Aerobic glycolysis is a hallmark of cancer and is regulated by growth factors, protein kinases and transcription factors. However, it remains poorly understood how these components interact to regulate aerobic glycolysis coordinately. Here, we show that sine oculis homeobox 1 (SIX1) phosphorylation integrates growth factors (e.g. TGFβ, EGF) to control aerobic glycolysis and determines its tumor-promoting activity. SIX1 is phosphorylated at serine 225 (S225) by growth factors-activated protein kinases ERK1/2 and its phosphorylation is responsible for glycolysis stimulated by some growth factors. SIX1 is dephosphorylated by the atypical protein phosphatase eyes absent 4 (EYA4). Phosphorylation blocks non-canonical ubiquitination and degradation of SIX1 through the E3 ubiquitin ligase FZR1. Unexpectedly, the non-canonical phosphorylation mimic SIX1 (S225K), but not the canonical phosphorylation mimic SIX1 (S225D/E), phenocopies the effects of SIX1 phosphorylation on glycolysis and cancer cell growth and metastasis in vitro and in mice. Compared to normal liver tissues, SIX1 phosphorylation at S225 (pS225) is upregulated in human liver cancer tissues. ERK1/2 expression is positively correlated with pS225 and EYA4 expression is negatively associated with pS225 in liver cancer specimens. Moreover, low expression of pS225 had longer disease-free survival and overall survival in patients with liver cancer. Thus, we identify a common mechanism underlying growth factors-mediated glycolysis, and provide a previously unidentified mode for non-classical phosphorylation mimics of a protein. Targeting growth factors/SIX1 signaling pathway may be beneficial to cancer treatment.

需氧糖酵解是癌症的标志，受生长因子、蛋白激酶和转录因子的调节。然而，人们对这些成分如何相互作用以协调调节有氧糖酵解仍然知之甚少。在这里，我们表明正弦眼同源框 1 （SIX1） 磷酸化整合了生长因子（例如 TGFβ、EGF）以控制有氧糖酵解并确定其促肿瘤活性。SIX1 在丝氨酸 225 （S225） 位点被生长因子活化的蛋白激酶 ERK1/2 磷酸化，其磷酸化负责某些生长因子刺激的糖酵解。SIX1 被非典型蛋白磷酸酶眼缺失 4 （EYA4） 去磷酸化。磷酸化阻断了 SIX1 通过 E3 泛素连接酶 FZR1 的非经典泛素化和降解。出乎意料的是，非经典磷酸化模拟物 SIX1 （S225K），而不是经典磷酸化模拟物 SIX1 （S225D/E），表型复制了 SIX1 磷酸化对体外和小鼠糖酵解、癌细胞生长和转移的影响。与正常肝组织相比，S225 （pS225） 位点的 SIX1 磷酸化在人肝癌组织中上调。在肝癌标本中，ERK1/2 表达与 pS225 呈正相关，EYA4 表达与 pS225 呈负相关。此外，pS225 的低表达在肝癌患者中具有更长的无病生存期和总生存期。因此，我们确定了生长因子介导的糖酵解的共同机制，并为蛋白质的非经典磷酸化模拟物提供了一种以前未确定的模式。靶向生长因子/SIX1 信号通路可能有益于癌症治疗。