Involvement of protein kinase Cdelta/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 (PARP-1) signaling pathway in histamine-induced up-regulation of histamine H1 receptor gene expression in HeLa cells

The histamine H(1) receptor (H1R) gene is up-regulated in patients with allergic rhinitis. However, the mechanism and reason underlying this up-regulation are still unknown. Recently, we reported that the H1R expression level is strongly correlated with the severity of allergic symptoms. Therefore, understanding the mechanism of this up-regulation will help to develop new anti-allergic drugs targeted for H1R gene expression. Here we studied the molecular mechanism of H1R up-regulation in HeLa cells that express H1R endogenously in response to histamine and phorbol 12-myristate 13-acetate (PMA). In HeLa cells, histamine stimulation caused up-regulation of H1R gene expression. Rottlerin, a PKCδ-selective inhibitor, inhibited up-regulation of H1R gene expression, but Go6976, an inhibitor of Ca(2+)-dependent PKCs, did not. Histamine or PMA stimulation resulted in PKCδ phosphorylation at Tyr(311) and Thr(505). Activation of PKCδ by H(2)O(2) resulted in H1R mRNA up-regulation. Overexpression of PKCδ enhanced up-regulation of H1R gene expression, and knockdown of the PKCδ gene suppressed this up-regulation. Histamine or PMA caused translocation PKCδ from the cytosol to the Golgi. U0126, an MEK inhibitor, and DPQ, a poly(ADP-ribose) polymerase-1 inhibitor, suppressed PMA-induced up-regulation of H1R gene expression. These results were confirmed by a luciferase assay using the H1R promoter. Phosphorylation of ERK and Raf-1 in response to PMA was also observed. However, real-time PCR analysis showed no inhibition of H1R mRNA up-regulation by a Raf-1 inhibitor. These results suggest the involvement of the PKCδ/ERK/poly(ADP-ribose) polymerase-1 signaling pathway in histamine- or PMA-induced up-regulation of H1R gene expression in HeLa cells.     

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation

The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers.     

B-MYB positively regulates serine-threonine kinase receptor-associated protein (STRAP) activity through direct interaction

Serine-threonine kinase receptor-associated protein (STRAP) functions as a regulator of both TGF-β and p53 signaling. However, the regulatory mechanism of STRAP activity is not understood. In this study, we report that B-MYB is a new STRAP-interacting protein, and that an amino-terminal DNA-binding domain and an area (amino acids 373-468) between the acidic and conserved regions of B-MYB mediate the B-MYB·STRAP interaction. Functionally, B-MYB enhances STRAP-mediated inhibition of TGF-β signaling pathways, such as apoptosis and growth inhibition, by modulating complex formation between the TGF-β receptor and SMAD3 or SMAD7. Furthermore, coexpression of B-MYB results in a dose-dependent increase in STRAP-mediated stimulation of p53-induced apoptosis and cell cycle arrest via direct interaction. Confocal microscopy showed that B-MYB prevents the normal translocation of SMAD3 in response to TGF-β1 and stimulates p53 nuclear translocation. These results suggest that B-MYB acts as a positive regulator of STRAP.     

RB1CC1 protein positively regulates transforming growth factor-beta signaling through the modulation of Arkadia E3 ubiquitin ligase activity

Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-β signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-β signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-β signaling, and knockdown of endogenous RB1CC1 attenuated TGF-β-induced expression of target genes as well as TGF-β-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-β signaling by restricting substrate specificity of Arkadia.     

Apolipoprotein E3 (ApoE3) but not ApoE4 protects against synaptic loss through increased expression of protein kinase C epsilon

Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid β oligomers (amylospheroids). Specific activators of PKCε, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCε inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCε mRNA and expression of the PKCε protein. Amyloid β specifically blocked the expression of PKCε but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCε pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD.     

Histone deacetylase inhibitor belinostat represses survivin expression through reactivation of transforming growth factor beta (TGFbeta) receptor II leading to cancer cell death

Survivin is a cancer-associated gene that functions to promote cell survival, cell division, and angiogenesis and is a marker of poor prognosis. Histone deacetylase inhibitors induce apoptosis and re-expression of epigenetically silenced tumor suppressor genes in cancer cells. In association with increased expression of the tumor suppressor gene transforming growth factor β receptor II (TGFβRII) induced by the histone deacetylase inhibitor belinostat, we observed repressed survivin expression. We investigated the molecular mechanisms involved in survivin down-regulation by belinostat downstream of reactivation of TGFβ signaling. We identified two mechanisms. At early time points, survivin protein half-life was decreased with its proteasomal degradation. We observed that belinostat activated protein kinase A at early time points in a TGFβ signaling-dependent mechanism. After longer times (48 h), survivin mRNA was also decreased by belinostat. We made the novel observation that belinostat mediated cell death through the TGFβ/protein kinase A signaling pathway. Induction of TGFβRII with concomitant survivin repression may represent a significant mechanism in the anticancer effects of this drug. Therefore, patient populations exhibiting high survivin expression with epigenetically silenced TGFβRII might potentially benefit from the use of this histone deacetylase inhibitor.     

Acridine yellow G blocks glioblastoma growth via dual inhibition of epidermal growth factor receptor and protein kinase C kinases

Amplification of the epidermal growth factor receptor (EGFR), frequently expressed as a constitutively active deletion mutant (EGFRvIII), occurs commonly in glioblastoma multiformes (GBM). However, blockade of EGFR is therapeutically disappointing for gliomas with PTEN deletion. To search for small molecules treating this aggressive cancer, we have established a cell-based screening and successfully identified acridine yellow G that preferentially blocks cell proliferation of the most malignant U87MG/EGFRvIII cells over the less malignant U87MG/PTEN cells. Oral administration of this compound markedly diminishes the brain tumor volumes in both subcutaneous and intracranial models. It directly inhibits EGFR and PKCs with IC(50) values of ~7.5 and 5 μM, respectively. It dually inhibits EGFR and PKCs, resulting in a blockade of mammalian target of rapamycin signaling and cell cycle arrest in the G(1) phase, which leads to activation of apoptosis in the tumors. Hence, combinatorial inhibition of EGFR and PKCs might provide proof of concept in developing therapeutic agents for treating malignant glioma and other human cancers.     

Mammalian Target of Rapamycin Complex 1 (mTORC1) Plays a Role in Pasteurella multocida Toxin (PMT)-induced Protein Synthesis and Proliferation in Swiss 3T3 Cells

Pasteurella multocida toxin (PMT) is a potent mitogen known to activate several signaling pathways via deamidation of a conserved glutamine residue in the α subunit of heterotrimeric G-proteins. However, the detailed mechanism behind mitogenic properties of PMT is unknown. Herein, we show that PMT induces protein synthesis, cell migration, and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (rpS6), in quiescent 3T3 cells. The extent of the phosphorylation is time and PMT concentration dependent, and is inhibited by rapamycin and Torin1, the two specific inhibitors of the mammalian target of rapamycin complex 1 (mTORC1). Interestingly, PMT-mediated mTOR signaling activation was observed in MEF WT but not in Gα_q/11 knock-out cells. These observations are consistent with the data indicating that PMT-induced mTORC1 activation proceeds via the deamidation of Gα_q/11, which leads to the activation of PLCβ to generate diacylglycerol and inositol trisphosphate, two known activators of the PKC pathway. Exogenously added diacylglycerol or phorbol 12-myristate 13-acetate, known activators of PKC, leads to rpS6 phosphorylation in a rapamycin-dependent manner. Furthermore, PMT-induced rpS6 phosphorylation is inhibited by PKC inhibitor, Gö6976. Although PMT induces epidermal growth factor receptor activation, it exerts no effect on PMT-induced rpS6 phosphorylation. Together, our findings reveal for the first time that PMT activates mTORC1 through the Gα_q/11/PLCβ/PKC pathway. The fact that PMT-induced protein synthesis and cell migration is partially inhibited by rapamycin indicates that these processes are in part mediated by the mTORC1 pathway.     

