz; Xia, Tai-he; Brown, Stephen C.

doi:10.1021/bi970833a

Cited by 127 publications

(132 citation statements)

References 46 publications

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“…As the three-dimensional model of residues 34 -445 predicted, purified recombinant hRasGRP4 was able to transfer ␥- 35 S-GTP to GDP-loaded H-Ras in a catalytic manner (Fig. 7a).…”

Section: Fig 7 Generation Of Recombinant Hrasgrp4 In Cos-7 Cellsmentioning

confidence: 89%

“…The solution was then incubated for another 15 min at room temperature to load H-Ras with non-radiolabeled GDP. Twenty l of the resulting solution was added to 75 l of reaction buffer (100 mM NaCl, 10 mM MgCl 2 , 20 mM Tris-HCl, pH 8.0, supplemented with 100 M AMP-PNP, 0.5 mg/ml bovine serum albumin, and 2 M guanosine 5Ј-␥- 35 S-triphosphate (ϳ11,000 cpm/pmol; Amersham Biosciences)), followed by 5 l of purified recombinant hRasGRP4 in the above buffer. The resulting samples were incubated at room temperature generally for 20 min.…”

Section: Rasgrp4mentioning

confidence: 99%

“…Residues 541-597 in hRasGRP4 correspond to its putative DAG-binding domain. This portion of the GEF was modeled based on the NMR structure of the Cys 2 domain in rat protein kinase C-␥ (Protein Data Bank code 1TBN) (35). The regions are ϳ20 and ϳ33% identical to the corresponding regions in Sos1 and protein kinase C-␥, respectively.…”

Section: Rasgrp4mentioning

confidence: 99%

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RasGRP4, a New Mast Cell-restricted Ras Guanine Nucleotide-releasing Protein with Calcium- and Diacylglycerol-binding Motifs

Yang¹,

Li²,

Wong³

et al. 2002

Journal of Biological Chemistry

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A cDNA was isolated from interleukin 3-developed, mouse bone marrow-derived mast cells (MCs) that contained an insert (designated mRasGRP4) that had not been identified in any species at the gene, mRNA, or protein level. By using a homology-based cloning approach, the ϳ2.6-kb hRasGRP4 transcript was also isolated from the mononuclear progenitors residing in the peripheral blood of normal individuals. This transcript information was then used to locate the RasGRP4 gene in the mouse and human genomes, to deduce its exon/ intron organization, and then to identify 10 single nucleotide polymorphisms in the human gene that result in 5 amino acid differences. The >15-kb hRasGRP4 gene consists of 18 exons and resides on a region of chromosome 19q13.1 that had not been sequenced by the Human Genome Project. Human and mouse MCs and their progenitors selectively express RasGRP4, and this new intracellular protein contains all of the domains present in the RasGRP family of guanine nucleotide exchange factors even though it is <50% identical to its closest homolog. Recombinant RasGRP4 can activate H-Ras in a cation-dependent manner. Transfection experiments also suggest that RasGRP4 is a diacylglycerol/phorbol ester receptor. Transcript analysis of an asthma patient, a mastocytosis patient, and the HMC-1 cell line derived from a MC leukemia patient revealed the presence of substantial amounts of non-functional forms of hRas-GRP4 due to an inability to remove intron 5 in the precursor transcript. Because only abnormal forms of hRas-GRP4 were identified in the HMC-1 cell line, this immature MC progenitor was used to address the function of RasGRP4 in MCs. HMC-1 leukemia cells differentiated and underwent granule maturation when induced to express a normal form of RasGRP4. Thus, RasGRP4 plays an important role in the final stages of MC development.

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“…As the three-dimensional model of residues 34 -445 predicted, purified recombinant hRasGRP4 was able to transfer ␥- 35 S-GTP to GDP-loaded H-Ras in a catalytic manner (Fig. 7a).…”

Section: Fig 7 Generation Of Recombinant Hrasgrp4 In Cos-7 Cellsmentioning

confidence: 89%

Section: Rasgrp4mentioning

confidence: 99%

See 1 more Smart Citation

RasGRP4, a New Mast Cell-restricted Ras Guanine Nucleotide-releasing Protein with Calcium- and Diacylglycerol-binding Motifs

Yang¹,

Li²,

Wong³

et al. 2002

Journal of Biological Chemistry

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show abstract

“…Recent investigations using NMR spectroscopy and x-ray crystallography have revealed the three-dimensional structure of C1B domains of PKC␣, PKC␥, and PKC␦ (17)(18)(19)(20). 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).…”

mentioning

confidence: 99%

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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“…Release of 14-3-3 and oxidation within the C1B domain of the PKCγ lead to membrane translocation and activation of PKCγ (Nguyen et al, 2004;Lin and Takemoto 2005). Structural modeling results predict that H101Y and G128D SCA14 mutations caused Zinc-finger conformational changes Xu et al, 1997, and Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

Protein kinase C γ mutations in the C1B domain cause caspase-3-linked apoptosis in lens epithelial cells through gap junctions

Lin

Shanks

Prakash

et al. 2007

Experimental Eye Research

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Failure to control oxidative stress is closely related to aging and to a diverse range of human diseases. We have reported that protein kinase C γ (PKCγ) acts as a primary oxidative stress sensor in the lens. PKCγ has a Zn-finger C1B stress switch domain, residues 101-150. Mutation, H101Y, in the C1B domain of PKCγ proteins causes a failure of the PKCγ oxidative stress response (Lin and Takemoto (2005) (100 μM, 3 h) activated a caspase-3 apoptotic pathway in the lens epithelial cells but was more severe in cells expressing PKCγ mutations. The presence of 18α-glycyrrhetinic acid (AGA), an inhibitor of gap junctions, decreased Cx43 and Cx50 protein levels and gap junction plaque number. This reduction in gap junctions by AGA resulted in inhibition of H 2 O 2 -induced apoptosis. Our results demonstrate that there is a dominant negative effect of PKCγ C1B mutations on endogenous PKCγ which results in loss of control of gap junctions. Modeled structures suggest that the severity of C1B mutation effects may be related to extent of loss of C1B structure. Mutations in the C1B domain of PKCγ result in increased apoptosis in lens epithelial cells. This can be prevented by a gap junction inhibitor. Thus, propagation of apoptosis from cellto-cell in lens epithelial cells may be through open gap junctions. The control of gap junctions requires PKCγ.

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NMR Structure of a Protein Kinase C-γ Phorbol-Binding Domain and Study of Protein−Lipid Micelle Interactions

Cited by 127 publications

References 46 publications

RasGRP4, a New Mast Cell-restricted Ras Guanine Nucleotide-releasing Protein with Calcium- and Diacylglycerol-binding Motifs

RasGRP4, a New Mast Cell-restricted Ras Guanine Nucleotide-releasing Protein with Calcium- and Diacylglycerol-binding Motifs

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

Protein kinase C γ mutations in the C1B domain cause caspase-3-linked apoptosis in lens epithelial cells through gap junctions

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