Phosphorylation regulates human T-cell leukemia virus type 1 Rex function

Kesic Matthew, Doueiri Rami, Ward Michael, Semmes, Green

Description

Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1) is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation. Results We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function. Conclusion We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into the regulation of Rex-1 function.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2009
• Nombre de lectures: 2
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Retrovirology

BioMedCentral

Open Access Research Phosphorylation regulates human Tcell leukemia virus type 1 Rex function 1,2 1,25 5 Matthew Kesic, Rami Doueiri, Michael Ward, O John Semmesand 1,2,3,4 Patrick L Green*

1 2 Address: Centerfor Retrovirus Research, The Ohio State University, Columbus, OH 43210, USA,Department of Veterinary Biosciences, The Ohio 3 State University, Columbus, OH 43210, USA,Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, 4 Columbus, OH 43210, USA,Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, Columbus, OH 43210, 5 USA andDepartment of Microbiology and Molecular Cell Biology and Center for Biomedical Proteomics, Eastern Virginia Medical School, Norfolk, Virginia 235070, USA Email: Matthew Kesic  [e-mail]; Rami Doueiri  [e-mail]; Michael Ward  [e-mail]; O John Semmes  [e-mail]; Patrick L Green*  [e-mail] * Corresponding author

Published: 17 November 2009Received: 22 June 2009 Accepted: 17 November 2009 Retrovirology2009,6:105 doi:10.1186/174246906105 This article is available from: http://www.retrovirology.com/content/6/1/105 © 2009 Kesic et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background:Human Tcell leukemia virus type 1 (HTLV1) is a pathogenic complex deltaretrovirus, which is the causative agent of adult Tcell leukemia/lymphoma (ATL) and HTLV 1associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolarlocalizing shuttling protein that acts posttranscriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV1 Rex (Rex1) is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex1 function may be regulated through sitespecific phosphorylation. Results:We conducted a phosphoryl mapping of Rex1 overexpressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex1 polypeptide and identified five novel phosphorylation sites at Thr22, Ser36, Thr37, Ser97, and Ser106. We also confirmed evidence of two previously identified residues, Ser70 and Thr174, but found no evidence of phosphorylation at Ser177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and sitedirected mutational analysis. Our results indicate that phosphorylation at Ser97 and Thr174 is critical for Rex1 function. Conclusion:We have mapped completely the sitespecific phosphorylation of Rex1 identifying a total of seven residues; Thr22, Ser36, Thr37, Ser70, Ser97, Ser106, and Thr174. Overall, this work is the first to completely map the phosphorylation sites in Rex1 and provides important insight into the regulation of Rex1 function.

Page 1 of 11 (page number not for citation purposes)