Regulation of Stat3 nuclear import by importin alpha5 and importin alpha7 via two different functional sequence elements

Regulated import of STAT proteins into the nucleus through the nuclear pores is a vital event. We previously identified Arg214/215 in the coiled-coil domain and Arg414/417 in the DNA binding domain involved in the ligand-induced nuclear translocation of Stat3. In this study, we investigated the mechanism for Stat3 nuclear transport. We report here that among five ubiquitously expressed human importin alphas, importin alpha5 and alpha7, but not importin alpha1, alpha3, and alpha4, bind to Stat3 upon cytokine stimulation. Similar results were observed for Stat1, but not for Stat5a and 5b, which were unable to interact with any of the importin alphas. The C-terminus of importin alpha5 is necessary but not sufficient for Stat3 binding. Truncation mutant of Stat3 (aa1-320) that contains Arg214/215 exhibits specific binding to importin alpha5, and an exclusive nuclear localization. Point mutations of Arg214/215 in this mutant destroy importin alpha5 binding and its nuclear localization. In contrast, the truncation mutant (aa320-770) including Arg414/417 fails to interact with importin alpha5 and is localized in the cytoplasm. However, both sequence elements are necessary for the full-length Stat3's interaction with importin alpha5. These results suggest that Arg214/215 is likely the binding site for importin alpha5, whereas Arg414/417 may not be involved in the direct binding, but necessary for maintaining the proper conformation of Stat3 dimer for importin binding. A model for Stat3 nuclear translocation is proposed based on these data.     

Dimer stability as a determinant of differential DNA binding activity of Stat3 isoforms

Stat3alpha and Stat3beta are two Stat3 isoforms with marked quantitative differences in their DNA binding activities. To examine the molecular basis of the differential DNA binding activities, we measured DNA binding strength and dimer stability, two possible mechanisms responsible for these differences. Stat3alpha and Stat3beta showed no difference in DNA binding strength, i.e. they had similar association and dissociation rates for DNA binding. However, competition analyses performed with dissociating reagents including an anti-phosphotyrosine antibody, SH2 domain protein, and a phosphopeptide demonstrated that Stat3beta dimers are more stable than Stat3alpha dimers. We report here that dimer stability of activated forms plays a critical role in determining DNA binding activity of Stat3 isoforms. We found that C-terminal deletions of Stat3alpha increased both DNA binding activity and dimer stability of Stat3alpha. Our findings suggest that the acidic C-terminal region of Stat3alpha does not interfere with the DNA binding of activated Stat3alpha dimers, but destabilizes the dimeric forms of Stat3alpha. We propose that dimer stability described in vitro may be the underlying mechanism of in vivo stability of activated Stat3 proteins, regulating dephosphorylation of tyrosine 705.     

Stat5B shuttles between cytoplasm and nucleus in a cytokine-dependent and -independent manner

In response to cytokine stimuli, Stats are phosphorylated and translocated to the nucleus to activate target genes. Then, most are dephosphorylated and returned to the cytoplasm. Using Ba/F3 cells, we found that the nuclear export of Stat5B by cytokine depletion was inhibited by leptomycin B (LMB), a specific inhibitor of nuclear export receptor chromosome region maintenance 1. Interestingly, LMB treatment in the absence of cytokine led to the accumulation of Stat5B in the nucleus, suggesting that Stat5B shuttles between the nucleus and the cytoplasm as a monomer without cytokine stimulation. This notion is supported by the observation that LMB-induced accumulation of Stat5B in the nucleus was also observed with Stat5B having a mutated tyrosine 699, which is essential for dimer formation. Using a series of mutant Stat5Bs, we identified a part of the coiled coil domain to be a critical region for monomer nuclear import and a more N-terminal region to be critical for the cytokine stimulation dependent import of Stat5B. Taken together, we propose a model in which Stat5B shuttles between the nucleus and cytoplasm by two different mechanisms, one being a factor-independent constitutive shuttling by monomeric form, and the other, a factor stimulation-dependent one regulated by tyrosine phosphorylation and subsequent dimerization.     

Stat3-dependent induction of interleukin-3 receptor expression in leukemia inhibitory factor-stimulated M1 mouse leukemia cells

M1 mouse leukemia cells differentiate to macrophages/monocytes by the stimulation of interleukin-6 (IL-6)/leukemia inhibitory factor (LIF). To identify new LIF-induced genes, we have performed representational difference analysis using M1 cells and cloned mouse interleukin-3 (IL-3) receptor beta subunit gene. The mRNA expression of both IL-3 receptor (IL-3R) alpha and beta subunits is upregulated after 1 h stimulation of LIF and remains to be elevated along the differentiation of M1 cells. This induction is almost completely suppressed in M1 cells expressing a dominant negative form of Stat3. Furthermore, we show that IL-3-induced Stat5 phosphorylation increases in LIF-stimulated M1 cells. These results suggest that Stat3 may play a role in the differentiation of myeloid cells by regulating IL-3R expression.     

