The mechanism of serotonin 1A receptor (5-HT1A-R) mediated activation of NF-κB has been studied in non-neural cells, but this pathway has not been elucidated in neuronal cells. We have used inducible expression of the 5-HT1A-R in the hippocampal neuron-derived HN2 cells to analyze the coupling of this receptor to NF-κB. A construct harboring luciferase cDNA driven by a minimal promoter under the control of an NF-κB-specific enhancer element was transfected into these 5-HT1A-R-expressing HN2 cells. Using luciferase expression in the transfected cells, we have studied 5-HT1A-R agonist evoked activation of NF-κB. Inhibition of either mitogen activated protein kinase (MAPK) pathway with PD98059 or protein kinase C (PKC) with GFX caused elevation of the basal level of NF-κB activity but did not affect 5-HT1A-R mediated activation of NF-κB. Furthermore, neither the basal level of NF-κB nor its activation by a 5-HT1A-R agonist was altered by dibutyrylcAMP. Thus, the MAPK pathway and PKC cause inhibition of the basal NF-κB activity and the 5-HT1A-R-linked NF-κB activation does not require MAPK, PKC, and cAMP. Intriguingly, Western blot analysis showed that 5-HT1A-R mediates activation of both CaMKII and PI-3K. This 5-HT1A-R-evoked the stimulation of CaMKII was reversed in the presence of a PI-3K inhibitor. Therefore, the likely mechanism of 5-HT1A-R mediated induction of NF-κB in neuronal cells involves activation of PI-3K upstream of CaMKII. This reveals a novel pathway that could be crucial in the functional activity of brain neurons.

The successful division of the cell depends on several morphological events that must be coordinated in a cell cycle dependent fashion. Crucial to this process is the remodeling of the cytoskeleton to both establish the bipolar spindle during mitosis, and cleave the cell into two daughters during cytokinesis. From the standpoint of the cytoskeleton, this process begins during interphase with the duplication of the centrosome; it is the two daughter centrosomes that will assemble the poles of the mitotic spindle and establish its necessary bipolarity. Following the cell cycle transition into mitosis, the spindle must assemble in order to properly align the sister chromatids at the center of the cell, and release the “wait anaphase checkpoint”. As the spindle transports the disjoined sister chromatids to the spindle poles, the cell must rapidly undergo cytokinesis, cleaving the cell into two – the plane of cleavage being established by the spindle itself. Understanding the temporal regulation and molecular basis for these events has come from extensive experiments using a variety of model systems, and has benefited from cell biological, molecular genetic, and biophysical approaches. One of the earliest and most important model systems for studying mitosis is the sea urchin zygote. With their large size, rapid/synchronous cell cycles, and advantages for conduct of biochemical and cytological investigations on the same system, fertilized sea urchin eggs have revealed many of the fundamental properties of centrosome duplication, cell division and cytokinesis. Here we review several key studies that have utilized the sea urchin zygote to explore mechanisms that coordinate and drive two of the major cytoskeletal events of mitotic division – centrosome duplication and cytokinesis.

Membrane fusion is important in many cell processes including membrane trafficking, mitotic reconstitution of organelles, viral infection and fertilization. Several fusion events occur just prior and subsequent to fertilization in the sea urchin, including the sperm acrosomal reaction, fusion of sperm and egg plasma membranes, exocytosis of cortical granules, reassembly of the sperm nuclear envelope and fusion of the male and female pronuclear envelopes leading to the zygote nucleus of the one-cell embryo. The study of male pronuclear membrane dynamics with cell-free extracts of fertilized sea urchin eggs has revealed several novel features, in particular a structural role arising from altering phospholipids prior to nuclear membrane formation. Fusion of chromatin-bound membrane vesicles in vitro can be triggered by either GTP hydrolysis or exogenous phosphatidyl inositol phospholipase C (PI-PLC). Recent data strongly implicate a role for diacylglycerol in nuclear envelope formation as a structural destabilizing lipid in membrane fusion. Moreover, the endogenous enzyme, PI-PLCγ, is >100-fold enriched in a nuclear envelope precursor vesicle population (MV1) that is required for nuclear envelope assembly. NMR and mass spectrometry analyses show that MV1 contains high levels of phosphoinositides, including the substrate of PLCγ, compared to the other nuclear envelope precursor membranes. MV1 exists in eggs as vesicles in the cortex distinct from the endoplasmic reticulum which contributes most of the nuclear envelope membrane. PLCγ is activated by a tyrosine kinase in response to GTP hydrolysis at an early stage of nuclear envelope formation suggesting a role in initiation of fusion and revealing aspects of a signaling mechanism leading to fusion. The binding of MV1 to two poles of the sperm nucleus offers spatial as well as temporal control of the initiation phase.

Secondary axis specification is a process that relies on asymmetric nuclearization of transcription factors in flies and vertebrates, such that the crucial factor is nuclear and therefore functional only in cells along one side of the embryo. In vertebrates, this transcription factor is β-catenin, which is canonically activated downstream from Wnt signals. However, the sea urchin uses asymmetric β-catenin nuclearization during specification of the primary animal-vegetal axis, rather than the secondary oral-aboral (OA) axis. OA specification relies instead on the asymmetric localization of p38 MAPK, a signaling kinase that directly modulates transcription factor activity. A number of genes are expressed in the oral territory downstream from p38, including Nodal and Goosecoid, both of which are associated with secondary axis specification in vertebrate embryos. Because the p38 asymmetry is the earliest known event in the specification of the OA axis, an outstanding question concerns identifying the apparatus upstream from p38 that regulates its asymmetrical activity. Intriguingly, this may be controlled by reactive oxygen species released from the mitochondria, which are asymmetrically distributed about the OA axis.

The signaling networks controlling calcium release and cortical granule exocytosis at fertilization are complex and multilayered, providing various points for regulatory input and quality control. Though it is clear that many of the mechanisms leading to both calcium release and cortical granule exocytosis are conserved, a great deal of variability exists between homologous signaling pathways in different species. The signaling pathways responsible for the release of calcium seen at fertilization vary from species to species, yet they center around the importance of IP3-mediated signaling. Similarly, while there are differences in the mechanisms of regulated secretion between species and between intracellular membrane trafficking events, particularly with respect to time and space, all seem to be dependent on the SNARE proteins and their regulator and effector proteins. What has been most helpful in these studies is the convergence of studies from many different species of eggs. With the amazing divergence of reproductive processes and mechanisms that exists throughout phylogeny, it is comforting to see such strong overlapping roles of key players in widely disparate eggs.

Early development of animal embryos involves establishing axial polarities that specify the anlage of major tissues in a 3-dimensional pattern. Cell fates are specified on this coordinate system through a combination of differential inheritance of maternal regulatory molecules and signaling interactions among cells. Correct patterning of cell fates along the primary axis of the sea urchin embryo depends on tightly regulating the ratio of activities of two nuclear regulatory proteins, SoxB1 and nuclear β–catenin. The latter acts at the top of the gene regulatory network that specifies mesoderm and endoderm and activates, directly or indirectly, signaling by Delta, Wnt8 and Nodal. In contrast, SoxB1 initially accumulates in all nuclei but is progressively eliminated from presumptive mesoderm and endoderm by β-catenin-dependent transcriptional repression and by localized protein turnover, a novel pathway acting downstream of canonical Wnt signaling. A precise temporal program for SoxB1 down regulation is crucial for endomesoderm development because SoxB1 interferes with β–catenin's transcriptional regulatory function. The mechanisms we are beginning to understand that govern the β–catenin-SoxB1 antagonism in sea urchin embryos are likely to have broad significance, since Sox factors are involved in regulating many developmental processes in many deuterostome embryos.

Pattern formation along the sea urchin A-V axis is initiated by the selective activation of the canonical Wnt signaling pathway in vegetal blastomeres. Activation of this pathway is essential for deployment of the endomesoderm gene regulatory network (EGRN), and for pattern formation along the entire A-V axis. During early embryogenesis the canonical Wnt signaling pathway is selectively activated by Dishevelled (Dsh), a critical activator of the Wnt pathway. Dsh is highly enriched in vesicular structures at the vegetal pole in eggs and early embryos, and selective activation of this protein leads to the nuclearization of β-catenin in the endomesoderm. Following activation of canonical Wnt signaling by Dsh, signaling by β-catenin and the Lef/Tcf transcription factors regulates endomesoderm specification by activating the EGRN. One critical early target of nuclear β-catenin is Wnt8, which is selectively expressed in the micromeres at the 16-cell stage and in the macromeres one cleavage division later. Wnt8 signaling is not required for the endomesoderm-inducing activity of the micromeres, but this protein regulates primary mesenchyme cell differentiation. Within the endomesodermal domain Wnt8 regulates the later specification of endoderm and mesoderm. These results have highlighted the important role of the canonical Wnt signaling pathway in patterning the A-V axis in the sea urchin embryo, and have strongly suggested that this axis is initially specified by a cytoplasmic/cytoarchitectural mechanism to activate Dsh in vegetal blastomeres. Additionally, this work along with work in vertebrates and cnidarians has shown that the canonical Wnt pathway plays a conserved role in early pattern formation in metazoan embryos.

The ectoenzyme dipeptidyl peptidase IV (DP IV; E.C. 3.4.14.5; CD26) plays a crucial role in T cell activation, representing a new type of costimulatory T cell structure. Effectors of DP IV-like activity, including naturally occurring and specific synthetic inhibitors, suppress DNA synthesis as well as cytokine production (IL-2, IL-10, IL-12, IFN-γ) of stimulated T cells. These effects are in part caused by the immunosuppressive cytokine TGF-β1, leading to blockade of the cell cycle at the restriction point G1/S via p27kip. At the same time, DP IV inhibitors provoke tyrosine phosphorylation and p38 MAP kinase activation. Elevated numbers of CD26+ T cells were observed in patients with autoimmune diseases such as rheumatoid arthritis or multiple sclerosis (MS). The expression of DP IV/CD26 in resting T cell clones derived from patients with MS was found to be 3- to 4-fold higher than on resting peripheral blood T cells from healthy persons. In myelin-specific T cells from MS patients, DP IV inhibitors suppress DNA synthesis, as well as IFN-γ, IL-4 and TNF-α production. Moreover, in experimental autoimmune encephalomyelitis (EAE), a well characterized CD4+ T cell-mediated autoimmune disease leading to CNS inflammation and demyelinization, administration of a DP IV/CD26 inhibitor prevents clinical and neuropathological signs of EAE and suppresses ongoing disease. This review summarizes evidence for the role of DP IV enzyme activity in regulation of T cell activation, outlines signal transduction mechanisms used or affected by this enzyme and provides support for the concept of a novel peptidase-based immunosuppressive approach to treat autoimmune diseases like MS.

Myostatin (Mstn) is a potent negative regulator of skeletal development shown to inhibit myoblast proliferation by impinging on cell cycle and suppressing the synthesis of MyoD. Moreover, Mstn causes muscle wasting and its expression is linked with several conditions of muscle loss, mainly dystrophy and cachexia. NF-κB is a transcription factor that is constitutively active in proliferating myoblasts and also plays a role in cell growth control and skeletal muscle differentiation. NF-κB inhibits myogenesis by promoting myoblast growth and inducing loss of MyoD, and NF-κB activity is required in states of muscle wasting. However, the extracellular factors that regulate NF-κB activity to modulate myogenesis are currently not known. Given the similarities in Mstn and NF-κB activities in muscle cells, we investigated the possibility that Mstn-induced regulation of myogenesis may signal via NF-κB. Using a variety of assays to monitor for NF-κB activity, we found that Mstn signaling does not activate NF-κB in differentiating C2C12 myoblasts, nor is the constitutive activity of NF-κB required for Mstn-mediated inhibition of myogenesis. Likewise, in pre-differentiated myotubes, Mstn signaling induces only a modest activation of NF-κB DNA binding activity. We also investigated whether NF-κB inhibition of myogenesis may occur through the regulation of Mstn. However, activation of NF-κB by TNFα or IL-1β failed to induce Mstn expression. These results thus highlight the distinctive differences by which Mstn and NF-κB signal to regulate myogenesis, a finding which broadens our understanding of how these pathways function in both development and disease.

