TGFbeta2 mediates rapid inhibition of calcium influx in identified cholinergic basal forebrain neurons.

Transforming growth factors betas (TGFbetas) are known to have important roles in neuronal survival and can be upregulated in disease. However, unlike many other trophic factors, nothing is known about the rapid neurotransmitter-like actions of TGFbeta in the CNS. We explored this by examining the effects of TGFbeta on calcium influx of large enzymatically dissociated basal forebrain neurons. We show that brief application of TGFbeta2, but not TGFbeta1, to fura-2AM-loaded neurons reversibly and acutely (within seconds) inhibited K(+)-evoked calcium influx. Moreover, using single-cell RT-PCR, we confirmed that the large TGFbeta2-responsive neurons presented a cholinergic phenotype. Investigation of the signaling mechanism underlying TGFbeta2 actions using whole-cell recordings of calcium currents revealed that TGFbeta2-mediated responses were insensitive to the nonhydrolyzable GTP analogue GTPgammaS. However, TGFbeta2-mediated calcium current reductions were prevented by intracellular perfusion of a Smad2/3 peptide antagonist. Together, these results suggest that TGFbeta2 can acutely regulate the excitability of basal forebrain cholinergic neurons through an atypical signaling mechanism.     

Glycoproteome profiling of transforming growth factor-beta (TGFbeta) signaling: nonglycosylated cell death-inducing DFF-like effector A inhibits TGFbeta1-dependent apoptosis

Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. TGFbeta binds to specific serine/threonine kinase receptors, which leads to activation of Smad-dependent and Smad-independent signaling pathways. O-Glycosylation is a dynamic PTM which has been observed in many regulatory proteins, but has not been studied in the context of TGFbeta signaling. To explore the effect of TGFbeta1 on protein O-glycosylation in human breast epithelial cells, we performed analyses of proteins which were affinity purified with Helix pomatia agglutinin (HPA). HPA lectin allowed enrichment of proteins containing GalNAc and GlcNAc linked to serine and threonine residues. Using 2-DE and MALDI-TOF-MS, we identified 21 HPA-precipitated proteins, which were affected by treatment of cells with TGFbeta1. Among these proteins, regulators of cell survival, apoptosis, trafficking, and RNA processing were identified. We found that TGFbeta1 inhibited the appearance of cell death-inducing DFF-like effector A (CIDE-A) in 2-D gels with HPA-precipitated proteins. CIDE-A is a cell death activator which promotes DNA fragmentation. We observed that TGFbeta1 did not affect expression of CIDE-A, but inhibited its glycosylation. We found that deglycosylation of CIDE-A correlated with enhanced nuclear export of the protein, and that high level of nonglycosylated CIDE-A inhibited TGFbeta1-dependent cell death. Thus, inhibition of the glycosylation of CIDE-A may be a mechanism to protect cells from apoptosis.     

Proteomic analyses of retina of excitatory amino acid carrier 1 deficient mice

Background  

Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter found in neuronal tissues and is extensively expressed in the retina. EAAC1 plays a role in a variety of neural functions, but its biological functions in the retina has not been fully determined. The purpose of this study was to identify proteins regulated by EAAC1 in the retina of mice. To accomplish this, we used a proteomics-based approach to identify proteins that are up- or down-regulated in EAAC1-deficient (EAAC1^-/-) mice.     

Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor

Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling.