With the current access to the whole genomes of various organisms and the completion of the first draft of the human genome, there is a strong need for a structure-function classification of protein families as an initial step in moving from DNA databases to a comprehensive understanding of human biology. As a result of the explosion in nucleic acid sequence information and the concurrent development of methods for high-throughput functional characterization of gene products, the genomic revolution also promises to provide a new paradigm for drug discovery, enabling the identification of molecular drug targets in a significant number of human diseases. This molecular view of diseases has contributed to the importance of combining primary sequence data with three-dimensional structure and has increased the awareness of computational homology modeling and its potential to elucidate protein function. In particular, when important proteins or novel therapeutic targets are identified-like the family of protein tyrosine phosphatases (PTPs) (reviewed in reference 53)-a structure-function classification of such protein families becomes an invaluable framework for further advances in biomedical science. Here, we present a comparative analysis of the structural relationships among vertebrate PTP domains and provide a comprehensive resource for sequence analysis of phosphotyrosine-specific PTPs.PTPs are a key group of signal transduction enzymes which, together with protein tyrosine kinases, control the levels of cellular protein tyrosine phosphorylation. Protein tyrosine kinases phosphorylate cellular substrates on tyrosine residues, and much progress has been made over the last 20 years in elucidating their significance in signal transduction (for reviews, see references 26, 30, 31, 33, 71, and 72). However, it is only recently that the complexities of the PTPs have been appreciated. Thus, today it is recognized that the capacity of PTPs to dephosphorylate phosphotyrosine residues selectively on their substrates plays a pivotal role in initiating, sustaining and terminating cellular signaling (for reviews, see references 1, 4, 19, 32, 35, 46, 55, and 83). It has been shown that both the catalytic domain and noncatalytic segments of the PTPs contribute to the definition of substrate specificity in vivo. Whereas noncatalytic domains may target the PTPs to specific intracellular compartments in which the effective local concentration of substrate is high (3, 19, 51), the PTP catalytic domains themselves confer site-selective protein dephosphorylation by recognizing both the phosphotyrosine residue to be dephosphorylated and its flanking amino acids in the substrate. The combination of structural studies, kinetic analysis of PTP domains (37,74,76,90,91,96), and studies involving substratetrapping mutants (20,23,89) as well as PTP chimeras (60, 82) has convincingly demonstrated that isolated PTP domains may exhibit exquisite substrate selectivity.The structurally conserved PTP domain defines membership of the PTP family, and ...

Aims/hypothesis Brain-derived neurotrophic factor (BDNF) is produced in skeletal muscle, but its functional significance is unknown. We aimed to determine the signalling processes and metabolic actions of BDNF. Methods We first examined whether exercise induced BDNF expression in humans. Next, C2C12 skeletal muscle cells were electrically stimulated to mimic contraction. L6 myotubes and isolated rat extensor digitorum longus muscles were treated with BDNF and phosphorylation of the proteins AMP-activated protein kinase (AMPK) (Thr 172 ) and acetyl coenzyme A carboxylase β (ACCβ) (Ser 79 ) were analysed, as was fatty acid oxidation (FAO).Finally, we electroporated a Bdnf vector into the tibialis cranialis muscle of mice. Results BDNF mRNA and protein expression were increased in human skeletal muscle after exercise, but muscle-derived BDNF appeared not to be released into the circulation. Bdnf mRNA and protein expression was increased in muscle cells that were electrically stimulated. BDNF increased phosphorylation of AMPK and ACCβ and enhanced FAO both in vitro and ex vivo. The effect of BDNF on FAO was AMPK-dependent, since the increase in FAO was abrogated in cells infected with an AMPK dominant negative adenovirus or treated with Compound C, an inhibitor of AMPK. Electroporation of a Bdnf expression

US adults take between approximately 2,000 and approximately 12,000 steps per day, a wide range of ambulatory activity that at the low range could increase risk for developing chronic metabolic diseases. Dramatic reductions in physical activity induce insulin resistance; however, it is uncertain if and how low ambulatory activity would influence peripheral insulin sensitivity. We aimed to explore if healthy, nonexercising subjects who went from a normal to a low level of ambulatory activity for 2 wk would display metabolic alterations including reduced peripheral insulin sensitivity. To do this, ten healthy young men decreased their daily activity level from a mean of 10,501+/-808 to 1,344+/-33 steps/day for 2 wk. Hyperinsulinemic-euglycemic clamps with stable isotopes and muscle biopsies, maximal oxygen consumption (VO2 max) tests, and blood samples were performed pre- and postintervention. A reduced number of daily steps induced a significant reduction of 17% in the glucose infusion rate (GIR) during the clamp. This reduction was due to a decline in peripheral insulin sensitivity with no effect on hepatic endogenous glucose production. The insulin-stimulated ratio of pAktthr308/total Akt decreased after step reduction, with a post hoc analysis revealing the most pronounced effect after 4 h of insulin infusion. In addition, the 2-wk period induced a 7% decline in VO2 max (ml/min; cardiovascular fitness). Lean mass of legs, but not arms and trunk, decreased concurrently. Taken together, one possible biological cause for the public health problem of Type 2 diabetes has been identified. Reduced ambulatory activity for 2 wk in healthy, nonexercising young men significantly reduced peripheral insulin sensitivity, cardiovascular fitness, and lean leg mass.