Tumor promoter binding of the protein kinase C C1 homology domain peptides of RasGRPs, chimaerins, and Unc13s

Irie, Kazuhiro; Masuda, Akiko; Shindo, Mayumi; Nakagawa, Yu; Ohigashi, Hajime

doi:10.1016/j.bmc.2004.07.008

Cited by 31 publications

(47 citation statements)

References 32 publications

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“…S3A). Among them, RasGRP3 was the most likely candidate because RasGRP3, but not RasGRP1, is capable of activating Rap1 (16) and RasGRP2 is unresponsive to TPA (33). We therefore knocked down RasGRP3 expression by using siRNAs and examined Rap1 activity in TPA-stimulated cells.…”

Section: Resultsmentioning

confidence: 99%

Crucial Role of Phospholipase Cε in Skin Inflammation Induced by Tumor-Promoting Phorbol Ester

Ikuta

Edamatsu

et al. 2008

Cancer Research

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In two-stage skin chemical carcinogenesis, phorbol ester 12-Otetradecanoylphorbol-13-acetate (TPA) acts as a promoter essential for clonal expansion of the initiated cells carrying the activated ras oncogenes. Although protein kinase C (PKC) isozymes are the main targets of TPA, their role in tumor promotion remains controversial. We previously reported that mice lacking a Ras/Rap effector phospholipase CE (PLCe À/À mice) exhibited marked resistance to tumor formation in the two-stage skin carcinogenesis. PLCe À/À mice also failed to exhibit basal layer cell proliferation and epidermal hyperplasia induced by TPA, suggesting a role of PLCE in tumor promotion. Here, we show that PLCe À/À mice exhibit resistance to TPA-induced skin inflammation as assessed by reduction in edema, granulocyte infiltration, and expression of a proinflammatory cytokine, interleukin-1A (IL-1A). On the other hand, the proliferative potentials of keratinocytes or dermal fibroblasts in culture remain unaffected by the PLCe background, suggesting that the PLCE's role in tumor promotion may be ascribed to augmentation of inflammatory responses. In dermal fibroblast primary culture, TPA can induce activation of the PLCE lipase activity, which leads to the induction of IL-1A expression. Experiments using small interfering RNA-mediated knockdown indicate that this activation is mediated by Rap1, which is activated by a TPA-responsive guanine nucleotide exchange factor RasGRP3. Moreover, TPA-induced activation of Rap1 and PLCE is inhibited by a PKC inhibitor GF109203X, indicating a crucial role of PKC in signaling from TPA to PLCE. These results imply that two TPA targets, RasGRP3 and PKC, are involved in TPA-induced inflammation through PLCE activation, leading to tumor promotion. [Cancer Res 2008;68(1):64-72]

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Section: Resultsmentioning

confidence: 99%

Crucial Role of Phospholipase Cε in Skin Inflammation Induced by Tumor-Promoting Phorbol Ester

Ikuta

Edamatsu

et al. 2008

Cancer Research

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“…These two observations rule out the possibility that the lack of translocation of these GAPs during anti-CD3-mediated TCR signaling could be due to the lack of DAG generation or, alternatively, to artifacts derived from the use of overexpressed proteins. Given that the isolated C1 domains of α2-and β2-chimaerin show affinities to DAG analogs similar or even higher than C1 domains of PKCs and RasGRP1 [32,33,60], we believe that the differential behavior of these GAPs is probably a reflection of the high-energy cost required for releasing the auto-inhibitory structure of chimaerins [56]. This interpretation is consistent with the observation that β2-chimaerin requires in vivo ≈100-fold higher doses of PMA than PKCα for its effective translocation to cellular membranes [34].…”

Section: Discussionmentioning

confidence: 99%

Role of chimaerins, a group of Rac-specific GTPase activating proteins, in T-cell receptor signaling

Caloca

Delgado

Alarcón

et al. 2008

Cellular Signalling

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Chimaerins are GTPase-activating proteins that inactivate the GTP-hydrolase Rac1 in a diacylglycerol-dependent manner. To date, the study of chimaerins has been done mostly in neuronal cells. Here, we show that α2-and β2-chimaerin are expressed at different levels in T-cells and that they participate in T-cell receptor signaling. In agreement with this, we have observed that α2-and β2-chimaerins translocate to the T-cell/B-cell immune synapse and, using both gain-and loss-offunction approaches, demonstrated that their catalytic activity is important for the inhibition of the T-cell receptor-and Vav1-dependent stimulation of the transcriptional factor NF-AT. Mutagenesisbased approaches have revealed the molecular determinants that contribute to the biological program of chimaerins during T-cell responses. Unexpectedly, we have found that the translocation of chimaerins to the T-cell/B-cell immune synapse does not rely on the canonical binding of diacylglycerol to the C1 region of these GTPase-activating proteins. Taken together, these results identify chimaerins as candidates for the downmodulation of Rac1 in T-lymphocytes and, in addition, uncover a novel regulatory mechanism that mediates their activation in T-cells.

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“…For PKC␣, the K i of 130C037 (343 Ϯ 35 nM) was 4-fold weaker than that for PKC␦ (91 Ϯ 16 nM), but its affinity for the better binding C1 domain, ␣C1a (610 Ϯ 170 nM), was 350-fold weaker than that for ␦C1b (1.78 Ϯ 0.51 nM). Although the analysis of structure activity relations for the binding of ligands to isolated C1 domains, as so elegantly done by Irie et al (14,24,33), should continue to be informative, particularly for identifying positive interactions, great caution should be exercised in interpreting negative results.…”

Section: Discussionmentioning

confidence: 99%

“…As a simplified example, the addition of extra positively charged residues at the end of the C1 domain, in a position where they will not affect the direct interaction of ligand with the binding cleft but where they can affect charged interactions with the negatively charged phospholipids, had a substantial effect on the measured binding affinity (33). In the intact PKC, moreover, multiple domains contribute to the membrane interaction, including the pseudosubstrate region, the C1 domains, and Ca 2ϩ -C2 domain complex.…”

Section: Discussionmentioning

confidence: 99%

A Novel Diacylglycerol-lactone Shows Marked Selectivity in Vitro among C1 Domains of Protein Kinase C (PKC) Isoforms α and δ as Well as Selectivity for RasGRP Compared with PKCα

Perry

Yang

et al. 2005

Journal of Biological Chemistry

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Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms ␣, ␤, ␥, ␦, and ⑀, with apparent K i values ranging from 340 nM for PKC␣ to 29 nM for PKC⑀. When binding to isolated C1 domains of PKC␣ and -␦, 130C037 showed good affinity (K i ‫؍‬ 1.78 nM) only for ␦C1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of ␦C1b. 130C037 bound intact RasGRP1 and RasGRP3 with K i values of 3.5 and 3.8 nM, respectively, reflecting 8-and 90-fold selectivity relative to PKC⑀ and PKC␣. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED 50 ‫؍‬ 286 nM), partial reduction of PKC⑀ (ED 50 > 10 M), and no effect on PKC␣. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKC⑀, and no translocation of PKC␣. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets. Diacylglycerol (DAG)1 is a lipid second messenger, produced through hydrolysis of phosphatidylinositol 4,5-bisphosphate following the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D (1). Most but not all effects of DAG reflect its interaction with proteins containing C1 domains, resulting in their activation and/or driving their membrane translocation. Reflecting the importance and diversity of its downstream effectors, DAG is involved in signal transduction of numerous physiological and pathological processes, including proliferation, differentiation, apoptosis, angiogenesis, and drug resistance (2). These functions have focused attention on C1 domain-containing proteins as molecular targets for cancer chemotherapy (3).The interaction between DAG and its receptors is typically mediated by a DAG-responsive motif called a "C1 domain" (4). The highly conserved C1 domain (ϳ50 amino acids) is a cysteine-rich zinc finger structure (5) that was first identified in protein kinase C (PKC) as the interaction site for DAG and the phorbol esters (6). The PKC family of serine/threonine protein kinases comprises the best studied mediators of DAG signaling. 8 of its 11 family members have DAG-responsive C1 domains: (i) the conventional PKCs (␣, ␤I, ␤II, and ␥) and (ii) the novel PKCs (␦, ⑀, , and ). Both the classic and novel PKCs conta...

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Tumor promoter binding of the protein kinase C C1 homology domain peptides of RasGRPs, chimaerins, and Unc13s

Cited by 31 publications

References 32 publications

Crucial Role of Phospholipase Cε in Skin Inflammation Induced by Tumor-Promoting Phorbol Ester

Crucial Role of Phospholipase Cε in Skin Inflammation Induced by Tumor-Promoting Phorbol Ester

Role of chimaerins, a group of Rac-specific GTPase activating proteins, in T-cell receptor signaling

A Novel Diacylglycerol-lactone Shows Marked Selectivity in Vitro among C1 Domains of Protein Kinase C (PKC) Isoforms α and δ as Well as Selectivity for RasGRP Compared with PKCα

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