Design and development of specific antisense probes against the major protein kinase B isoforms

Abstract

The study of cell signalling is important in elucidating the roles of many proteins in cellular functions. Signalling factors such as insulin act via receptor mediated transduction cascades to bring about a variety of effects including controlling translation, metabolic regulation and the cell cycle. Most of these effects are mediated by phosphorylation of serine, threonine or tyrosine residues of proteins and hence protein kinases have a crucial role to play in regulation One such important signal transduction protein is the serine/threonine kinase; protein kinase B (PKB) which is also referred to as Akt. PKB was discovered in 1990 and is a 60kDa cytosolic protein with 3 known isofbrms (a, p, y). PKB has been shown to be activated in response to a variety of & ctars including insulin, EGF and PDGF, and this activation has been shown to involve PI 3 kinase and the recently discovered kinase, PDKl. To date PKB has been proposed to be involved in the regulation of many cellular processes including: apoptosis, protein synthesis and insulin stimulated glucose/glycogen metabolism. Possible roles for PKB include the translocation of GLUT4 transporters to the plasma membrane, inhibition of glycogen synthase kinase-3 (GSK-3) and activation of S6 kinase. PKB has also been proposed to act as a survival factor. Direct evidence for these and other roles for PKB is still lacking and is now urgently sought. Towards this objective I have developed, 2 eSective antisense oligcmucleotides speciGc for eidier the a or p isofbrms of PKB, which have been shown to d^ Ids the levels of that iso & rm by over 90% in 3T3-L1 fibroblasts and adipoctyes. Optimum conditions (time/concentration) have now beei laid down for these 2 probes and by using various ccmtrol oligonucleotides (sense, random and mismatch) I have shown these probes to be both speciSc and very effective inhibitors of each target PKB isofbrm. Using these probes 1 have already established a critical role for PKBa in the differaitiation of cells (3T3-L1 fibroblasts to adipocytes). Use of these probes also suggests that PKBd and PKBP are not involved in the growth factor induced phosphorylation and activation of S6 kinase in the cell lines tested. A suitable peptide based assay for analysing the activation of GSK-3 was developed and preliminary studies into the position of GSK-3 in signalling pathways undertaken. A specific PKBy antisense probe has also been devised and is currently under test. As the developed antisense probes are the first inhibitors against PKB available, it is hoped they can be used not only to establish the roles of PKB in various complex cell processes, but also aid understanding of certain diseases which this cascade mav be involved in.

More information

Identifiers

Catalogue record

Export record

ASCII CitationAtomBibTeXData Cite XMLDublin CoreDublin CoreEP3 XMLEndNoteHTML CitationHTML CitationHTML ListJSONMETSMODSMPEG-21 DIDLOpenURL ContextObjectOpenURL ContextObject in SpanRDF+N-TriplesRDF+N3RDF+XMLRIOXX2 XMLReferReference ManagerSimple Metadata