Tyrosine phosphorylation is required for functional activation of disulfide-containing constitutively active STAT mutants.
Aberrant activation of STAT transcription factors has been implicated in a variety of cancers. Constitutively active forms of STAT1 and STAT3 (STAT1C and STAT3C) have been developed to determine the effects of STAT activation in isolation from other cytokine-stimulated signaling pathways. These mutants were created by engineering cysteine residues into the carboxy terminus of each STAT molecule, allowing a hypothesized disulfide bond to form between two unphosphorylated monomers. To determine whether the presence of cysteine residues is sufficient to allow for functional activation in the absence of tyrosine phosphorylation, we developed STAT1C and STAT3C mutants that are unable to be phosphorylated on the critical tyrosine residue. Without the tyrosine residue, cysteine containing constitutive STAT mutants failed to transactivate STAT target genes. Furthermore, transfection of STAT dominant negative mutants prevented the activation of STAT1C and STAT3C. Cytokine-induced activation of STAT1C and STAT3C was dramatically prolonged when compared to wild-type proteins and led to extended STAT-dependent gene activation. These data show that tyrosine phosphorylation is required for activation of STAT1C and STAT3C. Additionally, these findings suggest the existence of basal phosphorylation that is a dynamic process that involves both phosphorylation and dephosphorylation. The constitutive STAT mutants likely show heightened activity because of the cysteine residues stabilizing these dimers and preventing dephosphorylation, resulting in the accumulation of trancriptionally active STAT dimer complexes.
Liddle
Forrester J
FJ
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Alvarez
James V
JV
Poli
Valeria
V
Frank
David A
DA
D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology*; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000096:biosynthesis; D050791:STAT Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
D004220:Disulfides; D050791:STAT Transcription Factors; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D019000:Phosphotyrosine; D014443:Tyrosine