Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Beta2-chimaerin, a member of the GTPase-activating proteins for the small GTP-binding protein p21Rac, possesses a single cysteine-rich domain with high homology to those implicated in phorbol ester and diacylglycerol binding in protein kinase C (PKC) isozymes. We have expressed beta2-chimaerin in Sf9 insect cells using the baculovirus expression system and determined that, like PKCs, beta2-chimaerin binds phorbol esters with high affinity in the presence of phosphatidylserine as a cofactor. Scatchard plot analysis using the radioligand [3H]phorbol 12,13-dibutyrate revealed a dissociation constant of 1.9 +/- 0.2 nM for beta2-chimaerin. Likewise, beta2-chimaerin is a high affinity receptor for the bryostatins, a class of atypical PKC activators. A detailed comparison of structure-activity relations using several phorbol ester analogs revealed striking differences in binding recognition between beta2-chimaerin and PKCalpha. Although the diacylglycerol 1-oleoyl-2-acetylglycerol binds with similar potency to both beta2-chimaerin and PKCalpha, the mezerein analog thymeleatoxin has 56-fold less affinity for binding to beta2-chimaerin. To establish whether beta2-chimaerin responds to phorbol esters in cellular systems, we overexpressed beta2-chimaerin in COS-7 cells and monitored its subcellular distribution after phorbol ester treatment. Interestingly, as described previously for PKC isozymes, beta2-chimaerin translocates from cytosolic to particulate fractions as a consequence of phorbol ester treatment. Our results demonstrate that beta2-chimaerin is a novel target for the phorbol ester tumor promoters. The expansion of the family of phorbol ester receptors strongly suggests a potential for the "non-kinase" receptors as cellular mediators of the phorbol ester responses.
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PMID:Beta2-chimaerin is a high affinity receptor for the phorbol ester tumor promoters. 933 26

The members of the chimaerin family of Rac-GTPase-activating proteins possess a single C1 domain with high homology to those present in protein kinase C (PKC) isozymes. This domain in PKCs is involved in phorbol ester and diacylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, beta2-chimaerin, binds phorbol esters with high affinity. In this study we analyzed the properties of beta2-chimaerin as a DAG receptor by using a series of conformationally constrained cyclic DAG analogues (DAG lactones) as probes. We identified analogs that bind to beta2-chimaerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycerol. The potencies of these analogs approach those of the potent phorbol ester tumor promoters. The different DAG lactones show some selectivity for this novel receptor compared with PKCalpha. Cellular studies revealed that these DAG analogs induce translocation of beta2-chimaerin from cytosolic (soluble) to particulate fractions. Using green fluorescent protein-fusion proteins for beta2-chimaerin we determined that this novel receptor translocates to the perinuclear region after treatment with DAG lactones. Binding and translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of beta2-chimaerin and the C1b domain of PKCdelta also was confirmed by modeling analysis. Our results demonstrate that beta2-chimaerin is a high affinity receptor for DAG through binding to its C1 domain and supports the emerging concept that multiple pathways transduce signaling through DAG and the phorbol esters.
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PMID:beta2-chimaerin is a novel target for diacylglycerol: binding properties and changes in subcellular localization mediated by ligand binding to its C1 domain. 1051 40

The novel phorbol ester receptor beta2-chimaerin is a Rac-GAP protein possessing a single copy of the C1 domain, a 50-amino acid motif initially identified in protein kinase C (PKC) isozymes that is involved in phorbol ester and diacylglycerol binding. We have previously shown that, like PKCs, beta2-chimaerin binds phorbol esters with high affinity in a phospholipid-dependent manner (Caloca, M. J., Fernandez, M. N., Lewin, N. E., Ching, D., Modali, R., Blumberg, P. M., and Kazanietz, M. G. (1997) J. Biol. Chem. 272, 26488-26496). In this paper we report that like PKC isozymes, beta2-chimaerin is translocated by phorbol esters from the cytosolic to particulate fraction. Phorbol esters also induce translocation of alpha1 (n)- and beta1-chimaerins, suggesting common regulatory mechanisms for all chimaerin isoforms. The subcellular redistribution of beta2-chimaerin by phorbol esters is entirely dependent on the C1 domain, as revealed by deletional analysis and site-directed mutagenesis. Interestingly, beta2-chimaerin translocates to the Golgi apparatus after phorbol ester treatment, as revealed by co-staining with the Golgi marker BODIPY-TR-ceramide. Structure relationship analysis of translocation using a series of PKC ligands revealed substantial differences between translocation of beta2-chimaerin and PKCalpha. Strikingly, the mezerein analog thymeleatoxin is not able to translocate beta2-chimaerin, although it very efficiently translocates PKCalpha. Phorbol esters also promote the association of beta2-chimaerin with Rac in cells. These data suggest that chimaerins can be positionally regulated by phorbol esters and that each phorbol ester receptor class has distinct pharmacological properties and targeting mechanisms. The identification of selective ligands for each phorbol ester receptor class represents an important step in dissecting their specific cellular functions.
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PMID:Phorbol esters and related analogs regulate the subcellular localization of beta 2-chimaerin, a non-protein kinase C phorbol ester receptor. 1127 94

