Tyrosine phosphorylation is required for functional activation of disulfide-containing constitutively active STAT mutants
Tyrosine phosphorylation is required for functional activation of disulfide-containing constitutively active STAT mutants
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DOI：
10.1021/bi0525674
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发表时间：
2006-05-02
期刊：
影响因子：
2.9
通讯作者：
Frank, DA
中科院分区：
文献类型：
--
作者：
Liddle, FJ;Alvarez, JV;Frank, DA
STAT转录因子的异常激活与多种癌症有关。已经开发了组成型活性形式的STAT1和STAT3（STAT1C和STAT3C），以确定与其他精氨酸刺激的信号传导途径分离的STAT激活的作用。这些突变体是通过将半胱氨酸残基工程化到每个STAT分子的羧基末端来创建的，允许在两个未磷酸化的单体之间形成假设的二硫键。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允许功能活化，我们开发了STATIC和STAT3C突变体，其不能在关键的酪氨酸残基上磷酸化。在没有酪氨酸残基的情况下，含半胱氨酸的组成型STAT突变体不能反式激活STAT靶基因。此外，STAT显性负突变体的转染阻止了STATIC和STAT3C的激活。与野生型蛋白相比，细胞因子诱导的STATIC和STAT3C活化显著延长，并导致STAT依赖性基因活化延长。这些数据表明，STATIC和STAT3C的激活需要酪氨酸磷酸化。此外，这些发现表明存在基础磷酸化，这是一个动态过程，涉及磷酸化和去磷酸化。组成型STAT突变体可能显示出增强的活性，因为半胱氨酸残基稳定这些二聚体并防止去磷酸化，导致转录活性STAT二聚体复合物的积累。
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 STATIC 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 STATIC and STAT3C. Cytokine-induced activation of STATIC 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 STATIC 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.