Modulation of the Pol II CTD Phosphorylation Code by Rac1 and Cdc42 Small GTPases in Cultured Human Cancer Cells and Its Implication for Developing a Synthetic-Lethal Cancer Therapy.

Bo Zhang,Moira Sauane,Zhi-Liang Zheng,Yihong Zhao,Xuelin Zhong

doi:10.3390/cells9030621

Bo Zhang, Moira Sauane + Show 3 more

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https://doi.org/10.3390/cells9030621

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Abstract

Rho GTPases, including Rho, Cdc42, Rac and ROP subfamilies, are key signaling molecules in RNA polymerase II (Pol II) transcriptional control. Our prior work has shown that plant ROP and yeast Cdc42 GTPases similarly modulate Ser2 and Ser5 phosphorylation status of the C-terminal domain (CTD) of the Pol II largest subunit by regulating CTD phosphatase degradation. Here, we present genetic and pharmacological evidence showing that Cdc42 and Rac1 GTPase signaling modulates a similar CTD Ser2 and Ser5 phosphorylation code in cultured human cancer cells. While siRNA knockdown of Cdc42 and Rac1, respectively, in HeLa cells increased the level of CTD Ser phosphatases RPAP2 and FCP1, they both decreased the level of CTD kinases CDK7 and CDK13. In addition, the protein degradation inhibitor MG132 reversed the effect of THZ1, a CDK7 inhibitor which could decrease the cell number and amount of CDK7 and CDK13, accompanied by a reduction in the level of CTD Ser2 and Ser5 phosphorylation and DOCK4 and DOCK9 (the activators for Rac1 and Cdc42, respectively). Conversely, treatments of Torin1 or serum deprivation, both of which promote protein degradation, could enhance the effect of THZ1, indicating the involvement of protein degradation in controlling CDK7 and CDK13. Our results support an evolutionarily conserved signaling shortcut model linking Rho GTPases to Pol II transcription across three kingdoms, Fungi, Plantae and Animalia, and could lead to the development of a potential synthetic-lethal strategy in controlling cancer cell proliferation or death.

Highlights

Regulation of gene expression is essential for cellular activities in response to intracellular cues and extracellular signals
Using genetic and biochemical approaches, we have shown that elevation of the C-terminal domain (CTD) Ser2 and Ser5 phosphorylation status caused by activation of ROP2 and Cdc42 GTPases is mediated by degradation of CTD phosphatases, CPL1 in Arabidopsis or Fcp1 in yeast [8]
We provide pharmacological evidence that the CTD Ser2 and Ser5 phosphorylation status could be altered by inhibitors of Ras, as well as Rho, Rac and Cdc42, in human cancer cell lines, supporting a common theme for the Ras superfamily of GTPases-mediated modulation of the polymerase II (Pol II) CTD code

Summary

Introduction

Regulation of gene expression is essential for cellular activities in response to intracellular cues and extracellular signals. The classical model has been described in genetics and cell biology textbooks and literature and presented as a paradigm in oncogenic signaling networks. In this model, activation of Ras or Rho GTPases causes a cascade of MAP kinase phosphorylation events in the cytoplasm, leading to translocation of MAPK into the nucleus and activation of sequence-specific transcription factors. Given that the shortcut model does not involve the MAP kinase cascade and instead directly regulates Pol II or its components, this model has a potential of efficiently regulating transcription so as to rapidly bring about the broad changes in gene expression in response to dynamic intracellular and extracellular changes [6,7,8]

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