Akt , a serine threonine kinase, plays a critical role in various cellular processes concerned in apoptosis, proliferation, and diabetes . The primary function of Akt could be to market cell survival by phosphorylating and inhibiting many downstream professional apoptotic variables which include Undesirable, caspase , and forkhead transcription elements . An elevated Akt action has been found in a wide spectrum of human malignancies, like prostate, breast, lung, ovarian, pancreatic and colorectal cancers . To the other hand, inhibition of Akt activation is proven to induce cancer cell death in preclinical and clinical studies . These attributes have produced Akt a potential pharmacological target for cancer treatment and triggered comprehensive research for the comprehensive molecular mechanism of Akt activation along with the hunt for specific Akt kinase inhibitors . Three remarkably homologous Akt isomers have already been reported in mammals. Every isomer is composed of 3 distinctive regions including an N terminal pleckstrin homology domain , a C terminal regulatory domain , and also a central kinase domain .
The activation of Akt is mediated by membrane phosphatidylinositol trisphosphate , that is produced from , phosphatidylinositol bisphosphate by phosphoinositide kinase upon growth component stimulation. The binding of PIP for the Akt PH MK 801 kinase inhibitor domain anchors cytosolic Akt on the plasma membrane, in which Akt is activated by phosphorylation of T and S by phosphoinositide dependent protein kinases . It’s been very well established that the Akt membrane interaction is usually a crucial step for that activation. The interaction not only brings Akt into make contact with together with the membrane bound PDKs, but also ends in conformational changes of Akt which might be essential for its phosphorylation and activation by the upstream enzymes. It has been acknowledged that the PH domain blocks access in the upstream kinase to T, and this structural hindrance appears to get eliminated through the binding within the PH domain to PIP. This membranedependent conformational change is supported by Xray crystallography, NMR , in cell fluorescence lifetime imaging microscopy , and, far more just lately, chemical cross linking and mass spectrometry .
Before years, chemical cross linking mass spectrometry has emerged Wortmannin like a valuable device for probing protein conformation in physiologically appropriate conditions . The spatial distance knowledge supplied from the MS determined cross linking websites of amino acid residues has become proven to become precious for that elucida tion of protein structural changes associated with protein functions . In an try to understand the molecular basis of Akt activation, we’ve got just lately developed a method combining tandem mass spectrometry, lysine distinct chemical cross linking , and proteolytic O digestion to probe conformation within the complete length Akt for which the crystal framework is just not still obtainable.