HongBin Wang

Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of β2-chimaerin, a ‘non-protein kinase C’ phorbol ester receptor

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.

Protein kinase C and cancer: what we know and what we do not

Since their discovery in the late 1970s, protein kinase C (PKC) isozymes represent one of the most extensively studied signaling kinases. PKCs signal through multiple pathways and control the expression of genes relevant for cell cycle progression, tumorigenesis and metastatic dissemination. Despite the vast amount of information concerning the mechanisms that control PKC activation and function in cellular models, the relevance of individual PKC isozymes in the progression of human cancer is still a matter of controversy. Although the expression of PKC isozymes is altered in multiple cancer types, the causal relationship between such changes and the initiation and progression of the disease remains poorly defined. Animal models developed in the last years helped to better understand the involvement of individual PKCs in various cancer types and in the context of specific oncogenic alterations. Unraveling the enormous complexity in the mechanisms by which PKC isozymes have an impact on tumorigenesis and metastasis is key for reassessing their potential as pharmacological targets for cancer treatment.

Phorbol Esters and Related Analogs Regulate the Subcellular Localization of β2-Chimaerin, a Non-protein Kinase C Phorbol Ester Receptor

The novel phorbol ester receptor β2-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, β2-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, β2-chimaerin is translocated by phorbol esters from the cytosolic to particulate fraction. Phorbol esters also induce translocation of α1 (n)- and β1-chimaerins, suggesting common regulatory mechanisms for all chimaerin isoforms. The subcellular redistribution of β2-chimaerin by phorbol esters is entirely dependent on the C1 domain, as revealed by deletional analysis and site-directed mutagenesis. Interestingly, β2-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 β2-chimaerin and PKCα. Strikingly, the mezerein analog thymeleatoxin is not able to translocate β2-chimaerin, although it very efficiently translocates PKCα. Phorbol esters also promote the association of β2-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.

The RacGAP β2-Chimaerin Selectively Mediates Axonal Pruning in the Hippocampus

Axon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) β2-Chimaerin (β2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds β2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. β2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/β2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling.

Downregulation of Rap1GAP Contributes to Ras Transformation

ABSTRACT Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors.

Phospholipase Cγ/diacylglycerol‐dependent activation of β2‐chimaerin restricts EGF‐induced Rac signaling

Although receptor‐mediated regulation of small G‐proteins and the cytoskeleton is intensively studied, the mechanisms for attenuation of these signals are poorly understood. In this study, we have identified the Rac‐GAP β2‐chimaerin as an effector of the epidermal growth factor receptor (EGFR) via coupling to phospholipase Cγ (PLCγ) and generation of the lipid second messenger diacylglycerol (DAG). EGF redistributes β2‐chimaerin to promote its association with the small GTPase Rac1 at the plasma membrane, as determined by FRET. This relocalization and association with Rac1 were impaired by disruption of the β2‐chimaerin C1 domain as well as by PLCγ1 RNAi, thus defining β2‐chimaerin as a novel DAG effector. On the other hand, GAP‐deficient β2‐chimaerin mutants show enhanced translocation and sustained Rac1 association in the FRET assays. Remarkably, RNAi depletion of β2‐chimaerin significantly extended the duration of Rac activation by EGF, suggesting that β2‐chimaerin serves as a mechanism that self‐limits Rac activity in response to EGFR activation. Our results represent the first direct evidence of divergence in DAG signaling downstream of a tyrosine‐kinase receptor via a PKC‐independent mechanism.

Activation of Nuclear Factor-Kappa B (NFκB) in Prostate Cancer is Mediated by PKC Epsilon (PKCϵ)

Background: PKCϵ, a potential oncogene, is up-regulated in prostate cancer. Results: PKCϵ facilitates the formation of TNFR-I complex to regulate the NF-κB pathway via a C1 domain/diacylglycerol-dependent mechanism. Conclusion: PKCϵ is an upstream regulator of NF-κB signaling in prostate cancer. Significance: Mechanisms identified here may reveal novel PKCϵ effectors that contribute to prostate cancer progression and highlight the potential relevance of this pathway for therapeutic purposes. Protein kinase C ϵ (PKCϵ) has emerged as an oncogenic kinase and plays important roles in cell survival, mitogenesis and invasion. PKCϵ is up-regulated in most epithelial cancers, including prostate, breast, and lung cancer. Here we report that PKCϵ is an essential mediator of NF-κB activation in prostate cancer cells. A strong correlation exists between PKCϵ overexpression and NF-κB activation status in prostate cancer cells. Moreover, transgenic overexpression of PKCϵ in the mouse prostate causes preneoplastic lesions that display significant NF-κB hyperactivation. PKCϵ RNAi depletion or inhibition in prostate cancer cells diminishes NF-κB translocation to the nucleus with subsequent impairment of both activation of NF-κB transcription and induction of NF-κB responsive genes in response to the proinflammatory cytokine tumor necrosis factor α (TNFα). On the other hand, PKCϵ overexpression in normal prostate cells enhances activation of the NF-κB pathway. A mechanistic analysis revealed that TNFα activates PKCϵ via a C1 domain/diacylglycerol-dependent mechanism that involves phosphatidylcholine-phospholipase C. Moreover, PKCϵ facilitates the assembly of the TNF receptor-I signaling complex to trigger NF-κB activation. Our studies identified a molecular link between PKCϵ and NF-κB that controls key responses implicated in prostate cancer progression.

p23/Tmp21 Associates with Protein Kinase Cδ (PKCδ) and Modulates Its Apoptotic Function

There is emerging evidence that C1 domains, motifs originally identified in PKC isozymes and responsible for binding of phorbol esters and diacylglycerol, interact with the Golgi/endoplasmic reticulum protein p23 (Tmp21). In this study, we investigated whether PKCδ, a kinase widely implicated in apoptosis and inhibition of cell cycle progression, associates with p23 and determined the potential functional implications of this interaction. Using a yeast two-hybrid approach, we found that the PKCδ C1b domain associates with p23 and identified two key residues (Asp245 and Met266) implicated in this interaction. Interestingly, silencing p23 from LNCaP prostate cancer cells using RNAi markedly enhanced PKCδ-dependent apoptosis and activation of PKCδ downstream effectors ROCK and JNK by phorbol 12-myristate 13-acetate. Moreover, translocation of PKCδ to the plasma membrane by phorbol 12-myristate 13-acetate was enhanced in p23-depleted LNCaP cells. Notably, a PKCδ mutant that failed to interact with p23 triggered a strong apoptotic response when expressed in LNCaP cells. In summary, our data compellingly support the concept that C1 domains have dual roles both in lipid and protein associations and provide strong evidence that p23 acts as an anchoring protein that retains PKCδ at the perinuclear region, thus limiting the availability of this kinase for activation in response to stimuli.

p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

The ER/Golgi protein p23/Tmp21 acts as a C1 domain-docking protein that mediates perinuclear translocation of β-chimaerin. C1 domains from PKC isozymes can also interact with p23/Tmp21. Our study highlights the relevance of C1 domains in protein-protein interactions in addition to their well-established lipid-binding properties.

Transcriptional Regulation of Oncogenic Protein Kinase Cϵ (PKCϵ) by STAT1 and Sp1 Proteins

Background: PKCϵ, a kinase widely implicated in tumorigenesis and metastasis, is overexpressed in many cancers. Results: Transcription factors Sp1 and STAT1 control the expression of PKCϵ in cancer cells. Conclusion: Up-regulation of PKCϵ is mediated by dysregulated transcriptional mechanisms. Significance: Our results may have significant implications for the development of approaches to target PKCϵ and its effectors in cancer therapeutics. Overexpression of PKCϵ, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCϵ expression and its up-regulation in cancer, we cloned an ∼1.6-kb promoter segment of the human PKCϵ gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between −777 and −105 bp (region A) and −921 and −796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions −668/−659 and −269/−247 as well as STAT1 sites in positions −880/−869 and −793/−782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCϵ mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCϵ and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCϵ and its effectors in cancer therapeutics.