| The human T-cell leukemia virus type 1 (HTLV-1) encodes a viral oncoprotein termed Tax, which plays a major role in transforming HTLV-1-infected cells. Tax is a potent transcriptional activator that stimulates HTLV-1 viral and cellular gene transcription. In addition, Tax disrupts a number of cell signaling pathways involved in cell growth and survival. Glycogen synthase kinase-3beta (GSK-3beta) is a ubiquitously expressed serine/threonine kinase present in all eukaryotic cells, which functions as a critical regulator of a wide range of cell signaling pathways. As GSK-3beta is constitutively active in resting cells, it is primarily regulated through inhibition. Ser-9 phosphorylation is inhibitory to the kinase activity of GSK-3beta. Deregulation of GSK-3beta has been linked to many human diseases such as Alzheimer's disease and cancers.;It has been reported in Aida Ulloa's thesis that Tax inhibits GSK-3beta kinase activity toward both primed and non-primed substrates through direct association. To delineate the protein-protein interaction between Tax and GSK-3beta, we compared the amino acid sequence of Tax with a well-characterized short peptide deriving from the GSK-3beta interacting domain (GID) of Axin, and found that Tax contains a notable amino acid sequence homology to Axin GID. The region spanning Tax amino acids 185--205 has 24% sequence identity and 19% similarity with Axin GID. We named this region the putative Tax GID. We characterized the putative Tax GID biochemically, and discovered that a longer peptide (Tax aa. 138--205) of the putative Tax GID strongly inhibits GSK-3beta kinase activity in vitro. Bioinformatics computation was used to predict the secondary structure of the Tax GID, which was further used in our docking test to identify a potential binding interface in GSK-3beta. This was tested by GST pull-down and Co-IP assays using point and deletion mutants. In addition, the effects of Tax-GSK-3beta interaction on the downstream beta-catenin and NFAT pathways were characterized by luciferase reporter assays. However, unexpectedly, we observed that Tax expression has little effects on beta-catenin and NFAT transcriptional activation. |
