The nuclear pore proteins Nup88/214 and T-cell acute lymphatic leukemia-associated NUP214 fusion proteins regulate Notch signaling.

The Notch receptor is a key mediator of developmental programs and cell-fate decisions. Imbalanced Notch signaling leads to developmental disorders and cancer. To fully characterize the Notch signaling pathway and exploit it in novel therapeutic interventions, a comprehensive view on the regulation and requirements of Notch signaling is needed. Notch is regulated at different levels, ranging from ligand binding, stability to endocytosis. Using an array of different techniques, including reporter gene assays, immunocytochemistry, and ChIP-qPCR we show here, to the best of our knowledge for the first time, regulation of Notch signaling at the level of the nuclear pore. We found that the nuclear pore protein Nup214 (nucleoporin 214) and its interaction partner Nup88 negatively regulate Notch signaling in vitro and in vivo in zebrafish. In mammalian cells, loss of Nup88/214 inhibited nuclear export of recombination signal-binding protein for immunoglobulin κJ region (RBP-J), the DNA-binding component of the Notch pathway. This inhibition increased binding of RBP-J to its cognate promoter regions, resulting in increased downstream Notch signaling. Interestingly, we also found that NUP214 fusion proteins, causative for certain cases of T-cell acute lymphatic leukemia, potentially contribute to tumorigenesis via a Notch-dependent mechanism. In summary, the nuclear pore components Nup88/214 suppress Notch signaling in vitro, and in zebrafish, nuclear RBP-J levels are rate-limiting factors for Notch signaling in mammalian cells, and regulation of nucleocytoplasmic transport of RBP-J may contribute to fine-tuning Notch activity in cells.

The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels.

Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Nav), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Nav1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Cav2.1 and of Kv3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Nav1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs.

Myofibroblasts have an impact on expression, dimerization and signaling of different ErbB receptors in OSCC cells.

INTRODUCTION: Receptors of the ErbB family belong to the key players in cancer development and are targets of several therapeutic approaches. Their functional dependency on the tumor microenvironment, especially on CAFs is albeit still poorly understood. Our objective was to investigate the impact of CAF secretome on ErbB receptor expression and signaling behavior in OSCC. METHODS: Stimulation of PE/CA-PJ15 OSCC cells with conditioned media of TGF-ß1-activated fibroblasts was used as model system for CAF to cancer cell communication. Thereby costimulation with inhibitors against matrix metalloproteinases (MMPs), epidermal growth factor receptor (EGFR), MAPK/ERK kinase (MEK), phosphoinositide-3 kinase (PI3-K), signal transducer and activator of transcription 3 (Stat3) or knockdown of Her3 by siRNA was utilized for detailed investigation of the expression, dimerization and signaling pattern of ErbB in western blot and coimmunoprecipitation. RESULTS: Our results show that soluble factors in activated fibroblast secretome stimulate metalloproteinase activity in the membrane of cancer cells. Thereby ligands are released that activate EGFR and subsequently upregulates EGFR expression via the STAT3 pathway. Simultaneously, the expression of PKCɛ was enhanced via a PI3-kinase/Akt-mediated pathway and a negative feedback regulation loop on EGFR downstream signaling generated. Furthermore, the activated fibroblasts secretome stimulated the highly oncogenic hetero-dimerization between HER3 and p95HER2. That protein association is inversely dependent on the expression level of HER3. CONCLUSIONS: Our results demonstrate that the activated fibroblasts secretome can induce a counterbalanced regulation of protein expression, downstream signaling and the dimerization patterns of different ErbB receptor subtypes in the cancer cell. Thus, the combinatorial targeting of CAFs and selective ErbB receptor subtype inhibitors may provide a useful approach in cancer therapy.

The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors.

Disrupted-in-Schizophrenia 1 (DISC1) is necessary for the correct migration of cortical interneurons.

Disrupted-in-Schizophrenia 1 (DISC1) is a prominent susceptibility gene for major psychiatric disorders. Previous work indicated that DISC1 plays an important role during neuronal proliferation and differentiation in the cerebral cortex and that it affects the positioning of radial migrating pyramidal neurons. Here we show that in mice, DISC1 is necessary for the migration of the cortical interneurons generated in the medial ganglionic eminence (MGE). RT-PCR, in situ hybridizations, and immunocytochemical data revealed expression of DISC1 transcripts and protein in MGE-derived cells. To study the possible functional role of DISC1 during tangential migration, we performed in utero and ex utero electroporation to suppress DISC1 in the MGE in vivo and in vitro. Results indicate that after DISC1 knockdown, the proportion of tangentially migrating MGE neurons that reached their cortical target was strongly reduced. In addition, there were profound alterations in the morphology of DISC1-deficient neurons, which exhibited longer and less branched leading processes than control cells. These findings provide a possible link between clinical studies reporting alterations of cortical interneurons in schizophrenic patients and the current notion of schizophrenia as a neurodevelopmental disorder.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Activation by tyrosine phosphorylation as a prerequisite for protein kinase Cζ to mediate epidermal growth factor receptor signaling to ERK.

The atypical protein kinase Czeta (PKCzeta) was recently shown to mediate epidermal growth factor (EGF)-induced activation of extracellular signal-regulated kinase (ERK) in head and neck squamous carcinoma (HNSCC) cells. Here, it is shown that EGF may induce tyrosine phosphorylation of PKCzeta in several HNSCC cells, breast carcinoma cells, as well as mouse embryonic fibroblasts. In COS-7 cells overexpressing EGF receptor (EGFR) and PKCzeta as a tumor cell model, we show that PKCzeta tyrosine phosphorylation by EGF is induced by catalytic activation. Using a loss-of-function mutant of PKCzeta, we can show that the tyrosine residue 417 in PKCzeta plays an important role in both PKCzeta activation and the ability of PKCzeta to mediate activation of ERK. The importance of PKCzeta in EGF-induced ERK activation can also be shown in several HNSCC and breast carcinoma cell lines as well as in PKCzeta-deficient mouse embryonic fibroblasts. In addition, we present several lines of evidence suggesting the physical association of PKCzeta with EGFR and the importance of the EGFR tyrosine kinase c-Src and the Src-specific phosphorylation site pY845-EGFR in the tyrosine phosphorylation as well as catalytic activation of PKCzeta. This study characterizes PKCzeta as a novel mitogenic downstream mediator of EGFR and indicates PKCzeta as a therapeutic target in some carcinomas.

Masking of transmembrane-based retention signals controls ER export of gamma-secretase.

gamma-Secretase is critically involved in the Notch pathway and in Alzheimer's disease. The four subunits of gamma-secretase assemble in the endoplasmic reticulum (ER) and unassembled subunits are retained/retrieved to the ER by specific signals. We here describe a novel ER-retention/retrieval signal in the transmembrane domain (TMD) 4 of presenilin 1, a subunit of gamma-secretase. TMD4 also is essential for complex formation, conferring a dual role for this domain. Likewise, TMD1 of Pen2 is bifunctional as well. It carries an ER-retention/retrieval signal and is important for complex assembly by binding to TMD4. The two TMDs directly interact with each other and mask their respective ER-retention/retrieval signals, allowing surface transport of reporter proteins. Our data suggest a model how assembly of Pen2 into the nascent gamma-secretase complex could mask TMD-based ER-retention/retrieval signals to allow plasma membrane transport of fully assembled gamma-secretase.

Endoplasmic reticulum retention of the gamma-secretase complex component Pen2 by Rer1.

gamma-Secretase is involved in the production of amyloid beta-peptide, which is the principal component of amyloid plaques in the brains of patients with Alzheimer disease. gamma-Secretase is a complex composed of presenilin (PS), nicastrin, anterior pharynx-defective phenotype 1 (Aph1) and PS enhancer 2 (Pen2). We previously proposed a mechanism of complex assembly by which unassembled subunits are retained in the endoplasmic reticulum (ER) and only the fully assembled complex is exported from the ER. We have now identified Retention in endoplasmic reticulum 1 (Rer1) as a protein that is involved in the retention/retrieval of unassembled Pen2 to the ER. Direct binding of unassembled Pen2 to Rer1 is mediated by the first transmembrane domain of Pen2, and a conserved asparagine in this domain is required. Downregulation of Rer1 leads to increased surface localization of Pen2, whereas overexpression of Rer1 stabilizes unassembled Pen2. To our knowledge, Rer1 is the first identified interaction partner of mammalian transmembrane-based retention/retrieval signals.

Protein kinase Cepsilon may act as EGF-inducible scaffold protein for phospholipase Cgamma1.

Phospholipase Cgamma1 (PLCgamma1) represents a major downstream signalling component of the epidermal growth factor (EGF) receptor (EGFR) and is activated by tyrosine phosphorylation. Here we show for the first time that cellular knockdown of protein kinase Cepsilon (PKCepsilon) leads to decreased activation of PLCgamma1 by EGF and that EGF induces tyrosine phosphorylation of PKCepsilon as well as association of PKCepsilon with both EGFR and PLCgamma1. Using several mutants, co-immunoprecipitation and phosphopeptide-based pull-down experiments we found that in dependency on c-Src and EGF-stimulation PKCepsilon may bind to the c-Src-specific phosphorylation site pY845-EGFR. Furthermore, we identified a single tyrosine residue, PKCepsilon-Y573, within a consensus binding sequence of the C-terminal SH2 domain of PLCgamma1 which is critical for both tyrosine phosphorylation of PKCepsilon and its association with PLCgamma1. Thus, in particular cells and independent of the kinase activity PKCepsilon may form a signalling module with EGFR and PLCgamma1. Thereby the tyrosine phosphorylation of PLCgamma1 via the EGFR may be facilitated. This is a novel function of PKCepsilon upstream of PLCgamma1 and a novel paradigm for the EGF-induced formation of multi-protein complexes.