Mast cells play a central role in a wide range of immunological and pathological processes, but are most noted for their role in IgE-dependent allergic responses. Aggregation of the high-affinity receptor for IgE, FcηRI, stimulates mast cell degranulation, production of lipid mediators, and the synthesis and secretion of cytokines and chemokines. FcηRI-induced mast cell activation is subject to regulation by inhibitory receptors that transduce intracellular signals via associating phosphatases. The inositol 5-phosphatase SHIP has been implicated in FcγIIB-mediated inhibition of FcηRI-induced mast cell activation. However, SHIP also negatively regulates FcηRI signaling independent of FcγRIIB, suggesting the existence of additional receptors that mediate SHIP recruitment into sites where it mediates its inhibitory function. Here we show that SHIP associates with numerous phosphoproteins from pervanadate-stimulated mast cells. Based on their sensitivity to PNGase F treatment and cell surface biotinylation, some of these molecules may represent cell surface receptors. A prominent 120−130 kDa SHIP-binding phosphoprotein was identified in untreated RBL-2H3 cells and BMMC stimulated with stem cell factor. Based on its molecular weight, sensitivity to PNGase F, and reactivity with an anti-c-kit antibody, we conclude that this phosphoprotein is c-kit. Furthermore, tyrosine phosphorylation of SHIP is enhanced following SCF stimulation. Taken together, these data suggest that SHIP may function as a negative regulator of SCF signaling via direct association with phosphorylated c-kit.

Engagement of the B cell antigen receptor by antigen initiates a complex and interconnected cascade of signaling pathways that determine whether a B cell will divide, differentiate, or die. Both biochemical and genetic studies have defined the principal molecules, including the BCR components Igσ and Igβ, Src kinases, Syk, and Btk. Linker proteins such as BLNK have recently been shown to play a vital role in organizing proximal signaling molecules and coupling the BCR to distal signaling pathways. In this review, we will pay particular attention to how BCR-proximal kinases coordinate the activation of PLCγ2, leading to the initiation and amplification of BCR-mediated calcium flux and the activation of PI-3 kinase.

Signaling through the Transforming Growth Factor-β (TGFβ)/Smad pathway plays an important role in a variety of cellular processes including the control of cellular proliferation and oncogenesis. The TGFβ Inducible Early Gene (TIEG) is a primary response gene for TGFβ which controls the activity of the Smad pathway. In addition, studies from our laboratory have demonstrated that TIEG expression is lost during the progression of breast cancer. In the present study we utilized a tetracycline inducible TIEG overexpressing breast cancer cell line and TIEG null mouse embryo fibroblasts (MEFs) to establish whether TIEG plays a central role in eliciting the anti-proliferative effects of TGFβ. We demonstrate that, similar to TGFβ treatment, TIEG overexpression significantly decreases cellular proliferation. Consistent with a role in regulating cellular proliferation, TIEG overexpression increases the expression of the cyclin dependent kinase inhibitor p21. Interestingly, while cellular proliferation of wild-type MEFs is inhibited by TGFβ, proliferation of TIEG null MEFs is stimulated by TGFβ. Furthermore, TIEG null MEF cells display a decrease in Smad dependent transcription with a concomitant prolonged increase in Smad7 expression compared to wild-type cells. These data strongly suggest that TIEG plays a central role in the anti-proliferative response to TGFβ and may explain how a loss of TIEG expression contributes to the development of cancer.

