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Blood, 15 January 2006, Vol. 107, No. 2, pp. 708-715. Prepublished online as a Blood First Edition Paper on September 13, 2005; DOI 10.1182/blood-2005-07-2743. This Article Full Text Full Text (PDF) All Versions of this Article: 2005-07-2743v1 107/2/708    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Stark, M. Articles by Assaraf, Y. G. Search for Related Content PubMed PubMed Citation Articles by Stark, M. Articles by Assaraf, Y. G. Related Collections Neoplasia Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  NEOPLASIA Loss of Sp1 function via inhibitory phosphorylation in antifolate-resistant human leukemia cells with down-regulation of the reduced folate carrier Michal Stark, and Yehuda G. Assaraf From the Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel. The reduced folate carrier (RFC) is the dominant influx transporter for antifolates. A major mechanism of antifolate resistance is loss of RFC (SLC19A1) gene expression due to decreased GC-box-dependent transcription. However, despite the poor GC-box binding in multiple antifolate-resistant cell lines, normal Sp1 levels were retained. Here we explored the post-translational modifications that may disrupt Sp1 function. Phospho-affinity purification of nuclear proteins revealed that resistant cells contained approximately 8-fold more phosphorylated Sp1 than parental cells; treatment of nuclear proteins from these cells with alkaline phosphatase restored GC-box binding. As protein kinase A phosphorylates Sp1, resistant cells were treated with various cAMP-reactive agents, revealing no apparent effect on GC-box binding except for the general phosphodiesterase inhibitor IBMX. As cGMP levels also may be affected by IBMX, resistant cells were treated with 8-pCPT-cGMP, resulting in the complete restoration of GC-box binding, luciferase reporter activity, and RFC mRNA levels. This restoration was abolished in the presence of the protein phosphatase 2A inhibitor (PP2A) okadaic acid. Importantly, whereas resistant cells showed multiple phosphorylated Sp1 forms barely detectable in parental cells, treatment with 8-pCPT-cGMP resulted in their elimination; this disappearance, however, was prevented by the copresence of okadaic acid. These findings provide the first evidence that loss of RFC gene expression in antifolate-resistant cells is associated with an inhibitory Sp1 phosphorylation that can be eliminated by a cGMP-dependent activation of PP2A. (Blood. 2006;107:708-715) CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: J.-Y. Chuang, Y.-T. Wang, S.-H. Yeh, Y.-W. Liu, W.-C. Chang, and J.-J. Hung Phosphorylation by c-Jun NH2-terminal Kinase 1 Regulates the Stability of Transcription Factor Sp1 during Mitosis Mol. Biol. Cell, March 1, 2008; 19(3): 1139 - 1151. [Abstract] [Full Text] [PDF] S. G. Lee and D. L. Mykles Proteomics and signal transduction in the crustacean molting gland Integr. Comp. Biol., December 1, 2006; 46(6): 965 - 977. [Abstract] [Full Text] [PDF] Y. Zhang, M. Liao, and M. L. Dufau Phosphatidylinositol 3-Kinase/Protein Kinase C{zeta}-Induced Phosphorylation of Sp1 and p107 Repressor Release Have a Critical Role in Histone Deacetylase Inhibitor-Mediated Depression of Transcription of the Luteinizing Hormone Receptor Gene. Mol. Cell. Biol., September 1, 2006; 26(18): 6748 - 6761. [Abstract] [Full Text] [PDF] Y. Kaufman, I. Ifergan, L. Rothem, G. Jansen, and Y. G. Assaraf Coexistence of multiple mechanisms of PT523 resistance in human leukemia cells harboring 3 reduced folate carrier alleles: transcriptional silencing, inactivating mutations, and allele loss Blood, April 15, 2006; 107(8): 3288 - 3294. [Abstract] [Full Text] [PDF]

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