Differential sensitivity of Src-family kinases to activation by SH3 domain displacement

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo. © 2014 Moroco et al

Arctigenin Efficiently Enhanced Sedentary Mice Treadmill Endurance

Physical inactivity is considered as one of the potential risk factors for the development of type 2 diabetes and other metabolic diseases, while endurance exercise training could enhance fat oxidation that is associated with insulin sensitivity improvement in obesity. AMP-activated protein kinase (AMPK) as an energy sensor plays pivotal roles in the regulation of energy homeostasis, and its activation could improve glucose uptake, promote mitochondrial biogenesis and increase glycolysis. Recent research has even suggested that AMPK activation contributed to endurance enhancement without exercise. Here we report that the natural product arctigenin from the traditional herb Arctium lappa L. (Compositae) strongly increased AMPK phosphorylation and subsequently up-regulated its downstream pathway in both H9C2 and C2C12 cells. It was discovered that arctigenin phosphorylated AMPK via calmodulin-dependent protein kinase kinase (CaMKK) and serine/threonine kinase 11(LKB1)-dependent pathways. Mice treadmill based in vivo assay further indicated that administration of arctigenin improved efficiently mice endurance as reflected by the increased fatigue time and distance, and potently enhanced mitochondrial biogenesis and fatty acid oxidation (FAO) related genes expression in muscle tissues. Our results thus suggested that arctigenin might be used as a potential lead compound for the discovery of the agents with mimic exercise training effects to treat metabolic diseases

Polysialic acid bioengineering of cancer and neuronal cells by N-acyl sialic acid precursor treatment

Peer reviewed: YesNRC publication: Ye

Rapid reaction kinetic techniques

This chapter provides an overview of different methodologies to dissect the ATPase mechanism of motor proteins. The use of ATP is fundamental to how these molecular engines work and how they can use the energy to perform various cellular roles. Rapid reaction and single-molecule techniques will be discussed to monitor reactions in real time through the application of fluorescence intensity, anisotropy and FRET. These approaches utilise fluorescent nucleotides and biosensors. While not every technique may be suitable for your motor protein, the different ways to determine the ATPase mechanism should allow a good evaluation of the kinetic parameters