C-terminal Domain Phosphatase Research Articles

PP2C phosphatase Pic14 negatively regulates tomato Pto/Prf-triggered immunity by inhibiting MAPK activation.

Type 2C protein phosphatases (PP2Cs) are emerging as important regulators of plant immune responses, although little is known about how they might impact nucleotide-binding, leucine-rich repeat (NLR)-triggered immunity (NTI). We discovered that expression of the PP2C immunity-associated candidate 14 gene (Pic14) is induced upon activation of the Pto/Prf-mediated NTI response in tomato. Pto/Prf recognizes the effector AvrPto translocated into plant cells by the pathogen Pseudomonas syringae pv. tomato (Pst) and activate a MAPK cascade and other responses which together confer resistance to bacterial speck disease. Pic14 encodes a PP2C with an N-terminal kinase-interacting motif (KIM) and a C-terminal phosphatase domain. Upon inoculation with Pst-AvrPto, Pto/Prf-expressing tomato plants with loss-of-function mutations in Pic14 developed less speck disease, specifically in older leaves, compared to wild-type plants. Transient expression of Pic14 in leaves of Nicotiana benthamiana and tomato inhibited cell death typically induced by Pto/Prf and the MAPK cascade members M3Kα and Mkk2. The cell death-suppressing activity of Pic14 was dependent on the KIM and the catalytic phosphatase domain. Pic14 inhibited M3Kα- and Mkk2-mediated activation of immunity-associated MAPKs and Pic14 was shown to be an active phosphatase that physically interacts with and dephosphorylates Mkk2 in a KIM-dependent manner. Together, our results reveal Pic14 as an important negative regulator of Pto/Prf-triggered immunity by interacting with and dephosphorylating Mkk2.

Tracking Inhibition of Human Small C-Terminal Domain Phosphatase 1 Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.

Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

BackgroundGlioblastoma (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some GBMs, cell proliferation is fueled by a transcriptional regulator, repressor element-1 silencing transcription factor (REST).ResultsUsing CRISPR/Cas9, we identified GBM cell lines dependent on REST activity. We developed new small molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) to reduce REST protein level and transcriptional activity in glioblastoma cells. Top leads of the series like GR-28 exhibit potent cytotoxicity, reduce REST protein level, and suppress its transcriptional activity. Upon the loss of REST protein, GBM cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation using long-chain acyl-CoA synthetase inhibitor Triacsin C demonstrated substantial synergetic potential without inducing hepatotoxicity.ConclusionsOur results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST GBM.

Tumor Suppressor Properties of Small C-Terminal Domain Phosphatases in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for 80-90% of kidney cancers worldwide. Small C-terminal domain phosphatases CTDSP1, CTDSP2, and CTDSPL (also known as SCP1, 2, 3) are involved in the regulation of several important pathways associated with carcinogenesis. In various cancer types, these phosphatases may demonstrate either antitumor or oncogenic activity. Tumor-suppressive activity of these phosphatases in kidney cancer has been shown previously, but in general case, the antitumor activity may be dependent on the choice of cell line. In the present work, transfection of the Caki-1 cell line (ccRCC morphologic phenotype) with expression constructs containing the coding regions of these genes resulted in inhibition of cell growth in vitro in the case of CTDSP1 (p < 0.001) and CTDSPL (p < 0.05) but not CTDSP2. The analysis of The Cancer Genome Atlas (TCGA) data showed differential expression of some of CTDSP genes and of their target, RB1. These results were confirmed by quantitative RT-PCR using an independent sample of primary ccRCC tumors (n = 52). We observed CTDSPL downregulation and found a positive correlation of expression for two gene pairs: CTDSP1 and CTDSP2 (rs = 0.76; p < 0.001) and CTDSPL and RB1 (rs = 0.38; p < 0.05). Survival analysis based on TCGA data demonstrated a strong association of lower expression of CTDSP1, CTDSP2, CTDSPL, and RB1 with poor survival of ccRCC patients (p < 0.001). In addition, according to TCGA, CTDSP1, CTDSP2, and RB1 were differently expressed in two subtypes of ccRCC-ccA and ccB, characterized by different survival rates. These results confirm that CTDSP1 and CTDSPL have tumor suppressor properties in ccRCC and reflect their association with the more aggressive ccRCC phenotype.

The Sts Proteins: Modulators of Host Immunity.

