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

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.

Cyclin-Dependent Kinases and CTD Phosphatases in Cell Cycle Transcriptional Control: Conservation across Eukaryotic Kingdoms and Uniqueness to Plants

“…As discussed above, human CDK7 and its yeast ortholog Kin28 are believed to phosphorylate Ser5 and Ser7, but not Ser2 based on in vitro biochemical studies [ 44 ], while Arabidopsis counterparts CDKD1;1/1;2/1;3 also phosphorylate Ser2P, in addition to Ser5 and Ser7 [ 45 ]. However, using a potent and specific CDK7 inhibitor THZ1, we found a dramatic decrease of Ser2P, Ser5P, and Ser7P in human cells [ 46 ]. Despite this functional divergence, human CDK7 has emerged as a critical target in containing uncontrolled cell division in various tumor types [ 47 , 48 ].…”

“…Importantly, we have found that the Rho signaling shortcut to Pol II CTD Ser2P and Ser5P was controlled by proteasome-mediated degradation of CPL1 and CPL2 in Arabidopsis or Fcp1 in yeast [ 23 ]. Furthermore, Rho family GTPases (Cdc42 and Rac1) in human cells also seem to suppress CTD phosphatases in a GTPase-specific manner: Suppression of RPAP2 by Cdc42 signaling but not Rac1, and suppression of CTDP1 (FCP1) by Rac1 signaling but not Cdc42 [ 46 ]. This strongly suggests that the shortcut model of Pol II transcription is conserved from yeast to Arabidopsis and human.…”

“…As ROP GTPases act as a universal signaling switch for multiple hormones or developmental processes and various stress responses, it remains to be determined how the ROP2/4-CPL1/2 signaling shortcut is involved in many different processes. The finding that yeast Cdc42 GTPase signaling also promotes Fcp1 degradation [ 23 ] and the observation that human Rac1 and Cdc42 activity differentially inhibits CTDP1 (FCP1) and RPAP2 [ 46 ] may provide clues to this signaling specificity. For example, the shortcut model may involve different members of Rho GTPases and target any of three groups of CTD phosphatases (and CDKs, as well).…”

Rac-maninoff and Rho-vel: The symphony of Rho-GTPase signaling at excitatory synapses

“…Herein, DOCK4 and DOCK9 activate Rac1 and Cdc42, which each target a specific phosphatase for degradation. This action prevents the dephosphorylation and inhibition of a major subunit of RNA polymerase, resulting in enhanced transcription [ 120 ]. Here, DOCK4 and DOCK9 are not known to be complexed, but since they do interact, it is a possibility.…”

A novel and ubiquitous miRNA-involved regulatory module ensures precise phosphorylation of RNA polymerase II and proper transcription

“…It is important for CDKs to be activated at the proper time and modulated to a precise level during various stages of the cell cycle to ensure normal growth. The Ras superfamily of small GTPases, including Ras and Rho families, act as a signaling switch of phosphorylation state by regulating the expression of CDKs and CTD phosphatases FCP1 or RPAP2 through an indirect effect [ 19 ]. However, the direct regulator of CDKs expression remains enigmatic, the rapid and precise regulation is necessary to ensure transcriptional accuracy.…”