The Cbl family of ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent lysosomal targeting. Here, we have investigated the role of Cbl ubiquitin ligase activity in the negative regulation of a non-receptor tyrosine kinase, the Src-family kinase Fyn. Using primary embryonic fibroblasts from Cbl(+/+) and Cbl(-/-) mice, we demonstrate that endogenous Cbl mediates the ubiquitination of Fyn and dictates the rate of Fyn turnover. By analyzing CHO-TS20 cells with a temperature-sensitive ubiquitin activating enzyme, we demonstrate that intact cellular ubiquitin machinery is required for Cbl-induced degradation of Fyn. Analyses of Cbl mutants, with mutations in or near the RING finger domain, in 293T cells revealed that the ubiquitin ligase activity of Cbl is essential for Cbl-induced degradation of Fyn by the proteasome pathway. Finally, use of a SRE-luciferase reporter demonstrated that Cbl-dependent negative regulation of Fyn function requires the region of Cbl that mediates the ubiquitin ligase activity. Given the conservation of structure between various Src-family kinases and the ability of Cbl to interact with multiple members of this family, Cbl-dependent ubiquitination could serve a general role to negatively regulate activated Src-family kinases.

The phosphorylation of serine-/threonine-residues of insulin receptor substrate-1 (IRS-1) by protein kinase C-ζ (PKC-ζ), which leads to an increased tyrosine dephosphorylation of IRS-1, has been recently identified as a novel hyperinsulinemia-induced negative feed-back regulation of the insulin signalling pathway. To identify the underlying molecular mechanism, we investigated the serine-/threonine-residues in IRS-1 phosphorylated by PKC-ζ. Because the activity of the tyrosine phosphatase SHP-2, which dephosphorylates IRS-1, is regulated by the binding to the C-terminal part of IRS-1 (IRS-1c) this region was studied. In the in vitro assay the atypical PKC ζ phosphorylates IRS-1c comparable to PKC-θ and -β1, whereas PKC-β2 showed lower and PKC-i and PKC-α showed negligible phosphorylation. We observed one major 32P-motif labelled by PKC-ζ, containing four phosphorylated serine/threonine residues (Ser 1215, 1216, 1220 and Thr 1221) near the C-terminal end of IRS-1. These sites are closely adjacent to tyrosine 1222, which represents one of the two binding sites for the protein tyrosine phosphatase SHP-2. In conclusion, the PKC-ζ-dependent in vitro phosphorylation of IRS-1 at a site closely situated to a crucial binding site of the tyrosine phosphatase SHP-2 suggests the modulation of the dephosphorylation activity of SHP-2 by PKC-ζ. Furthermore, our findings can contribute to the explanation of the complex positive and negative regulatory action of PKC-ζ on insulin signalling.

Controlled upregulation of N-cadherin expression and function has been shown to be essential for bone morphogenetic protein-2 (BMP-2)-induced chondrogenic differentiation of high-density micromass cultures of the murine multipotential mesenchymal cell line, C3H10T1/2. In this report, we have examined the nature of the N-cadherin-related mechanisms involved in BMP-2-mediated chondrogenesis. BMP-2 treatment altered the expression of catenins, the cytoplasmic components of the adherens junction, and their interactions with N-cadherin. Within 24 h of BMP-2 treatment, immunoprecipitation analysis showed that the association of catenins with N-cadherin was significantly lower. With long-term (9 and 13 days) BMP-2 treatment, β-catenin accumulated in a nuclear localization, with reduced association with the adenomatous polyposis tumor suppressor protein (APC), a factor known to direct β-catenin degradation via ubiquitination. The functional importance of N-cadherin-β-catenin interaction was further investigated by the effect of transfection-mediated over-expression of two forms of β-catenin: wild-type β-catenin inhibited, while an amino terminal truncated (adhesive deficient) β-catenin enhanced BMP-2-induced chondrogenesis. These data indicate that BMP-2 induction of chondrogenesis in the mesenchymal cell line C3H10T1/2 depends on functional alterations in specific cell adhesion and signaling pathways involving N-cadherin-catenin interaction.