The suppressor of TCR signaling (Sts) proteins, Sts-1 and Sts-2, are a pair of closely related signaling molecules that belong to the histidine phosphatase (HP) family of enzymes by virtue of an evolutionarily conserved C-terminal phosphatase domain. HPs derive their name from a conserved histidine that is important for catalytic activity and the current evidence indicates that the Sts HP domain plays a critical functional role. Sts-1HP has been shown to possess a readily measurable protein tyrosine phosphatase activity that regulates a number of important tyrosine-kinase-mediated signaling pathways. The in vitro catalytic activity of Sts-2HP is significantly lower than that of Sts-1HP, and its signaling role is less characterized. The highly conserved unique structure of the Sts proteins, in which additional domains, including one that exhibits a novel phosphodiesterase activity, are juxtaposed together with the phosphatase domain, suggesting that Sts-1 and -2 occupy a specialized intracellular signaling niche. To date, the analysis of Sts function has centered predominately around the role of Sts-1 and -2 in regulating host immunity and other responses associated with cells of hematopoietic origin. This includes their negative regulatory role in T cells, platelets, mast cells and other cell types, as well as their less defined roles in regulating host responses to microbial infection. Regarding the latter, the use of a mouse model lacking Sts expression has been used to demonstrate that Sts contributes non-redundantly to the regulation of host immunity toward a fungal pathogen (C. albicans) and a Gram-negative bacterial pathogen (F. tularensis). In particular, Sts-/- animals demonstrate significant resistance to lethal infections of both pathogens, a phenotype that is correlated with some heightened anti-microbial responses of phagocytes derived from mutant mice. Altogether, the past several years have seen steady progress in our understanding of Sts biology.

MiR-574-5p promotes cell proliferation by negatively regulating small C-terminal domain phosphatase 1 in esophageal squamous cell carcinoma.

Objective(s):Esophageal cancer is one of the most common cancers with high incidence and mortality rates, especially in China. MicroRNA (miRNA) can be used as a prognostic marker for various human cancers. This study aims to detect suitable miRNA markers for esophageal squamous cell carcinoma (ESCC). Materials and Methods: Our previous gene expression data of ESCC cells and the data from GSE43732 and GSE112840 were analyzed. The expression of miR-574-5p in ESCC patients and controls was analyzed by real-time quantitative PCR. The effect of miR-574-5p on proliferation was detected by real-time cell analysis (RTCA) and EdU proliferation assay after cell transfections. The target gene small C-terminal domain phosphatase 1 (CTDSP1) of miR-574-5p was validated by luciferase reporter assay and western blotting.Results:In the current study, the bioinformatics analysis found miR-574-5p up-regulated in ESCC. The qPCR assay of 26 ESCC and 13 adjacent/ normal tissues confirmed these results. We further demonstrated that miR-574-5p overexpression promoted cell proliferation. Then the dual-luciferase reporter assay and the rescue experiment suggested that CTDSP1 was a direct target of miR-574-5p.Conclusion:MiR-574-5p played an oncological role in ESCC by interacting and negatively regulating CTDSP1. These results provided a deeper understanding of the effect of miR-574-5p on ESCC.

Studying the nature of ascending-descending-floating-sinking of Chinese medicines based on gonadotropin-releasing hormone.

to explore the nature of ascending-descending-floating-sinking of Traditional Chinese Medicine on normal rats using gonadotropin-releasing hormone (GnRh). Normal male Sprague-Dawley rats were orally administered six floating Chinese medicines and seven sinking Chinese medicines for 14 d. then, GnRh and relevant indicators were detected. Initially, the different effects of floating and sinking drugs on the body were explored. To verify the effects of floating and sinking drugs on the body, normal rats were orally administered Mahuang (Herba Ephedra Sinica) and Tinglizi (Semen Lepidii Apetali) for 14 d. Then, GnRh antagonists were administered. We observed the changes of relevant indicators and clarified the correlation between GnRh and the laws of floating and sinking. Floating Chinese medicines significantly increased the testicular coefficients; GnRh level in the serum; the protein level of GnRh receptor (GnRhR) in the testis; and the mRNA levels of androgen receptor (AR), small C-terminal domain phosphatase (SCP) 3, SCP2, SYCE1, SMC1B, SMC3, and Rec8 (P < 0.01 or < 0.05). Sinking Chinese medicines did not react similarly, while GnRh antagonists blocked the regulatory effect of Mahuang (Herba Ephedra Sinica) but did not affect Tinglizi (Semen Lepidii Apetali). GnRh may be closely related to the nature of ascending-descending-floating-sinking of Chinese medicine. Floating Chinese medicines may promote their medicinal properties by regulating GnRH level; however, sinking Chinese medicines did not affect GnRh level.

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

Cell cycle control is vital for cell proliferation in all eukaryotic organisms. The entire cell cycle can be conceptually separated into four distinct phases, Gap 1 (G1), DNA synthesis (S), G2, and mitosis (M), which progress sequentially. The precise control of transcription, in particular, at the G1 to S and G2 to M transitions, is crucial for the synthesis of many phase-specific proteins, to ensure orderly progression throughout the cell cycle. This mini-review highlights highly conserved transcriptional regulators that are shared in budding yeast (Saccharomyces cerevisiae), Arabidopsis thaliana model plant, and humans, which have been separated for more than a billion years of evolution. These include structurally and/or functionally conserved regulators cyclin-dependent kinases (CDKs), RNA polymerase II C-terminal domain (CTD) phosphatases, and the classical versus shortcut models of Pol II transcriptional control. A few of CDKs and CTD phosphatases counteract to control the Pol II CTD Ser phosphorylation codes and are considered critical regulators of Pol II transcriptional process from initiation to elongation and termination. The functions of plant-unique CDKs and CTD phosphatases in relation to cell division are also briefly summarized. Future studies towards testing a cooperative transcriptional mechanism, which is proposed here and involves sequence-specific transcription factors and the shortcut model of Pol II CTD code modulation, across the three eukaryotic kingdoms will reveal how individual organisms achieve the most productive, large-scale transcription of phase-specific genes required for orderly progression throughout the entire cell cycle.

Mammalian Ssu72 phosphatase preferentially considers tissue-specific actively transcribed gene expression by regulating RNA Pol II transcription.

Reversible phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) is essential for gene expression control. How altering the phosphorylation of the CTD contributes to gene expression in mammalian systems remains poorly understood.Methods: Primary mouse embryonic fibroblasts, hepatocytes, and embryonic stem cells were isolated from conditional Ssu72f/f mice. To knockout the mouse Ssu72 gene, we infected the cells with adenoviruses of incorporated luciferase and Cre recombinase, respectively. RNA sequencing, ChIP sequencing, ChIP assay, immunoblot analyses, qRT-PCR assay, and immunostaining were performed to gain insights into the functional mechanisms of Ssu72 loss in Pol II dynamics.Results: Using primary cells isolated from Ssu72 conditional knockout and transgenic mice, we found that mammalian Ssu72-mediated transcriptional elongation rather than polyadenylation or RNA processing contributed to the transcriptional regulation of various genes. Depletion of Ssu72 resulted in aberrant Pol II pausing and elongation defects. Reduced transcriptional elongation efficiency tended to preferentially affect expression levels of actively transcribed genes in a tissue-specific manner. Furthermore, Ssu72 CTD phosphatase seemed to regulate the phosphorylation levels of CTD Ser2 and Thr4 through accurate modulation of P-TEFb activity and recruitment.Conclusions: Our findings demonstrate that mammalian Ssu72 contributes to the transcription of tissue-specific actively transcribed gene expression by regulating reciprocal phosphorylation of Pol II CTD.

A Molecular switch for FLOWERING LOCUS C activation determines flowering time in Arabidopsis.

Plants have evolved sophisticated mechanisms to ensure flowering in favorable conditions for reproductive success. In the model plant Arabidopsis thaliana, FLOWERING LOCUS C (FLC) acts as a central repressor of flowering and the major determinant for winter cold requirement for flowering. FLC is activated in winter annuals by the FRIGIDA (FRI) activator complex containing FRI, FLC EXPRESSOR (FLX), and FLX-LIKE 4 (FLX4), among which FLX and FLX4 are also essential for establishing basal FLC expression in summer annuals. Here we show that a plant RNA polymerase II C-terminal domain phosphatase, C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (CPL3), interacts with and dephosphorylates FLX4 through their scaffold protein FLX to inhibit flowering. CPL3-mediated dephosphorylation of FLX4 serves as a key molecular switch that enables binding of dephosphorylated FLX4 to the FLC locus to promote FLC expression, thus repressing flowering in both winter and summer annuals of Arabidopsis. Our findings reveal a molecular switch underlying the activation of FLC for flowering time control.

A nuclear proteome localization screen reveals the exquisite specificity of Gpn2 in RNA polymerase biogenesis

ABSTRACT The GPN proteins are a conserved family of GTP-binding proteins that are involved in the assembly and subsequent import of RNA polymerase II and III. In this study, we sought to ascertain the specificity of yeast GPN2 for RNA polymerases by screening the localization of a collection of 1350 GFP-tagged nuclear proteins in WT or GPN2 mutant cells. We found that the strongest mislocalization occurred for RNA polymerase II and III subunits and only a handful of other RNAPII associated proteins were altered in GPN2 mutant cells. Our screen identified Ess1, an Rpb1 C-terminal domain (CTD) prolyl isomerase, as mislocalized in GPN2 mutants. Building on this observation we tested for effects of mutations in other factors which regulate Rpb1-CTD phosphorylation status. This uncovered significant changes in nuclear-cytoplasmic distribution of Rpb1-GFP in strains with disrupted RNA polymerase CTD kinases or phosphatases. Overall, this screen shows the exquisite specificity of GPN2 for RNA polymerase transport, and reveals a previously unappreciated role for CTD modification in RNAPII nuclear localization.

Abstract PS18-49: A transcriptome approach to reveal CTDP1 knockdown induced G1 cell cycle arrest in triple negative breast cancer cells

Abstract CTDP1 (C-terminal domain phosphatase 1) is the only phosphatase domain with a BRCT-domain and known to be associated with transcription and mitosis in eukaryotic systems. Our previous research showed that CTDP1 participates in the DNA damage response by mediating interstrand DNA repair through the Fanconi anemia pathway. This study also revealed that CTDP1 is an essential gene in breast cancer cell lines but not in normal breast derived epithelial MCF10A cells. This project aims to identify the changes in transcripts, kinases and interactors leading to the cell death induced by CTDP1 knockdown in the triple-negative breast cancer cell lines. Since RNA Pol II is one of the major targets of CTDP1 phosphatase activity, we initially hypothesized that the cellular changes were mediated by transcriptional regulation. The global transcriptomic profiling of shCTDP1 MDA-MB-231 cells detected more than 29000 gene products, where 1000 and 616 genes were found consistently up- and down-regulated, respectively. The downregulation of cell cycle-associated genes CCNA2, TOP2A and POLA1 in the MDA-MB-231 cells were confirmed with q-RT-PCR. While the upregulated genes did not show enrichment in the Reactome pathway database, the downregulated genes were significantly associated with DNA damage response, mitosis and transcription, which is consistent with the known functions of CTDP1. The transcription factor binding motifs analysis with iRegulon reveals that the E2F1 transcription activity decreased as CTDP1 was knocked down in MDA-MB-231. The subsequent cell cycle analysis supports this prediction and showed G1 arrest as CTDP1 is knocked down in MDA-MB-231 and MDA-MB-453, but not in MCF10A. A kinase array analysis was also conducted to further identify kinases activated or deactivated as CTDP1 is knocked down. A nested network analysis of the predicted transcription factors, kinases and CTDP1 interactors is used to identify potential pathways contributing to cell death of the triple-negative breast cancer cells upon loss of CTDP1 expression. Citation Format: Henry Chun Hin Law, Nicholas Woods. A transcriptome approach to reveal CTDP1 knockdown induced G1 cell cycle arrest in triple negative breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-49.

Methods for Identification of Substrates/Inhibitors of FCP/SCP Type Protein Ser/Thr Phosphatases

Protein phosphorylation is the most widespread type of post-translational modification and is properly controlled by protein kinases and phosphatases. Regarding the phosphorylation of serine (Ser) and threonine (Thr) residues, relatively few protein Ser/Thr phosphatases control the specific dephosphorylation of numerous substrates, in contrast with Ser/Thr kinases. Recently, protein Ser/Thr phosphatases were reported to have rigid substrate recognition and exert various biological functions. Therefore, identification of targeted proteins by individual protein Ser/Thr phosphatases is crucial to clarify their own biological functions. However, to date, information on the development of methods for identification of the substrates of protein Ser/Thr phosphatases remains scarce. In turn, substrate-trapping mutants are powerful tools to search the individual substrates of protein tyrosine (Tyr) phosphatases. This review focuses on the development of novel methods for the identification of Ser/Thr phosphatases, especially small C-terminal domain phosphatase 1 (Scp1), using peptide-displayed phage library with AlF4−/BeF3−, and discusses the identification of putative inhibitors.

WDR82/PNUTS-PP1 Prevents Transcription-Replication Conflicts by Promoting RNA Polymerase II Degradation on Chromatin

Transcription-replication (T-R) conflicts cause replication stress and loss of genome integrity. However, the transcription-related processes that restrain such conflicts are poorly understood. Here, we demonstrate that the RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatase protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS)-PP1 inhibits replication stress. Depletion of PNUTS causes lower EdU uptake, S phase accumulation, and slower replication fork rates. In addition, the PNUTS binding partner WDR82 also promotes RNAPII-CTD dephosphorylation and suppresses replication stress. RNAPII has a longer residence time on chromatin after depletion of PNUTS or WDR82. Furthermore, the RNAPII residence time is greatly enhanced by proteasome inhibition in control cells but less so in PNUTS- or WDR82-depleted cells, indicating that PNUTS and WDR82 promote degradation of RNAPII on chromatin. Notably, reduced replication is dependent on transcription and the phospho-CTD binding protein CDC73 after depletion of PNUTS/WDR82. Altogether, our results suggest that RNAPII-CTD dephosphorylation is required for the continuous turnover of RNAPII on chromatin, thereby preventing T-R conflicts.

Diverse and conserved roles of the protein Ssu72 in eukaryotes: from yeast to higher organisms.

Gene transcription is a complex biological process that involves a set of factors, enzymes and nucleotides. Ssu72 plays a crucial role in every step of gene transcription. RNA polymerase II (RNAPII) occupies an important position in the synthesis of mRNAs. The largest subunit of RNAPII, Rpb1, harbors its C-terminal domain (CTD), which participates in the initiation, elongation and termination of transcription. The CTD consists of heptad repeats of the consensus motif Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 and is highly conserved among different species. The CTD is flexible in structure and undergoes conformational changes in response to serine phosphorylation and proline isomerization, which are regulated by specific kinases/phosphatases and isomerases, respectively. Ssu72 is a CTD phosphatase with catalytic activity against phosphorylated Ser5 and Ser7. The isomerization of Pro6 affects the binding of Ssu72 to its substrate. Ssu72 can also indirectly change the phosphorylation status of Ser2. In addition, Ssu72 is a member of the 3'-end cleavage and polyadenylation factor (CPF) complex. Together with other CPF components, Ssu72 regulates the 3'-end processing of premature mRNA. Recent studies have revealed other roles of Ssu72, including its roles in balancing phosphate homeostasis and controlling chromosome behaviors, which should be further explored. In conclusion, the protein Ssu72 is an enzyme worthy of attention, not confined to its role in gene transcription.

Peripheral Inhibition of Small C‐Terminal Domain Phosphatase 1 With Napthoquinone Analogs

Small C‐terminal domain phosphatase 1(SCP1)'s biological function is significant in many cellular activities. Still, a recent study on neuroglioma cells emphasized the requirement of negative regulation of SCP1 in cancer invasion suppression. Due to the structural conservation of C‐terminal domain (CTD) phosphatases, we aimed to determine the peripherally targeting inhibitors, which reciprocally bind to an eccentric site on SCP1 using a multidisciplinary approach. From biochemical screening, we have identified two potential inhibitors, which showed twofold to threefold selectivity toward SCP1 compared to Dullard. Dullard was utilized as a negative control as it is a small CTD phosphatase that contains structural similarities to SCP1. Besides, from in silico approaches like protein–ligand docking and molecular dynamics analyses, we have successfully discovered two allosteric inhibitors of napthoquinone family compounds explicitly binding to the unique hydrophobic pocket of SCP1 from 1000 molecular dockings and 125 ns of dynamics simulation studies, respectively.

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.

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.

Tumor suppressor properties of the small C-terminal domain phosphatases in non-small cell lung cancer.

Non-Small Cell Lung Cancer (NSCLC) is responsible for the majority of deaths caused by cancer. Small C-terminal domain (CTD) phosphatases (SCP), CTDSP1, CTDSP2 and CTDSPL (CTDSPs) belong to SCP/CTDSP subfamily and are involved in many vital cellular processes and tumorigenesis. High similarity of their structures suggests similar functions. However their role in NSCLC remains insufficiently understood. For the first time we revealed the suppressor function of CTDSPs leading to a significant growth slowdown and senescence of A549 lung adenocarcinoma (ADC) cells in vitro. Their tumor-suppressive activity can be realized through increasing the proportion of the active form of Rb protein dephosphorylated at Ser807/811, Ser780, and Ser795 (P<0.05) thereby negatively regulating cancer cell proliferation. Moreover, we observed that a frequent (84%, 39/46) and highly concordant (Spearman’s rank correlation coefficient (rs) = 0.53–0.62, P≤0.01) down-regulation of CTDSPs and RB1 is characteristic of primary NSCLC samples (n=46). A clear difference in their mRNA levels was found between lung ADCs with and without lymph node metastases, but not in squamous cell carcinomas (SCCs) (P≤0.05). Based on The Cancer Genome Atlas (TCGA) data and the results obtained using the CrossHub tool, we suggest that the well-known oncogenic cluster miR-96/182/183 could be a common expression regulator of CTDSPs. Indeed, according to our qPCR, the expression of CTDSPs negatively correlates with these miRs, but positively correlates with their intronic miR-26a/b. Our results reflect functional association of CTDSP1, CTDSP2, and CTDSPL, expand knowledge about their suppressor properties through Rb dephosphorylation and provide new insights into the regulation of NSCLC growth.

Identification of a Specific Inhibitor of Human Scp1 Phosphatase Using the Phosphorylation Mimic Phage Display Method

Protein phosphatases are divided into tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. While substrate trapping mutants are frequently used to identify substrates of Tyr phosphatases, a rapid and simple method to identify Ser/Thr phosphatase substrates is yet to be developed. The TFIIF-associating component of RNA polymerase II C-terminal domain (CTD) phosphatase/small CTD phosphatase (FCP/SCP) phosphatase family is one of the three types of Ser/Thr protein phosphatases. Defects in these phosphatases are correlated with the occurrence of various diseases such as cancer and neuropathy. Recently, we developed phosphorylation mimic phage display (PMPD) method with AlF4−, a methodology to identify substrates for FCP/SCP type Ser/Thr phosphatase Scp1. Here, we report a PMPD method using BeF3− to identify novel substrate peptides bound to Scp1. After screening peptide phages, we identified peptides that bound to Scp1 in a BeF3−-dependent manner. Synthetic phosphopeptide BeM12-1, the sequence of which was isolated at the highest frequency, directly bound to Scp1. The binding was inhibited by adding BeF3−, indicating that the peptide binds to the active center of catalytic site in Scp1. The phosphorylated BeM12-1 worked as a competitive inhibitor of Scp1. Thus, PMPD method may be applicable for the identification of novel substrates and inhibitors of the FCP/SCP phosphatase family.

PTEN interacts with RNA polymerase II to dephosphorylate polymerase II C-terminal domain.

Gene transcription is a highly complex and strictly regulated process. RNA polymerase II (Pol II) C-terminal domain (CTD) undergoes massive cycles of phosphorylation and dephosphorylation during the process of gene transcription. These post-translational modifications of CTD provide an interactive platform for various factors required for transcription initiation, elongation, termination, and co-transcriptional RNA processing. Pol II CTD kinases and phosphatases are key regulators and any deviation may cause genome-wide transcriptional dysregulation leading to various pathological conditions including cancer. PTEN, a well known tumor suppressor, is one of the most commonly somatically altered in diverse malignancies. When mutated in the germline, PTEN causes cancer predisposition. Numerous studies have demonstrated that PTEN regulates the expression of hundreds of genes, however, no mechanism is known so far. PTEN is a dual specificity phosphatase, using both lipid and protein as substrates. In the present study, we demonstrate that PTEN interacts with the RNA Pol II and that PTEN expression is inversely correlated with global phosphorylation of Pol II CTD. Furthermore, PTEN dephosphorylates Pol II CTD in vitro with a significant specificity for Ser5p. Interestingly, ChIP-seq data analysis revealed that PTEN globally binds to promoter proximal regions, and PTEN loss increases genome-wide Pol II Ser5p occupancy, suggest that PTEN is a Pol II CTD phosphatase. Our observations demonstrate an unexplored function of PTEN with the potential of global transcriptional regulation, adding a new dimension to somatic carcinogenesis and germline cancer predisposition.