Small C-terminal Domain Phosphatase Research Articles

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.

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.

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.

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.

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.

Phosphatase activity of small C-terminal domain phosphatase 1 (SCP1) controls the stability of the key neuronal regulator RE1-silencing transcription factor (REST)

The RE1-silencing transcription factor (REST) is the major scaffold protein for assembly of neuronal gene silencing complexes that suppress gene transcription through regulating the surrounding chromatin structure. REST represses neuronal gene expression in stem cells and non-neuronal cells, but it is minimally expressed in neuronal cells to ensure proper neuronal development. Dysregulation of REST function has been implicated in several cancers and neurological diseases. Modulating REST gene silencing is challenging because cellular and developmental differences can affect its activity. We therefore considered the possibility of modulating REST activity through its regulatory proteins. The human small C-terminal domain phosphatase 1 (SCP1) regulates the phosphorylation state of REST at sites that function as REST degradation checkpoints. Using kinetic analysis and direct visualization with X-ray crystallography, we show that SCP1 dephosphorylates two degron phosphosites of REST with a clear preference for phosphoserine 861 (pSer-861). Furthermore, we show that SCP1 stabilizes REST protein levels, which sustains REST's gene silencing function in HEK293 cells. In summary, our findings strongly suggest that REST is a bona fide substrate for SCP1 in vivo and that SCP1 phosphatase activity protects REST against degradation. These observations indicate that targeting REST via its regulatory protein SCP1 can modulate its activity and alter signaling in this essential developmental pathway.

Emerging roles of nuclear phosphatase SCP4 in CKD-associated muscle wasting

Cachexia with wasting of muscle protein is a serious complication of chronic kidney disease (CKD). Muscle protein phosphorylation is a potential therapeutic target. Liu etal. reported that small C-terminal domain phosphatase (SCP) 4 was increased in muscles of patients and mice with CKD. Importantly, knockdown of SCP4 significantly ameliorated muscle wasting in CKD mice. Inhibition of SCP4 may represent a novel therapeutic intervention for muscle wasting in patients with CKD.

Two-track virtual screening approach to identify both competitive and allosteric inhibitors of human small C-terminal domain phosphatase 1.

Despite a wealth of persuasive evidence for the involvement of human small C-terminal domain phosphatase 1 (Scp1) in the impairment of neuronal differentiation and in Huntington's disease, small-molecule inhibitors of Scp1 have been rarely reported so far. This study aims to the discovery of both competitive and allosteric Scp1 inhibitors through the two-track virtual screening procedure. By virtue of the improvement of the scoring function by implementing a new molecular solvation energy term and by reoptimizing the atomic charges for the active-site Mg2+ ion cluster, we have been able to identify three allosteric and five competitive Scp1 inhibitors with low-micromolar inhibitory activity. Consistent with the results of kinetic studies on the inhibitory mechanisms, the allosteric inhibitors appear to be accommodated in the peripheral binding pocket through the hydrophobic interactions with the nonpolar residues whereas the competitive ones bind tightly in the active site with a direct coordination to the central Mg2+ ion. Some structural modifications to improve the biochemical potency of the newly identified inhibitors are proposed based on the binding modes estimated with docking simulations.

The nuclear phosphatase SCP4 regulates FoxO transcription factors during muscle wasting in chronic kidney disease

Chronic kidney disease (CKD) and related inflammatory responses stimulate protein-energy wasting, a complication causing loss of muscle mass. Primarily, muscle wasting results from accelerated protein degradation via autophagic/lysosomal and proteasomal pathways, but mechanisms regulating these proteolysis pathways remain unclear. Since dephosphorylation of FoxOs regulates ubiquitin/proteasome protein metabolism, we tested whether a novel nuclear phosphatase, the small C-terminal domain phosphatase (SCP) 4, regulates FoxOs signaling and, in turn, muscle wasting. In cultured mouse myoblast cells, SCP4 overexpression stimulated proteolysis, while knockdown of SCP4 prevented the proteolysis stimulated by inflammatory cytokines. SCP4 overexpression led to nuclear accumulation of FoxO1/3a followed by increased expression of catabolic factors including myostatin, Atrogin-1, and MuRF-1, and induction of lysosomal-mediated proteolysis. Treatment of C2C12 myotubes with proinflammatory cytokines stimulated SCP4 expression in an NF-κB-dependent manner. In skeletal muscle of mice with CKD, SCP4 expression was up-regulated. Similarly, in skeletal muscle of patients with CKD, SCP4 expression was significantly increased. Knockdown of SCP4 significantly suppressed FoxO1/3a-mediated expression of Atrogin-1 and MuRF-1 and prevented muscle wasting in mice with CKD. Thus, SCP4 is a novel regulator of FoxO transcription factors and promotes cellular proteolysis. Hence, targeting SCP4 may prevent muscle wasting in CKD and possibly other catabolic conditions.

