IntroductionRegulation of signal transducer and activator of transcription 1 (STAT1) involves posttranslational modifications such as phosphorylation of Tyr701 and Ser727 as well as arginine methylation. 1,2 STAT1 signaling is negatively regulated by protein tyrosine phosphatases (PTPs), suppressors of cytokine signaling (SOCS), and protein inhibitor of activated STAT1 (PIAS) proteins. 1-3 The family of PIAS proteins consists of 5 members that have been implicated in the regulation of several nuclear proteins. PIAS1 and PIAS3 were identified as interaction partners for STAT1 and STAT3, respectively, and they were found to inhibit the DNA-binding activity of activated STATs. [3][4][5] The other members, PIASx␣/ARIP3 (androgen receptor-interacting protein 3), PIASx/ Miz1, and PIASy, function as transcriptional coregulators for steroid hormone receptors. [6][7][8] Recently, several PIAS proteins have been shown to function as E3-type small ubiquitin-like modifier (SUMO) ligases. 9-12 SUMO-1, -2, and -3 are small modifier proteins that are covalently conjugated to specific lysine residues of target proteins. 13 A number of nuclear proteins, such as transcription factors AR, p53, and c-Jun, become modified by SUMO-1 conjugation, and this reversible posttranslational modification has been implicated in regulation of proteinprotein interactions, protein stability, localization, or activity. 9,13,14 However, it is currently unknown whether STAT factors are modified through sumoylation. Study design ReagentsAntibodies used include anti-SUMO-1 (mouse anti-GMP-1) (Zymed, San Francisco, CA); anti-HA (clone 16B12) (Berkeley-Antibody, Richmond, CA); anti-Flag (anti-Flag M2) (Sigma Aldrich, St Louis, MO); anti-STAT1 (N terminus) (Transduction Laboratories, BD Biosciences); anti-STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA) biotinylated antimouse (Dako, Glustrup, Denmark) and streptavidin-biotin horseradish peroxidase conjugate (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Human interferon-␥ (huIFN-␥) was purchased from Immugenex, Los Angeles, CA. Plasmid constructsSUMO-1-Flag and SUMO-1-Flag-Flag were a kind gift from Dr H. Yasuda. 15 The SUMO-1, Flag-PIAS1, Flag-PIAS1mut (PIAS1⌬310-407), Flag-PIAS3, Flag-ARIP3 Flag-ARIP3mut (ARIP3⌬347-418) plasmids 4,9 as well as STAT1-WT-HA and GAS-luc reporter construct have been previously described. 16 The STAT1 Lys703Arg mutation was created from STAT1-WT-HA using direct polymerase chain reaction (PCR) mutagenesis with the following primers: 5ЈGGAACTGGATATATCAGGACTGAGTTGATTTCTGTGTCTG-3Ј and 5Ј-CAGACACAGAAATCAACTCAGTCCTGATATATCCAGTTCC-3Ј. Transfections, luciferase assay, and immunodetectionCOS-7 cells were electroporated using a Bio-Rad (Hercules, CA) gene pulser at 260 V and 960 microfarads (F) and lysed in Triton X lysis buffer supplemented with 5 mM N-ethylmaleimide (NEM) (Sigma Aldrich). HeLa cells were transfected using a calcium phosphate method. For luciferase assay, U3A cells were transfected using a calcium phosphate method as described. 16 Immunodetection wa...
Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatasemediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response. Mechanistically, PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and protein kinase C. In malignant gliomas, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (n = 222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells and suggest an important functional role for PME-1 in the disease progression of human astrocytic gliomas.
IntroductionSignal transducers and activators of transcription 1 (STAT1) is critical for interferon-gamma (IFN-␥)-mediated immune responses, and its activation requires phosphorylation on tyrosine 701 by the receptorassociated Janus kinases (JAKs). 1 The function of STAT1 is also modulated by other posttranslational modifications, such as phosphorylation of Ser727 and, more recently, sumoylation at Lys703. [2][3][4] Small ubiquitin-like modifier (SUMO)-1, -2, -3, and -4 are protein moieties covalently conjugated to specific lysine residues on substrate proteins through an enzymatic pathway. 5 Recently, the regulatory enzymes in these reactions have been characterized, and protein inhibitors of activated STAT (PIAS) proteins, initially identified as regulators of STAT and androgen receptor activation, were shown to function as E3-type SUMO ligases. 6,7 Sumoylation mediates divergent effects on transcription factors and can cause transcriptional repression or enhanced activation. 8 Previously, we and others showed that PIAS proteins promote the sumoylation of STAT1 at Lys703, but the effect of the Lys703 mutation on reporter gene responses varied, leaving the functional role of this modification elusive. 3,4 This study aimed to analyze the functional role of sumoylation in STAT1, and our results indicate that sumoylation mediates a negative, promoter-dependent regulatory function in STAT1-mediated transcription. Study design Reagents PlasmidsThe SUMO-1, STAT1-wild-type-HA (STAT1-WT-HA), and STAT1-KR-HA plasmids have been previously described. 3 STAT1-I702R-HA (Ile3Arg) and STAT1-E705A-HA (Glu3Ala) were constructed from STAT1-WT-HA using polymerase chain reaction (PCR) mutagenesis with the following primers: 5Ј-GGAACTGGATAT-AGGAAGACTGAGTTGATTTCTGTGTCTGAA-3Ј and 5Ј-TTCAGA-CACAGAAATCAACTCAGTCTTCCTATATCCAGTTCC-3Ј; 5Ј-GGA-ACTGGATATATCAAGACTGCGTTGATTTCTGTGTCTGAA-3Ј and 5Ј-TTCAGACACAGAAATCAACGCAGTCTTGATATATCCAGTTCC-3Ј. Transfections and immunodetectionCOS-7 cells were transfected using Fugene6 reagent and lysed in Triton-X lysis buffer supplemented with 5 mM NEM (N-ethylmaleimide; SigmaAldrich). HeLa cells were transfected using the calcium phosphate method. Immunodetection was performed as described. 3 Quantitative RT-PCR and EMSATotal RNA was extracted using TRIZOL (Gibco-BRL, Carlsbad, CA). Reverse transcription (RT) was performed using a First strands cDNA synthesis kit (MBI Fermentas, Burlington, ON, Canada). The primers for For personal use only. on May 9, 2018. by guest www.bloodjournal.org From glyceraldehyde phosphate dehydrogenase (GAPDH), interferon regulatory factor 1 (IRF1), guanylate-binding protein 1 (GBP1), and transporters associated with antigen presentation 1 (TAP1) real-time PCR were previously described. 2 Detection of IFN-␥-activated sequence (GAS)-binding proteins by electrophoretic mobility shift assay (EMSA) has been previously described. 9 Immunofluorescence detectionStably transfected U3A clones were serum starved and stimulated with 100 ng/mL huIFN-␥. Cells were fixed in p-formaldehyde (4%) a...
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