Liddle Forrester J, Alvarez James V, Poli Valeria, Frank David A
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Biochemistry. 2006 May 2;45(17):5599-605. doi: 10.1021/bi0525674.
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.
STAT转录因子的异常激活与多种癌症有关。已经构建了组成型活性形式的STAT1和STAT3（STAT1C和STAT3C），以确定在与其他细胞因子刺激的信号通路分离的情况下STAT激活的影响。这些突变体是通过在每个STAT分子的羧基末端设计半胱氨酸残基而产生的，这使得两个未磷酸化的单体之间形成一个假定的二硫键。为了确定半胱氨酸残基的存在是否足以在没有酪氨酸磷酸化的情况下实现功能激活，我们构建了无法在关键酪氨酸残基上磷酸化的STAT1C和STAT3C突变体。没有酪氨酸残基，含半胱氨酸的组成型STAT突变体无法反式激活STAT靶基因。此外，转染STAT显性负性突变体可阻止STAT1C和STAT3C的激活。与野生型蛋白相比，细胞因子诱导的STAT1C和STAT3C激活显著延长，并导致STAT依赖性基因激活延长。这些数据表明酪氨酸磷酸化是STAT1C和STAT3C激活所必需的。此外，这些发现表明存在基础磷酸化，这是一个涉及磷酸化和去磷酸化的动态过程。组成型STAT突变体可能由于半胱氨酸残基稳定了这些二聚体并阻止去磷酸化，导致转录活性STAT二聚体复合物的积累，从而表现出更高的活性。
