Glucose and insulin induce Ca2+ signaling in nesfatin-1 neurons in the hypothalamic paraventricular nucleus

Nucleobindin-2 derived nesfatin-1 in the hypothalamic paraventricular nucleus (PVN) plays a role in inhibition of feeding. The neural pathways downstream of PVN nesfatin-1 have been extensively investigated. However, regulation of the PVN nesfatin-1 neurons remains unclear. Since starvation decreases and refeeding stimulates nesfatin-1 expression specifically in the PVN, this study aimed to clarify direct effects of meal-evoked metabolic factors, glucose and insulin, on PVN nesfatin-1 neurons. High glucose (10mM) and insulin (10(-13)M) increased cytosolic calcium concentration ([Ca(2+)](i)) in 55 of 331 (16.6%) and 32 of 249 (12.9%) PVN neurons, respectively. Post [Ca(2+)](i) measurement immunocytochemistry identified that 58.2% of glucose-responsive and 62.5% of insulin-responsive neurons were immunoreactive to nesfatin-1. Furthermore, a fraction of the glucose-responsive nesfatin-1 neurons also responded to insulin, and vice versa. Some of the neurons that responded to neither glucose nor insulin were recruited to [Ca(2+)](i) increases by glucose and insulin in combination. Our data demonstrate that glucose and insulin directly interact with and increase [Ca(2+)](i) in nesfatin-1 neurons in the PVN, and that the nesfatin-1 neuron is the primary target for them in the PVN. The results suggest that high glucose- and insulin-induced activation of PVN nesfatin-1 neurons serves as a mechanism through which meal ingestion stimulates nesfatin-1 neurons in the PVN and thereby produces satiety.     

The scaffold protein c-Jun NH2-terminal kinase-associated leucine zipper protein regulates cell migration through interaction with the G protein G(alpha 13)

Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH(2)-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein G(alpha13), showed little or no effect on the cell migration defect. Furthermore, although a constitutively active G(alpha13) enhanced the migration of control HeLa cells, the G(alpha13)-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with G(alpha13).     

Sirt2 Deacetylase Is a Novel AKT Binding Partner Critical for AKT Activation by Insulin

AKT/PKB kinases transmit insulin and growth factor signals downstream of phosphatidylinositol 3-kinase (PI3K). AKT activation involves phosphorylation at two residues, Thr³⁰⁸ and Ser⁴⁷³, mediated by PDK1 and the mammalian target of rapamycin complex 2 (mTORC2), respectively. Impaired AKT activation is a key factor in metabolic disorders involving insulin resistance, whereas hyperactivation of AKT is linked to cancer pathogenesis. Here, we identify the cytoplasmic NAD⁺-dependent deacetylase, Sirt2, as a novel AKT interactor, required for optimal AKT activation. Pharmacological inhibition or genetic down-regulation of Sirt2 diminished AKT activation in insulin and growth factor-responsive cells, whereas Sirt2 overexpression enhanced the activation of AKT and its downstream targets. AKT was prebound with Sirt2 in serum or glucose-deprived cells, and the complex dissociated following insulin treatment. The binding was mediated by the pleckstrin homology and the kinase domains of AKT and was dependent on AMP-activated kinase. This regulation involved a novel AMP-activated kinase-dependent Sirt2 phosphorylation at Thr¹⁰¹. In cells with constitutive PI3K activation, we found that AKT also associated with a nuclear sirtuin, Sirt1; however, inhibition of PI3K resulted in dissociation from Sirt1 and increased association with Sirt2. Sirt1 and Sirt2 inhibitors additively inhibited the constitutive AKT activity in these cells. Our results suggest potential usefulness of Sirt1 and Sirt2 inhibitors in the treatment of cancer cells with up-regulated PI3K activity and of Sirt2 activators in the treatment of insulin-resistant metabolic disorders.     

Regulation of N-cadherin-based cell-cell interaction by JSAP1 scaffold in PC12h cells

We previously reported that the level of c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell-cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell-cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin.     

The Arabidopsis At1g45130 and At3g52840 genes encode beta-galactosidases with activity toward cell wall polysaccharides

The Arabidopsis genes At1g45130 and At3g52840 encode the β-galactosidase isozymes Gal-5 and Gal-2 that belong to Glycosyl Hydrolase Family 35 (GH 35). The two enzymes share 60% sequence identity with each other and 38–81% with other plant β-galactosidases that are reported to be involved in cell wall modification. We studied organ-specific expression of the two isozymes. According to our western blot analysis using peptide-specific antibodies, Gal-5 and Gal-2 are most highly expressed in stem and rosette leaves. We show by dot-immunoblotting that Gal-5 and Gal-2 are associated with the cell wall in Arabidopsis. We also report expression of the recombinant enzymes in P. pastoris and describe their substrate specificities. Both enzymes hydrolyze the synthetic substrate para-nitrophenyl-β-d-galactopyranoside and display optimal enzyme activity between pH 4.0 and 4.5, similar to the pH optimum reported for other well-characterized plant β-galactosidases. Both Gal-5 and Gal-2 show a broad specificity for the aglycone moiety and a strict specificity for the glycone moiety in that they prefer galactose and its 6-deoxy analogue, fucose. Both enzymes cleave β-(1, 4) and β-(1, 3) linkages in galacto-oligosaccharides and hydrolyze the pectic fraction of Arabidopsis cell wall. These findings suggest that Gal-5 and Gal-2 could be involved in the modification of cell wall polysaccharides.     

Ablation of the scaffold protein JLP causes reduced fertility in male mice

The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH₂-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the G_α13 and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Asuka Iwanaga and Guangmin Wang contributed equally to this study.     

JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cell migration

c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130(Cas)) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130(Cas), which required p130(Cas) hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130(Cas) pathway by expression of a dominant-negative form of p130(Cas) or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1.FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors.