| pmid | 10973496 | ||||
|---|---|---|---|---|---|
| title | Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain. | ||||
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| journal | Proc Natl Acad Sci U S A | ||||
| year | 2000 | ||||
| full_text_available | true | ||||
| full_text_extraction_method | html | ||||
| pmcid | PMC27039 | ||||
| doi | 10.1073/pnas.190318397 |
Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain.
Authors: Begitt A, Meyer T, van Rossum M, Vinkemeier U Journal: Proc Natl Acad Sci U S A (2000) DOI: 10.1073/pnas.190318397 PMC: PMC27039
- Proc Natl Acad Sci U S A. 2000 Sep 12;97(19):10418-23. doi: 10.1073/pnas.190318397.
Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain.
Begitt A(1), Meyer T, van Rossum M, Vinkemeier U.
Author information: (1)Nachwuchsgruppe Zelluläre Signalverarbeitung, Forschungsinstitut für Molekulare Pharmakologie, and Freie Universität, Institut für Kristallographie, D-10315 Berlin, Germany.
Signal transducer and activator of transcription (Stat) proteins are latent transcription factors that reside in the cytoplasm before activation. On cytokine-induced tyrosine phosphorylation, these molecules dimerize and accumulate transiently in the nucleus. No specific signals mediating these processes have been identified to date. In this report, we examine the nuclear export of Stat1. We find that treatment of cells with the export inhibitor leptomycin B does not affect steady-state localization of Stat1 but impedes nuclear export after IFNgamma-induced nuclear accumulation. We identify a conserved leucine-rich helical segment in the coiled-coil domain of Stat1, which is responsible for the efficient nuclear export of this protein. Mutation of two hallmark leucines within this segment greatly attenuate the back transport of Stat1 in the cytoplasm. When fused to a carrier protein, the Stat1 export sequence can mediate nuclear export after intranuclear microinjection. We show that prolonging the nuclear presence of Stat1 by inhibiting nuclear export reduces the transcriptional response to stimulation with IFNgamma. These data suggest that Stats are actively exported from the nucleus via several separate pathways and link this activity to transcriptional activation.
DOI: 10.1073/pnas.190318397 PMCID: PMC27039 PMID: 10973496 [Indexed for MEDLINE]
Abstract
Signal transducer and activator of transcription (Stat) proteins are latent transcription factors that reside in the cytoplasm before activation. On cytokine-induced tyrosine phosphorylation, these molecules dimerize and accumulate transiently in the nucleus. No specific signals mediating these processes have been identified to date. In this report, we examine the nuclear export of Stat1. We find that treatment of cells with the export inhibitor leptomycin B does not affect steady-state localization of Stat1 but impedes nuclear export after IFNγ-induced nuclear accumulation. We identify a conserved leucine-rich helical segment in the coiled-coil domain of Stat1, which is responsible for the efficient nuclear export of this protein. Mutation of two hallmark leucines within this segment greatly attenuate the back transport of Stat1 in the cytoplasm. When fused to a carrier protein, the Stat1 export sequence can mediate nuclear export after intranuclear microinjection. We show that prolonging the nuclear presence of Stat1 by inhibiting nuclear export reduces the transcriptional response to stimulation with IFNγ. These data suggest that Stats are actively exported from the nucleus via several separate pathways and link this activity to transcriptional activation.
Discussion
Currently the details of the dynamic redistribution of Stat proteins triggered by cytokine activation are largely unknown. Although the striking nuclear accumulation of Stats and their subsequent clearing from the nuclear compartment were noted early on ( 24 ), no signals that regulate these processes have been identified so far. Using a Stat1-GFP fusion protein, we find no evidence for a significant contribution of proteasome-mediated degradation or de novo cytoplasmic synthesis to the nucleocytoplasmic redistribution of Stat1. Exposure of cells to the nuclear export inhibitor LMB, on the other hand, clearly attenuates the reappearance of Stat1 in the cytoplasm and prolongs nuclear accumulation. The results obtained by microinjection and transfection of Stat1-derived peptides fused to reporter proteins indicate the existence of a transferable nuclear export signal in the coiled-coil domain, amino acids 302–314, of Stat1.
The activity of the isolated Stat1 NES can be blocked completely by the export inhibitor LMB (Fig. 2 ). Moreover, the kinetics of export inhibition by LMB of the full-length wild-type protein resemble closely the time course of nuclear clearance of a loss-of-function NES mutant of Stat1. Additionally, the mutant no longer displays sensitivity to the effects of the export inhibitor LMB (compare Figs. 1 and 5 ). Interestingly, neither disruption of the Stat1 NES nor prolonged treatment with LMB for up to 15 h (Figs. 1 and 5 and data not shown) lead to nuclear accumulation of the protein in unstimulated cells. This might be taken as an indication that Stat1 is cytoplasmic in the resting state and does not shuttle permanently in and out of the nucleus, with the net export exceeding import. A clear picture will have to await the characterization of the residual export activity of the NES mutant. As to the nature of this activity, LMB insensitivity indicates the existence of additional export mechanisms that do not rely on the export receptor CRM1. The continued, albeit impeded, nuclear export of a NES-minus Stat1 molecule reveals a first glimpse of a rather intricate degree of regulatory means determining nuclear transport of Stat1. Obviously, the prompt removal from the nucleus of this transcription factor is assured by parallel and independent mechanisms to achieve maximum capacity.
An important aspect concerning export rate modulations is the role of cytokine regulation of nucleocytoplasmic travel of Stat1. Although nuclear entry is triggered by Stat activation on cytokine treatment of the cell, we do not know whether the export is at the same time also subject to cytokine control. It is conceivable that the rate of export is influenced by the accessibility of the nuclear export signal. As shown in Fig. 4 c , only the leucine in position 308 of the hallmark leucine residues of the NES is accessible from the outside and therefore available for interactions with molecules of the export machinery. Experiments exploring the role of a nuclear tyrosine phosphatase in the signal inactivation of Stats as well as the time course of dephosphorylation of the mutant protein presented here suggested that monomeric Stat1 is the substrate for export ( 8 ). We cannot therefore be certain whether the position of the NES in the actual “export-conformation” is reflected accurately in the structure representation of Fig. 4 , which is modeled on the available dimeric protein ( 14 ).
Our data show that the mutant Stat1 is promptly tyrosine dephosphorylated (Fig. 6 a ). This result was anticipated, because the current model of Stat1 inactivation localizes the inactivating phosphatase in the cell nucleus ( 7 ). Importantly, the transcription data highlight a direct coupling between the speed of nuclear export and the magnitude of transcription activation. Inhibition of Stat1 export, either by means of mutation of Stat1 or by blocking chemically the export receptor, is accompanied by a lowered transcriptional output (Fig. 6 c ). This suggests that the time until tyrosine dephosphorylation limits the “lifespan” of the transcriptionally active Stat1, whereas the length of time that the protein stays in the nucleus determines the overall transcriptional yield. The reduced pool of cytoplasmic Stat1 that is available for rephosphorylation might therefore be the limiting factor for the transcriptional response.
A comparison of the Stat1 NES sequence with the homologous amino acids of the other family members suggests the presence of a NES motif in all of them (Fig. 4 a ). The key feature of the Stat1 NES, two leucines spaced by three residues, is present almost without exception. The surrounding sequence is far less conserved, as commonly found in other NESs, but additional hydrophobic and bulky residues are generally found. These amino acid sequence characteristics as well as LMB sensitivity place the Stat1 NES among the class of leucine-rich CRM1-dependent export signals. A comparison with other members of this class indicates that the Stat1 export signal is missing one typical motif of classic NESs, which is an arrangement sometimes termed the “core tetramer” at the C terminus of the NES ( 23 ). Usually, mutation of two leucines in the core tetramer disrupts NES function. This does not hold true for the Stat1 NES, which lacks the cognate core tetramer motif. That is not without precedent, because not all leucine-rich NES sequences described so far conform to the general consensus ( 19 , 25 , 26 ).
Taken together, the data presented in this paper indicate that Stat1 returns to the cytoplasm from the nucleus after cytokine stimulation via a CRM1-dependent pathway. The experiments leading to the localization of a conserved NES motif in the coiled-coil domain of STAT1 reveal the existence of active nuclear export for a member of the Stat family of transcription factors. Notably, a reduced rate of back transport of Stat1 into the cytoplasmic compartment resulted in an aborted transcriptional response to stimulation with IFNγ. In addition, the present findings suggest a multipathway mechanism for the export of Stat1 from the nucleus.