The Small C-terminal Domain Phosphatase 1 Inhibits Cancer Cell Migration and Invasion by Dephosphorylating Ser(P)68-Twist1 to Accelerate Twist1 Protein Degradation

Twist1 is a basic helix-loop-helix transcription factor that strongly promotes epithelial-to-mesenchymal transition, migration, invasion, and metastasis of cancer cells. The MAPK-phosphorylated Twist1 on its serine 68 (Ser(P)(68)-Twist1) has a significantly enhanced stability and function to drive cancer cell invasion and metastasis. However, the phosphatase that dephosphorylates Ser(P)(68)-Twist1 and destabilizes Twist1 has not been identified and characterized. In this study, we screened a serine/threonine phosphatase cDNA expression library in HEK293T cells with ectopically coexpressed Twist1. We found that the small C-terminal domain phosphatase 1 (SCP1) specifically dephosphorylates Ser(P)(68)-Twist1 in both cell-free reactions and living cells. SCP1 uses its amino acid residues 43-63 to interact with the N terminus of Twist1. Increased SCP1 expression in cells decreased Ser(P)(68)-Twist1 and total Twist1 proteins, whereas knockdown of SCP1 increased Ser(P)(68)-Twist1 and total Twist1 proteins. Furthermore, the levels of SCP1 are negatively correlated with Twist1 protein levels in several cancer cell lines. SCP1-dephosphorylated Twist1 undergoes fast degradation via the ubiquitin-proteasome pathway. Importantly, an increase in SCP1 expression in breast cancer cells with either endogenous or ectopically expressed Twist1 largely inhibits the Twist1-induced epithelial-to-mesenchymal transition phenotype and the migration and invasion capabilities of these cells. These results indicate that SCP1 is the phosphatase that counterregulates the MAPK-mediated phosphorylation of Ser(68)-Twist1. Thus, an increase in SCP1 expression and activity may be a useful strategy for eliminating the detrimental roles of Twist1 in cancer cells.

Erratum to: Cellular Prion Protein Promotes Neuronal Differentiation of Adipose-Derived Stem Cells by Upregulating miRNA-124.

The cellular prion protein (PrP(C)) is a highly conserved glycoprotein anchored by glycosylphosphatidylinositol (GPI) to the cell surface and is also the source of pathogenic agent of scrapie prion protein (PrP(Sc)). Numerous researches have suggested putative physiological roles for PrP(C), including protection from ischemic and excitotoxic lesions, and participation in cell signaling and differentiation. Here, we demonstrated that PrP(C) positively regulates neuronal differentiation of mouse adipose-derived stem cells (ADSCs). The small C-terminal domain phosphatase 1 (SCP1) expression was knocked down by gene silencing. The mRNA expression of miRNA-124 and PrP(C) was measured with quantitative PCR. Western blot analysis was used to detect the protein levels of nestin, βIII-tubulin, and SCP1, and dual-luciferase reporter assay was performed to test the target of miRNA-124. The expression level of PrP(C) was found to increase steadily during neuron-like differentiation process, and PrP(C) knockout resulted in the reduction of neuron-like cell markers. We further showed that miRNA-124 could directly target SCP1-3'-untranslated region to decrease small C-terminal domain phosphatase 1 (SCP1) SCP1, and that miRNA-124 expression is regulated by PrP(C). Our results suggest that PrP(C) may play a key role in the neuronal differentiation of ADSC through modulating miRNA-124-SCP1 axis. To date, this is the first time strong evidence for the involvement of PrP(C) in the neuronal differentiation of ADSC is reported.

Cellular Prion Protein Promotes Neuronal Differentiation of Adipose-Derived Stem Cells by Upregulating miRNA-124.

