Nuclear phosphatases and the proteasome in suppression of STAT1 activity in hepatocytes q Dongxu Liu, a Jennifer Scafidi, a Anne E. Prada, b Kamyar Zahedi, b and Alvin E. Davis III a, * a The Center for Blood Research, Harvard Medical School, 800 Huntington Avenue, Boston, MA 02115, USA b The Division of Nephrology, Children’s Hospital Research Foundation, University of Cincinnati, Cincinnati, OH 45229, USA Received 25 October 2002 Abstract IFN-c induction of C1 inhibitor (C1INH) is mediated by an IFN-c-activated sequence (GAS), via binding of signal transducer and activator of transcription 1 (STAT1). These studies focused on the factors responsible for down-regulation of nuclear STAT1 in hepatocytes, the primary site of synthesis of C1INH. The activity of nuclear STAT1 following stimulation with IFN-c was sustained with the phosphatase inhibitor, pervanadate, or the proteasome inhibitor, lactacystin. Pervanadate prolonged STAT1 activation and blocked the inactivation of nuclear STAT1. Binding of ubiquitin to phosphorylated STAT1 was detectable in cells treated with lactacystin. Staurosporine only moderately decreased the prolongation of nuclear phosphorylated STAT1 after pretreatment with pervanadate or lactacystin. An antisense mitogen-activated protein kinase phosphatase (MKP-1) oligonucleotide prolonged the accumulation of phosphorylated STAT1. These data are consistent with the hypothesis that down-regulation of IFN-c-mediated nuclear STAT1 binding in hepatocytes involves both dephosphorylation by MKP-1 and degradation via proteolysis by the ubiquitin-dependent proteasome pathway. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: C1 inhibitor; Interferon-c; Signal transducer and activator of transcription-1; Phosphatases; Mitogen-activated protein kinase phosphatase; Proteasome; Ubiquitin A wide variety of stimuli, including cytokines and growth factors, regulate many aspects of cellular growth, differentiation, and activation via the signaling pathway involving the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) [1– 5]. By binding to their cell surface receptors, many dif- ferent cytokines and growth factors specifically activate one or more members of the family of latent STATs [1]. IFN-a induces the activation of JAK1 and TYK2 pro- tein kinases followed by the phosphorylation of STAT1 and STAT2, whereas IFN-c induces activation of JAK1 and JAK2 followed by the phosphorylation of only STAT1 [6]. Upon phosphorylation, STAT1 forms ho- modimers, translocates to the nucleus, and binds to IFN-c-activated sequences (GAS). Transcription acti- vation by STAT1 can be terminated by the action of nuclear protein tyrosine phosphatases (12–14, 35). It has been demonstrated that a nuclear tyrosine phosphatase plays a major role in STAT1 inactivation in human fi- broblasts [7,8]. The proteasome, a multi-subunit prote- ase that degrades ubiquitin-tagged proteins, degrades a number of transcription factors including c-fos and c-jun [9,10]. Evidence for involvement of the proteasome in STAT1 down-regulation included the demonstration that the proteasome inhibitor, lactacystin, stabilized IFN-c induced activated STAT1 in HeLa cells [11]. However, other data using fibroblasts have suggested that the role of this pathway is minor [8]. It is possible that the relative roles of these potential mechanisms may vary in different cell types. Treatment of hepatocytes, monocytes, endothelial cells, or fibroblasts, with IFN-a or c increases expression Biochemical and Biophysical Research Communications 299 (2002) 574–580 www.academicpress.com BBRC q Abbreviations: C1INH, C1 inhibitor; IFN, interferon; STAT1, signal transducers and activator of transcription 1; GAS, IFN-c- activated sequence; JAK, Janus kinase; MKP-1, mitogen-activated protein kinase phosphatase 1; EMSA, electrophoretic mobility shift assay; DMEM, DulbeccoÕs minimum essential medium; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide. * Corresponding author. Fax: 1-617-278-3490. E-mail address: aldavis@cbr.med.harvard.edu (A.E. Davis III). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)02694-3