The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein-protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.

Novel glucagon-like peptide-1 (GLP-1) analog (Val8)GLP-1 results in significant improvements of glucose tolerance and pancreatic β-cell function after 3-week daily administration in obese diabetic (ob/ob) mice

This study evaluates the antidiabetic potential of an enzyme-resistant analog, (Val8)GLP-1. The effects of daily administration of a novel dipeptidyl peptidase IV-resistant glucagon-like peptide-1 (GLP-1) analog, (Val8)GLP-1, on glucose tolerance and pancreatic β-cell function were examined in obese-diabetic (ob/ob) mice. Acute intraperitoneal administration of (Val8)GLP-1 (6.25-25 nmol/kg) with glucose increased the insulin response and reduced the glycemic excursion in a dose-dependent manner. The effects of (Val8)GLP-1 were greater and longer lasting than native GLP-1. Once-daily subcutaneous administration of (Val8)GLP-1 (25 nmol/kg) for 21 days reduced plasma glucose concentrations, increased plasma insulin, and reduced body weight more than native GLP-1 without a significant change in daily food intake. Furthermore, (Val8)GLP-1 improved glucose tolerance, reduced the glycemic excursion after feeding, increased the plasma insulin response to glucose and feeding, and improved insulin sensitivity. These effects were consistently greater with (Val8)GLP-1 than with native GLP-1, and both peptides retained or increased their acute efficacy compared with initial administration. (Val8)GLP-1 treatment increased average islet area 1.2-fold without changing the number of islets, resulting in an increased number of larger islets. These data demonstrate that (Val8)GLP-1 is more effective and longer acting than native GLP-1 in obese-diabetic ob/ob mice.

Synthesis and iron binding studies of myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate, and iron binding studies of all myo-inositol tetrakisphosphates

The first syntheses of the natural products myo-inositol 1,2,3-trisphosphate and (+/-)-myo-inositol 1,2-bisphosphate are described. The protected key intermediates 4,5,6-tri-O-benzoyl-myo-inositol and (+/-)-3,4,5,6-tetra-O-benzyl-myo-inositol were phosphorylated with dibenzyl N,N-di-isopropylphosphoramidite in the presence of 1H-tetrazole and subsequent oxidation of the phosphite. The crystal structures of the synthetic intermediates (+/-)-1-O-(tert-butyldiphenylsilyl)-2,3,O-cyclohexylidene-myo-inos itol and (+/-)-4,5,6-tri-O-benzoyl-1-O-(tert-butyldiphenylsilyl)-2,3-O-cycl ohexylidene- myo-inositol are reported. myo-Inositol 1,2,3-trisphosphate (+/-)-myo-inositol 1,2-bisphosphate, and all isomeric myo-inositol tetrakisphosphates were evaluated for their ability to alter HO. production in the iron-catalysed Haber-Weiss reaction. The results demonstrated that a 1,2,3-grouping of phosphates in myo-inositol was necessary for inhibition also that (+/-)-myo-inositol 1,2-bisphosphate potentiated HO. production. myo-Inositol 1,2,3-trisphosphate resembled myo-inositol hexakisphosphate (phytic acid) in its ability to act as a siderophore by promoting iron-uptake into Pseudomonas aeruginosa.

A comparison of the apoptotic and cytotoxic effects of hexanedione derivatives on human non-neuronal lines and the neuroblastoma line SH-SY5Y

The effects of the alpha-diketone derivatives 2,3- and 3,4-hexanediones were investigated in three non-neuronal cell lines (MCF7, HepG2 and CaCo-2) as well as in the neuroblastoma line, SH-SY5Y. The MTT reduction assay was employed to determine the necrotic effects of the alpha-diketones and the neurotoxin 2,5-hexanedione over 4, 24 and 48 hr exposures. Flow cytometry was also used to study the effects of the three isomers on the cell cycle of the SH-SY5Y line only. With 2,5-hexanedione, the mean MTT IC50 decreased more than 10-fold from 4 to 48 hr. The toxicities of both alpha-diketones were similar, with a more than 18-fold increase in sensitivity of the SH-SY5Y at 24 hr compared to that of 4 hr. With flow cytometry at 48 hr, SH-SY5Y apoptosis with 2,5-hexanedione rose throughout the concentration range evaluated (0-30 mM) while 2,3- and 3,4-hexanediones showed apoptosis over the concentration range 1-1.6 mM, with 3,4-hexanedione being the more potent compared to the 2,3-isomer. At 1.6 mM nearly all the cells had entered apoptosis in the presence of the 3,4-isomer, (94.9 ± 1.4%) but only 57.5 ±4.1% of the 2,3-isomer-treated cells had reached that stage. The 2,3-and 3,4-isomers in diets alone may not pose a serious threat to human health. Further studies may be necessary to evaluate the effects of other dietary components on their toxicity. These alpha-diketones also display a degree of toxic selectivity towards neuroblastoma cells, which may have therapeutic implications.