The cellular prion protein (PrP(C)) is a highly conserved glycoprotein anchored by glycosylphosphatidylinositol (GPI) to the cell surface and is also the source of pathogenic agent of scrapie prion protein (PrP(Sc)). Numerous researches have suggested putative physiological roles for PrP(C), including protection from ischemic and excitotoxic lesions, and participation in cell signaling and differentiation. Here, we demonstrated that PrP(C) positively regulates neuronal differentiation of mouse adipose-derived stem cells (ADSCs). The small C-terminal domain phosphatase 1 (SCP1) expression was knocked down by gene silencing. The mRNA expression of miRNA-124 and PrP(C) was measured with quantitative PCR. Western blot analysis was used to detect the protein levels of nestin, βIII-tubulin, and SCP1, and dual-luciferase reporter assay was performed to test the target of miRNA-124. The expression level of PrP(C) was found to increase steadily during neuron-like differentiation process, and PrP(C) knockout resulted in the reduction of neuron-like cell markers. We further showed that miRNA-124 could directly target SCP1-3'-untranslated region to decrease small C-terminal domain phosphatase 1 (SCP1) SCP1, and that miRNA-124 expression is regulated by PrP(C). Our results suggest that PrP(C) may play a key role in the neuronal differentiation of ADSC through modulating miRNA-124-SCP1 axis. To date, this is the first time strong evidence for the involvement of PrP(C) in the neuronal differentiation of ADSC is reported.

Human SCP4 is a chromatin-associated CTD phosphatase and exhibits the dynamic translocation during erythroid differentiation.

The C-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit contains tandem repeats of the heptapeptide, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD is subject to dynamic phosphorylation during transcription, mainly at serine residues (Ser2, Ser5 and Ser7). Regulation of CTD phosphorylation by specific kinases and phosphatases is crucial for coordinating transcription with RNA processing and histone modification. Human small CTD phosphatase 4 (SCP4), also called CTDSPL2 or HSPC129, is a putative CTD phosphatase belonging to the FCP/SCP family and implicated in control of ε- and γ-globin gene expression. Here, we report the biochemical and functional characterization of SCP4. SCP4 exhibited Ser5-preferential CTD phosphatase activity in vitro, while small interfering RNA-mediated SCP4 knockdown in HeLa cells increased phosphorylation levels of Pol II at Ser5 and Ser7, but not at Ser2. Furthermore, cell fractionation, chromatin immunoprecipitation and immunofluorescence assays revealed an exclusive localization for SCP4 in the chromatin, particularly at transcriptionally silenced chromosomal regions. Interestingly, SCP4 was gradually released from the chromatin fraction during hemin-induced erythroid differentiation of K562 cells, with concomitant cytoplasmic accumulation. Therefore, SCP4 is a unique chromatin-associated, Ser5-preferential CTD phosphatase that preferentially distributes to transcriptionally silenced gene regions and may participate in gene regulation during erythroid differentiation.

Small C-terminal Domain Phosphatase 3 Dephosphorylates the Linker Sites of Receptor-regulated Smads (R-Smads) to Ensure Transforming Growth Factor β (TGFβ)-mediated Germ Layer Induction in Xenopus Embryos

Germ layer induction is one of the earliest events shortly after fertilization that initiates body formation of vertebrate embryos. In Xenopus, the maternally deposited transcriptional factor VegT promotes the expression of zygotic Nodal/Activin ligands that further form a morphogen gradient along the vegetal-animal axis and trigger the induction of the three germ layers. Here we found that SCP3 (small C-terminal domain phosphatase 3) is maternally expressed and vegetally enriched in Xenopus embryos and is essential for the timely induction of germ layers. SCP3 is required for the full activation of Nodal/Activin and bone morphogenetic protein signals and functions via dephosphorylation in the linker regions of receptor-regulated Smads. Consistently, the linker regions of receptor-regulated Smads are heavily phosphorylated in fertilized eggs, and this phosphorylation is gradually removed when embryos approach the midblastula transition. Knockdown of maternal SCP3 attenuates these dephosphorylation events and the activation of Nodal/Activin and bone morphogenetic protein signals after midblastula transition. This study thus suggested that the maternal SCP3 serves as a vegetally enriched, intrinsic factor to ensure a prepared status of Smads for their activation by the upcoming ligands during germ layer induction of Xenopus embryos.

Selective Dephosphorylation by SCP1 and PP2A in Phosphorylated Residues of SMAD2

E-mail: ykim@kku.ac.krReceived July 1, 2014, Accepted July 31, 2014Key Words : Small CTD phosphatases, SCP1, SMAD2, Dephosphorylation, SMAD phosphataseProtein phosphatase is important for cellular events, and afamily of protein phosphatases, the so-called C-terminaldomain (CTD) of RNA polymerase II (RNAPII) phos-phatases, has recently attracted attention. The CTD is thelargest subunit of RNAPII and consists of a tandem repeatedheptapeptide (Y

Over-expression in E. coli and purification of functional full-length murine small C-terminal domain phosphatase (SCP1, or Golli-interacting protein)

During myelination in the central nervous system, proteins arising from the gene in the oligodendrocyte lineage (golli) participate in diverse events in signal transduction and gene regulation. One of the interacting partners of the Golli-isoform BG21 was discovered by yeast-2-hybrid means and was denoted the Golli-interacting-protein (GIP). In subsequent in vitro studies of recombinant murine GIP, it was not possible to produce a full-length version of recombinant murine rmGIP in functional form under native conditions, primarily because of solubility issues, necessitating the study of a hexahistidine-tagged, truncated form ΔN-rmGIP. This protein is an acidic phosphatase belonging to the family of RNA-polymerase-2, small-subunit, C-terminal phosphatases (SCP1), and studies of the human ortholog hSCP1 have also been performed on truncated forms. Here, a new SUMO-expression and purification protocol has been developed for the preparation of a functional, full-length mSCP1/GIP (our nomenclature henceforth), with no additional purification tags. Both full-length mSCP1/GIP and the truncated murine form (now denoted ΔN-rmSCP1/GIP) had similar melting temperatures, indicating that the integrity of the catalytic core per se was minimally affected by the N-terminus. Characterization of mSCP1/GIP activity with the artificial substrate p-NPP (p-nitrophenylphosphate) yielded kinetic parameters comparable to those of ΔN-rmSCP1/GIP and the truncated human ortholog ΔN-hSCP1. Similarly, mSCP1/GIP dephosphorylated a more natural CTD-peptide substrate (but not protein kinase C-phosphorylated BG21) with comparable kinetics to ΔN-hSCP1. The successful production of an active, full-length mSCP1/GIP will enable future evaluation of the functional role of its N-terminus in protein–protein interactions (e.g., BG21) that regulate its phosphatase activity.

Regulatory effect of the glial Golli-BG21 protein on the full-length murine small C-terminal domain phosphatase (SCP1, or Golli-interacting protein)

The gene in the oligodendrocyte lineage (golli) encodes a number of proteins essential for myelination, comprising Golli and classic isoforms that are expressed in a developmentally-regulated manner. The Golli-interacting-protein (GIP) was previously discovered in a search for potential interacting partners of the Golli-isoform BG21, and was realised to be an acidic phosphatase belonging to the family of RNA-polymerase-2, small-subunit, C-terminal phosphatases (viz., SCP1). Here, we refer to this protein as mSCP1/GIP. In subsequent in vitro studies of recombinant murine SCP1/GIP, the inability to produce an active full-length version of the protein under native conditions necessitated the study of a truncated form ΔN-rmSCP1/GIP, but with inconclusive results regarding its interaction with BG21 [13]. We have since developed a new SUMO-expression and purification protocol for the preparation of a functional, full-length mGIP/SCP1, with no additional purification tags. Here, the interaction between mSCP1/GIP (with intact N-terminus) and BG21 is shown to be different than for the truncation mutant studied previously. Specifically, this interaction shows a dual effect on the enzymatic activity of mSCP1/GIP by BG21: BG21 enhanced mSCP1/GIP phosphatase activity (Ka=30μM), whereas PKCα-phosphorylated BG21 inhibited its activity (Ki=2.9μM), suggesting a potential role of BG21 as a molecular switch (“quick-brake mechanism”) on mSCP1/GIP. The successful production of an active, full-length mSCP1/GIP thus demonstrates a role for its N-terminus in regulation of phosphatase activity, in events such as the regulation of transcription in oligodendrocytes.