Ligand-independent and EGF receptor-supported transactivation: lessons from beta2-adrenergic receptor signalling.

Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) is currently understood to be mediated by matrix metalloproteases (MMPs) and the release of EGF-like ligands. This ligand-mediated process also suggests that downstream of EGFR the signalling in response to GPCR ligands or EGF appears to be indistinguishable. Here we provide evidence that transactivation of EGFR by the beta2-adrenergic receptor (beta2-AR) is independent of MMPs and results in an incomplete downstream signalling involving extracellular signal-activated kinase (ERK) but not PLCgamma1 and Akt. In contrast, beta2-AR has the ability to activate PLCgamma1 when the EGFR is primed either by co-stimulation with EGF or by increased basal activity due to over-expression. In that way but not via the beta2-AR-mediated transactivation the EGFR docking sites pY992 and pY1173 may be generated which are critical for PLCgamma1. This EGFR-supported transactivation is strongly dependent on EGFR tyrosine kinase, c-Src, and the c-Src-specific EGFR tyrosine residue 845 and represents a novel paradigm of EGFR transactivation.

Activated EGF receptor may balance ERK-inhibitory network signalling pathways.

In the COS-7 cell signalling network high levels of cAMP produced, for example, by co-stimulation of beta2-adrenergic receptor (beta2-AR) and bradykinin B2 receptor (BKR) may affect epidermal growth factor receptor (EGFR)-mediated activation of extracellular signal-stimulated kinase (ERK). In contrast, co-stimulation of either beta2-AR or B2R with EGFR leads to synergistic activation of ERK. Due to triple stimulation of these receptors the synergistic effects on ERK activation as well as cAMP accumulation are diminished. Here we demonstrate that EGF is capable of inducing Src-mediated phosphorylation of the tyrosine residues 177 and 347 of BKR. Their replacement by phenylalanine led to BKR mutants which are unable to activate the cAMP pathway. Using these mutants we can show that EGF attenuates but does not completely inhibit the BKR/cAMP pathway which is counteracting the EGFR signalling to ERK. Our findings suggest that the EGFR may control the cellular network rather by balancing mechanisms then by switch on/off reactions.

Muscarinic M2 receptors mediate transactivation of EGF receptor through Fyn kinase and without matrix metalloproteases.

Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) has been attributed to the activation of matrix metalloproteases (MMPs) and the release of EGF family ligands such as HB-EGF. This mode of transactivation leads to signalling downstream of EGFR which is indistinguishable from that induced by the ligand. Here we provide evidence that in the COS-7 cell model EGFR transactivation via the muscarinic M2 receptor (M2R) is independent of MMPs and results in an incomplete EGFR signalling including ERK and Akt but not PLCgamma1. Using dominant-negative mutants of c-Src and Fyn and Src-deficient SYF cells as well as by co-immunoprecipitation studies, we can demonstrate that the M2R-mediated transactivation of EGFR specifically involves Fyn but not c-Src or Yes. This specific role of Fyn can be verified in SH-SY5Y human neuroblastoma cells with endogenously expressed M2 receptors.

Silencing of retroviral vector transduced LacZ reporter gene by frameshift mutation.

Moloney murine leukemia virus-based vector expressing Escherichia coli beta-galactosidase (lacZ) as reporter gene and the transposon Tn5 neomycin resistance (neo) gene was transduced at low-multiplicity of infections into NIH 3T3 cells. Geneticin (G418)-resistant cells were recloned and cell lines containing beta-galactosidase positive or beta-galactosidase negative cells were obtained. Both positive and negative cell lines contained a single proviral copy at distinct integration sites. RNA complementary to lacZ was detected in beta-galactosidase positive as well as in one of three investigated beta-galactosidase negative cell lines. DNA sequence analysis of proviral LacZ gene in beta-galactosidase negative cell line C6 showed a single nucleotide insertion at position 1567 resulting in reading frame shift and translational stop codon at position 1629. This mutation explains the enzyme inactivation. The absence of beta-galactosidase after retroviral transduction of LacZ reproter gene may be a consequence of definite mutation but not a consequence of ineffective transduction or transcriptional inactivation of transgene.