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J. Biol. Chem., Vol. 281, Issue 6, 3244-3253, February 10, 2006

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Activation of the RNA-dependent Protein Kinase PKR Promoter in the Absence of Interferon Is Dependent Upon Sp Proteins^*

Sonali Das, Simone V. Ward¹, Robert S. Tacke, Guntrum Suske, and Charles E. Samuel²

From the Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California 93106 and Institute of Molecular Biology and Tumor Research, Philipps-University, Emil-Mannkopff-Strasse 2, D-35033 Marburg, Germany

The protein kinase regulated by RNA (PKR) is interferon (IFN)-inducible and plays important roles in many cellular processes, including virus multiplication, cell growth, and apoptosis. The TATA-less PKR promoter possesses a novel 15-bp DNA element (kinase conserved sequence (KCS)) unique to the human and mouse PKR genes that is conserved in sequence and position. We found that Sp1 and Sp3 of the Sp family of transcription factors bind at the KCS element. Their involvement was analyzed in the activation of basal and IFN-inducible PKR promoter activity. Both the small and large isoforms of Sp3 co-purified with KCS protein binding activity (KBP) by using nuclear extracts from HeLa cells not treated with IFN. Two forms of the KCS-binding protein complex were demonstrated by electrophoretic mobility shift assay analysis; one contained Sp1 and the other Sp3. In mouse cells null for all Sp3 isoforms, PKR expression was reduced to 50% that of wild-type cells in the absence of IFN. The IFN-inducible expression of PKR, however, was Sp3-independent but STAT1- and JAK1-dependent. Overexpression of Sp1 in human U cells resulted in increased PKR promoter activity. In Drosophila SL2 cells lacking Sp proteins, both Sp1 and Sp3 large but not small isoforms activated PKR promoter expression, with the Sp1-mediated activation dominant. Mutational analysis of the PKR promoter region indicated a cooperative interaction between two different Sp sites, one of which is within the KCS element. These results establish that, in the absence of IFN treatment, activation of PKR basal expression is mediated by Sp1 and Sp3 proteins in a cooperative manner.

Received for publication, September 28, 2005 , and in revised form, November 21, 2005.

^* This work was supported in part by NIAID Research Grant AI-20611 from the United States Public Health Service, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Laboratory of Genetics, Salk Institute for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, CA 92037.

² To whom correspondence should be addressed. Tel.: 805-893-3097; Fax: 805-893-5780; E-mail: samuel{at}lifesci.ucsb.edu.

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