Activity and protein kinase C regulate synaptic accumulation of N-methyl-D-aspartate (NMDA) receptors independently of GluN1 splice variant

NMDA receptors are calcium-permeable ionotropic receptors that detect coincident glutamate binding and membrane depolarization and are essential for many forms of synaptic plasticity in the mammalian brain. The obligatory GluN1 subunit of NMDA receptors is alternatively spliced at multiple sites, generating forms that vary in N-terminal N1 and C-terminal C1, C2, and C2' cassettes. Based on expression of GluN1 constructs in heterologous cells and in wild type neurons, the prevalent view is that the C-terminal cassettes regulate synaptic accumulation and its modulation by homeostatic activity blockade and by protein kinase C (PKC). Here, we tested the role of GluN1 splicing in regulated synaptic accumulation of NMDA receptors by lentiviral expression of individual GluN1 splice variants in hippocampal neurons cultured from GluN1 (-/-) mice. High efficiency transduction of GluN1 at levels similar to endogenous was achieved. Under control conditions, the C2' cassette mediated enhanced synaptic accumulation relative to the alternate C2 cassette, whereas the presence or absence of N1 or C1 had no effect. Surprisingly all GluN1 splice variants showed >2-fold increased synaptic accumulation with chronic blockade of NMDA receptor activity. Furthermore, in this neuronal rescue system, all GluN1 splice variants were equally rapidly dispersed upon activation of PKC. These results indicate that the major mechanisms mediating homeostatic synaptic accumulation and PKC dispersal of NMDA receptors occur independently of GluN1 splice isoform.     

Suppression of latent transforming growth factor (TGF)-beta1 restores growth inhibitory TGF-beta signaling through microRNAs

Cancer cells secreting excess latent TGF-β are often resistant to TGF-β induced growth inhibition. We observed that RNAi against TGF-β1 led to apoptotic death in such cell lines with features that were, paradoxically, reminiscent of TGF-β signaling activity and that included transiently enhanced SMAD2 and AKT phosphorylation. A comprehensive search in Hela cells for potential microRNA drivers of this mechanism revealed that RNAi against TGF-β1 led to induction of pro-apoptotic miR-34a and to a globally decreased oncomir expression. The reduced levels of the oncomirs miR-18a and miR-24 accounted for the observed derepression of two TGF-β1 processing factors, thrombospondin-1, and furin, respectively. Our data suggest a novel mechanism in which latent TGF-β1, thrombospondin 1, and furin form a microRNA-mediated regulatory feedback loop. For cells with high levels of latent TGF-β, this provides a potentially widespread mechanism of escape from TGF-β-mediated growth arrest at the earliest point in the signaling pathway, TGF-β processing.     

Delayed phosphorylation of classical protein kinase C (PKC) substrates requires PKC internalization and formation of the pericentrion in a phospholipase D (PLD)-dependent manner

It was previously demonstrated that sustained activation (30-60 min) of protein kinase C (PKC) results in translocation of PKC α and βII to the pericentrion, a dynamic subset of the recycling compartment whose formation is dependent on PKC and phospholipase D (PLD). Here we investigated whether the formation of the pericentrion modulates the ability of PKC to phosphorylate substrates, especially if it reduces substrate phosphorylation by sequestering PKC. Surprisingly, using an antibody that detects phosphosubstrates of classical PKCs, the results showed that the majority of PKC phosphosubstrates are phosphorylated with delayed kinetics, correlating with the time frame of PKC translocation to the pericentrion. Substrate phosphorylation was blocked by PLD inhibitors and was not observed in response to activation of a PKC βII mutant (F663D) that is defective in interaction with PLD and in internalization. Phosphorylation was also inhibited by blocking clathrin-dependent endocytosis, demonstrating a requirement for endocytosis for the PKC-dependent major phosphorylation effects. Serotonin receptor activation by serotonin showed a similar response to phorbol 12-myristate 13-acetate, implicating a potential role of delayed kinetics in G protein-coupled receptor signaling. Evaluation of candidate substrates revealed that the phosphorylation of the PKC substrate p70S6K kinase behaved in a similar manner. Gradient-based fractionation revealed that the majority of these PKC substrates reside within the pericentrion-enriched fractions and not in the plasma membrane. Finally, proteomic analysis of the pericentrion-enriched fractions revealed several proteins as known PKC substrates and/or proteins involved in endocytic trafficking. These results reveal an important role for PKC internalization and for the pericentrion as key determinants/amplifiers of PKC action.     

Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1 (BmLsd1)

Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via the fatty acid biosynthetic pathway. This pathway is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-amino acid peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets, which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells. PBAN stimulates lipolysis of the stored lipid droplet triacylglycerols (TAGs) and releases the precursor for final modification. PBAN exerts its physiological function via the PG cell-surface PBAN receptor, a G protein-coupled receptor that belongs to the neuromedin U receptor family. The PBAN receptor-mediated signal is transmitted via a canonical store-operated channel activation pathway utilizing Gq-mediated phospholipase C activation (Hull, J. J., Kajigaya, R., Imai, K., and Matsumoto, S. (2007) Biosci. Biotechnol. Biochem. 71, 1993-2001; Hull, J. J., Lee, J. M., Kajigaya, R., and Matsumoto, S. (2009) J. Biol. Chem. 284, 31200-31213; Hull, J. J., Lee, J. M., and Matsumoto, S. (2010) Insect Mol. Biol. 19, 553-566). Little, however, is known about the molecular components regulating TAG lipolysis in PG cells. In the current study we found that PBAN signaling involves phosphorylation of an insect PAT family protein named B. mori lipid storage droplet protein-1 (BmLsd1) and that BmLsd1 plays an essential role in the TAG lipolysis associated with bombykol production. Unlike mammalian PAT family perilipins, however, BmLsd1 activation is dependent on phosphorylation by B. mori Ca(2+)/calmodulin-dependent protein kinase II rather than protein kinase A.     

Specificity and Structure of a High Affinity Activin Receptor-like Kinase 1 (ALK1) Signaling Complex

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.     

CIN85 modulates the down-regulation of Fc gammaRIIa expression and function by c-Cbl in a PKC-dependent manner in human neutrophils

We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-β-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of FcγRIIa signaling.     

Epidermal growth factor receptor (EGFR)-mediated positive feedback of protein-tyrosine phosphatase epsilon (PTPepsilon) on ERK1/2 and AKT protein pathways is required for survival of human breast cancer cells

Increased tyrosine phosphorylation has been correlated with human cancer, including breast cancer. In general, the activation of tyrosine kinases (TKs) can be antagonized by the action of protein-tyrosine phosphatases (PTPs). However, in some cases PTPs can potentiate the activation of TKs. In this study, we have investigated the functional role of PTPε in human breast cancer cell lines. We found the up-regulation and activation of receptor PTPε (RPTPε) in MCF-7 cells and MDA-MB-231 upon PMA, FGF, and serum stimulation, which depended on EGFR and ERK1/2 activity. Diminishing the expression of PTPε in human breast cancer cells abolished ERK1/2 and AKT activation, and decreased the viability and anchorage-independent growth of the cells. Conversely, stable MCF-7 cell lines expressing inducible high levels of ectopic PTPε displayed higher activation of ERK1/2 and anchorage-independent growth. Our results demonstrate that expression of PTPε is up-regulated and activated in breast cancer cell lines, through EGFR, by sustained activation of the ERK1/2 pathway, generating a positive feedback regulatory loop required for survival of human breast cancer cells.     

Regulation of the ATP-sensitive potassium channel subunit, Kir6.2, by a Ca2+-dependent protein kinase C

The activity of ATP-sensitive potassium (K(ATP)) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of K(ATP) channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca(2+)-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity.