Regulation of STAT1 nuclear export by Jak1

Abstract

Signal transducer and activator of transcription 1 (STAT1) mediates gene expression in response to cytokines and growth factors. Activation of STAT1 is achieved through its tyrosine phosphorylation, a process that involves Jak tyrosine kinases. Here we show that STAT1, although phosphorylated on Y701, is unable to localize in the nucleus in the absence of Jak1 or Jak1 kinase activity. In contrast, the nuclear accumulation of STAT1 in Tyk2-deficient cells remains intact. Nuclear presence of tyrosine-phosphorylated STAT1 could be restored in Jak1-deficient cells by leptomycin B, an inhibitor of nuclear export. Amino acids 197 to 205 of STAT1 were found to encode a leucine-rich nuclear export signal (NES). An L-->A mutation within the NES restored nuclear retention of STAT1 in Jak1-deficient cells. Impaired binding of the transcriptional coactivator CBP to tyrosine-phosphorylated STAT1 derived from Jak1-deficient cells offers a model for the intermolecular regulation of the nuclear export sequence.

FIG. 1

Priming with h/v restores IFN-dependent activation of STAT1 in Jak1^−/− and Tyk2^−/− cells. Parental 2fTGH (A), Tyk2^−/− U1A (B), and Jak1^−/− U4A (C) cells were either left untreated (lanes 1), stimulated for 30 min with 500 U of IFN-α per ml (lanes 2) or pretreated with h/v for 30 min followed by an additional 30 min of incubation without (lanes 3) or with (lanes 4) IFN-α (500 U/ml). Total cell extracts (CTL) were prepared and subject to EMSA using end-labeled GRR as a probe.

FIG. 2

Tyrosine-phosphorylated STAT1 does not accumulate in the nucleus in Jak1^−/− cells. (A) Parental 2fTGH, Tyk2^−/− U1A, and Jak1^−/− U4A cells were treated as in Fig. 1. Subcellular distribution of STAT1 was detected using a monoclonal antibody against STAT1. (B) Jak1^−/− U4A cells reconstituted with wild-type (WT) or kinase-inactive Jak1 were treated as in Fig. 1, and subcellular distribution of STAT1 was analyzed. (C and D) Wild-type (WT) and Jak1^−/− HeLa cells were stimulated with h/v and IFN-α (C) or EGF (2 ng/ml) (D) for 30 min, and the compartmentalization of STAT1 was analyzed. The + and − signs indicate the presence and absence, respectively, of STAT1 tyrosine phosphorylation after the indicated treatments.

FIG. 3

Nuclear accumulation of tyrosine-phosphorylated STAT1 in Jak1^−/− cells after addition of LMB. Parental 2fTGH and Jak1^−/− U4A cells were primed with h/v as in Fig. 1 without or with prior addition (90 min) of 100 nM LMB. Cells were stimulated with IFN-α (500 U/ml) for 30 min and STAT1 localization was analyzed as in Fig. 2. The + and − signs indicate the presence and absence, respectively, of STAT1 tyrosine phosphorylation after the indicated treatments.

FIG. 4

Amino acids 197 to 205 of STAT1 encode a functional NES. (A) Primary human foreskin fibroblasts were transiently transfected with either parental GFP plasmid (top left), GFP-STAT1^519–528 (top right), or GFP-STAT1^197–205 (bottom panels). In addition, cells expressing GFP-STAT1^197–205 were also incubated with 100 nM LMB for 90 min prior to visualization of the fusion proteins (bottom right). (B) Wild-type (WT) and Jak1^−/− HeLa cells were transiently transfected with either STAT1-GFP or STAT1 (L¹⁹⁹→A)-GFP, and localization of the fusion proteins after stimulation with h/v and IFN-β (1,000 U/ml) was determined. (C) The crystal structure of tyrosine-phosphorylated STAT1 bound to DNA was downloaded from the Protein Data Bank of the Brookhaven National Laboratory (1, 4), and the positions of individual amino acids were identified using Chemscape Chime. The highly exposed NES (boxed) is located at the start of the second α-helix.

FIG. 5

Impaired interaction of STAT1 and CBP and STAT1-mediated gene induction in Jak1^−/− cells. (A) Wild-type (WT) and Jak1^−/− HeLa cells were stimulated with h/v and IFN-α, and the lysates were incubated with glutathione-agarose-conjugated GST fusion proteins representing the KIX domain (lanes 1 and 4), the SE region (lanes 2 and 5), or the C/H3 domain (lanes 3 and 6) of CBP. Affinity-precipitated proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride, and probed for the presence of STAT1. (B) Wild-type and Jak1^−/− HeLa cells were stimulated with h/v and IFN-α in the presence of LMB, and ISG54 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were analyzed by RNase protection assay.