N-Benzyladriamycin-14-valerate (AD 198) is a semisynthetic anthracycline with experimental antitumor activity superior to that of doxorubicin (DOX). AD 198, unlike DOX, only weakly binds DNA, is a poor inhibitor of topoisomerase II, and circumvents anthracycline-resistance mechanisms, suggesting a unique mechanism of action for this novel analogue. The phorbol ester receptors, protein kinase C (PKC) and beta2-chimaerin, were recently identified as selective targets for AD 198 in vitro. In vitro, AD 198 competes with [3H]PDBu for binding to a peptide containing the isolated C1b domain of PKC-delta (deltaC1b domain). In the present study molecular modeling is used to investigate the interaction of AD 198 with the deltaC1b domain. Three models are identified wherein AD 198 binds into the groove formed between amino acid residues 6-13 and 21-27 of the deltaC1b domain in a manner similar to that reported for phorbol-13-acetate and other ligands of the C1 domain. Two of the identified models are consistent with previous experimental data demonstrating the importance of the 14-valerate side chain of AD 198 in binding to the C1 domain as well as current data demonstrating that translocation of PKC-alpha to the membrane requires the 14-valerate substituent. In this regard, the carbonyl of the 14-valerate participates in hydrogen bonding to the deltaC1b while the acyl chain is positioned for stabilization of the membrane-bound protein-ligand complex in a manner analogous to the acyl chains of the phorbol esters. These studies provide a structural basis for the interaction of AD 198 with the deltaC1b domain and a starting point for the rational design of potential new drugs targeting PKC and other proteins with C1 domains.
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PMID:Molecular models of N-benzyladriamycin-14-valerate (AD 198) in complex with the phorbol ester-binding C1b domain of protein kinase C-delta. 1129 49

Anthracycline antibiotics like doxorubicin (DOX) are known to exert their antitumor effects primarily via DNA intercalation and topoisomerase II inhibition. By contrast, the noncross-resistant cytoplasmically localizing DOX analogue, N-benzyladriamycin-14-valerate (AD 198), only weakly binds DNA and does not inhibit topoisomerase II, yet it displays superior antitumor activity, strongly suggesting a distinct cytotoxic mechanism. In recent modeling studies, we reported a structural similarity between AD 198 and commonly accepted ligands for the C1-domain of protein kinase C (PKC), and we hypothesized that the unique biological activity of AD 198 may derive, in part, through this kinase. Consistent with this hypothesis, the present biochemical studies demonstrate that AD 198 competes with [3H]phorbol-12,13-dibutyrate ([3H]PDBu) for binding to phorbol-responsive PKC isoforms, the isolated C1b domain of PKC-delta (delta C1b), and the nonkinase phorbol ester receptor, beta2-chimaerin. In NIH/3T3 cells, AD 198 competitively blocks PKC activation by C1-ligands. Importantly, neither DOX nor N-benzyladriamycin, the principal AD 198 metabolite, inhibits basal or phorbol-stimulated PKC activity or appreciably competes for [3H]PDBu binding. In CEM cells, structure activity studies with 14-acyl congeners indicate that the rapid induction of apoptosis correlates with competition for [3H]PDBu binding, strongly implicating phorbol-binding proteins in drug activity. Collectively, these studies support the conclusion that AD 198 is a C1-ligand and that C1-ligand receptors are selective drug targets. These studies provide the impetus for continuing efforts to understand the molecular basis for the unique biological activity of AD 198 and provide for the design of analogues with improved affinity for C1-domains and potentially greater antitumor activity.
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PMID:Interaction of the novel anthracycline antitumor agent N-benzyladriamycin-14-valerate with the C1-regulatory domain of protein kinase C: structural requirements, isoform specificity, and correlation with drug cytotoxicity. 1247 66

The biological and functional properties of beta2-chimaerin, a novel phorbol ester/diacylglycerol receptor unrelated to protein kinase C isozymes, are largely unknown. It has previously been established that beta2-chimaerin accelerates the hydrolysis rate of GTP from Rac1 in vitro, leading to the inactivation of this GTPase, which plays important roles in the control of actin cytoskeleton organization, proliferation, motility, and invasiveness. To explore the potential role of beta2-chimaerin in invasion and metastasis, we generated stable transfectants for its catalytic domain (the beta-GAP domain) in F3II murine mammary carcinoma cells. Reduced Rac-GTP levels were observed upon stimulation with epidermal growth factor in the beta-GAP clones compared with control cells. Moreover, a marked alteration in actin polymerization in response to epidermal growth factor was observed in the beta-GAP clones, suggesting impairment of Rac-dependent responses. The beta-GAP transfectants also evidenced slower growth rates and a striking reduction in their migratory properties. Adenoviral delivery of the beta-GAP domain into F3II cells also led to reduced proliferative and migratory responses. Importantly, significant differences were found between beta-GAP transfectants and control cells regarding their tumorigenic and metastatic properties after s.c. inoculation in syngeneic BALB/c mice. Tumors originating from beta-GAP transfectants showed a significantly lower growth rate and reduced invasive ability; in addition, a lower incidence of spontaneous lung metastases was observed. Our results indicate that beta2-chimaerin impairs key steps in the metastatic cascade and provide evidence for a rational modulation of the Rac signaling pathway in cancer treatment.
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PMID:Inhibition of aggressiveness of metastatic mouse mammary carcinoma cells by the beta2-chimaerin GAP domain. 1272 51

The regulation and function of beta2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to beta2-chimaerin with high affinity and promote its subcellular distribution. beta2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. beta2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the beta2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of beta2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor beta2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.
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PMID:Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of beta2-chimaerin, a 'non-protein kinase C' phorbol ester receptor. 1287 55

Chimaerins are high affinity receptors for phorbol esters and diacylglycerol (DAG), unrelated to the protein kinase C isozymes. These receptors have deep implications in tumour biology since they regulate the activity of Rac1, a small GTP-binding protein of the Ras superfamily. It has been demonstrated that chimaerins have GTPase activating protein (GAP) activity, leading to the acceleration of GTP hydrolysis from Rac1 and therefore facilitating the transition to its inactive state. Rac regulates various cellular events, including gene transcription, cell cycle, adhesion and migration. It has also been described that Rac is implicated in the intracellular response to the binding of specific extracellular matrix proteins to integrin receptors. In this work, we analysed cell morphology, actin cytoskeleton reorganisation and metalloprotease (MMP) secretion in response to matrix proteins in mouse mammary carcinoma cells transfected with the beta2-chimaerin GAP domain. Overexpression of beta2-chimaerin induced important cytoskeletal rearrangements in response to matrix stimuli. Transfectant cells also showed activation of MMP-9 activity after stimulation with collagen IV and epidermal growth factor. The restitution of normal Rac levels by beta2-chimaerin activity induced an increase in the sensitivity of tumour cells to extracellular factors, suggesting a regression of the malignant phenotype.
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PMID:Role of beta2-chimaerin in the behaviour of murine mammary carcinoma cells in response to extracellular matrix components. 1558 33

Recent structural analysis of crystalline beta2-chimaerin shows the central protein kinase C-like, diacylglycerol (DAG)-binding C1 domain to be masked by its intramolecular interactions with the N-terminal SH2 and GAP domains, and linker regions. A mechanism of activation has been derived from modelling of a GAP-Rac GTPase complex--the auto-inhibitory constraints are released via membrane engagement, unmasking the C1 domain to enable DAG binding and subsequent GAP stimulation of Rac GTPase catalytic activity.
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PMID:C1, see them all. 1581 91

The proliferation and migration of vascular smooth muscle cells (SMC) are important aspects of atherogenesis. Activated growth factor signaling in injured vessels subsequently promotes a number of intracellular events resulting in the phenotypic modulation of SMC. Here, we investigated the role of beta2-chimaerin, a non-protein kinase C phorbol ester receptor with Rac-GTPase-activating protein activity, in growth factor-stimulated SMC. The endogenous expression of beta2-chimaerin was detected in cultured human SMC by reverse transcription-polymerase chain reaction and immunohistochemistry. Next, the overexpression of HA-tagged wild-type human beta2-chimaerin was attempted using cultured rat SMC with a recombinant adenovirus (Adv-beta2-Chim). Adv-LZ encoding beta-galactosidase (LacZ) was used as the control. The proliferation of SMC stimulated by platelet-derived growth factor (PDGF-BB, 10 ng/ml), as measured by cell-counting and 5-bromo-2'deoxyuridine incorporation assay, was suppressed by infection with Adv-beta2-Chim (50-200 MOI), but not with control viruses. PDGF-induced SMC migration was inhibited by approximately 25% after infection with Adv-beta2-Chim (200 MOI) using a modified Boyden's chamber assay with a fibronectin-coated membrane. Confocal microscopy revealed that PDGF stimulation altered the sub-cellular localization of beta2-chimaerin. The administration of 12-O-tetradecanoyl phorbol 13-acetate also induced changes in the sub-cellular localization of beta2-chimaerin, which was not affected by a presence of the PKC inhibitor (GF109203X). Finally, PDGF-induced Rac1 activation was found to be inhibited in the Adv-beta2-Chim-infected cells. Thus, we demonstrated that beta2-chimaerin regulates the proliferation and migration of SMC downstream of growth factor signaling pathway via the regulation of Rac1 activity. The signaling mediated by beta2-chimaerin may play a role in the regulation of SMC phenotypes, thereby implicating human atherogenesis.
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PMID:Regulation of vascular smooth muscle proliferation and migration by beta2-chimaerin, a non-protein kinase C phorbol ester receptor. 1652 10


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