Interdomain interaction of Stat3 regulates its Src homology 2 domain-mediated receptor binding activity

Activation of Stat proteins by cytokines is initiated by their Src homology 2 (SH2) domain-mediated association with the cytokine receptors. Previously, we identified an essential role of the coiled-coil domain of Stat3 in binding of the receptor peptides derived from the interleukin-6 receptor subunit, gp130. In this study, we further investigated the molecular basis of this regulation. We found that the C-terminal domain of Stat3 negatively regulates its receptor binding activity only in the absence of the first alpha-helix of the coiled-coil domain, which leads to a hypothesis of intramolecular interaction. Physical interactions between the coiled-coil domain and the C-terminal domain, as well as the SH2 domain, were indeed detected. Furthermore, a sub-region of the C-terminal domain (amino acids 720-740), which is also involved in the interaction with the coiled-coil domain, was demonstrated to be critical for the regulation of the receptor binding. Correspondingly, phosphorylation on Ser-727 within this region inhibits this interaction. In agreement with the peptide binding results, both the coiled-coil domain and the C-terminal sub-region are necessary for the functional recruitment of Stat3 to the cellular gp130 in response to interleukin-6, suggesting that the interdomain interaction is a prerequisite for the SH2-mediated receptor binding in interleukin-6 signaling.     

Nucleocytoplasmic shuttling by nucleoporins Nup153 and Nup214 and CRM1-dependent nuclear export control the subcellular distribution of latent Stat1

Interferon stimulation of cells leads to the tyrosine phosphorylation of latent Stat1 and subsequent transient accumulation in the nucleus that requires canonical transport factors. However, the mechanisms that control the predominantly cytoplasmic localization in unstimulated cells have not been resolved. We uncovered that constitutive energy- and transport factor-independent nucleocytoplasmic shuttling is a property of unphosphorylated Stat1, Stat3, and Stat5. The NH(2)- and COOH-terminal Stat domains are generally dispensable, whereas alkylation of a single cysteine residue blocked cytokine-independent nuclear translocation and thus implicated the linker domain into the cycling of Stat1. It is revealed that constitutive nucleocytoplasmic shuttling of Stat1 is mediated by direct interactions with the FG repeat regions of nucleoporin 153 and nucleoporin 214 of the nuclear pore. Concurrent active nuclear export by CRM1 created a nucleocytoplasmic Stat1 concentration gradient that is significantly reduced by the blocking of energy-requiring translocation mechanisms or the specific inactivation of CRM1. Thus, we propose that two independent translocation pathways cooperate to determine the steady-state distribution of Stat1.     

Kindlin-2 (Mig-2): a co-activator of beta3 integrins

Integrin activation is essential for dynamically linking the extracellular environment and cytoskeletal/signaling networks. Activation is controlled by integrins' short cytoplasmic tails (CTs). It is widely accepted that the head domain of talin (talin-H) can mediate integrin activation by binding to two sites in integrin beta's CT; in integrin beta(3) this is an NPLY(747) motif and the membrane-proximal region. Here, we show that the C-terminal region of integrin beta(3) CT, composed of a conserved TS(752)T region and NITY(759) motif, supports integrin activation by binding to a cytosolic binding partner, kindlin-2, a widely distributed PTB domain protein. Co-transfection of kindlin-2 with talin-H results in a synergistic enhancement of integrin alpha(IIb)beta(3) activation. Furthermore, siRNA knockdown of endogenous kindlin-2 impairs talin-induced alpha(IIb)beta(3) activation in transfected CHO cells and blunts alpha(v)beta(3)-mediated adhesion and migration of endothelial cells. Our results thus identify kindlin-2 as a novel regulator of integrin activation; it functions as a coactivator.     

DNA binding of in vitro activated Stat1 alpha, Stat1 beta and truncated Stat1: interaction between NH2-terminal domains stabilizes binding of two dimers to tandem DNA sites.

Stat1 alpha, Stat1 beta and a proteolytically defined truncated Stat1 (132-713, Stat1tc) have been prepared from recombinant sources. All three proteins were specifically phosphorylated on Tyr701 in vitro and the phosphoprotein purified to homogeneity. This was achieved by employing a new isolation scheme that does not include DNA affinity steps and readily allows for the isolation of tens of milligrams of activated Stat protein. The purified phosphoprotein was free of traces of unphosphorylated polypeptide as detected by mass spectrometry. The phosphorylated Stat1 preparations bound to various DNA recognition sites with the same Keq of approximately 1 x 10(-9) M; distinction between 'weak' and 'strong' binding sites is determined by the very rapid dissociation (< 30 s, t1/2) from 'weak' sites compared with 'strong' sites (approximately 3 min, t1/2). Reports of 'weak' tandem binding sites in a natural gene caused us to examine binding to tandem sites leading to the finding that the Stat1 alpha or beta (38 amino acids shorter on the C terminus) bound to two tandem sites (but not two head-to-head sites) with a higher stability than to a single recognition site. The N-terminally truncated protein Stat1tc did not show this cooperative binding, thus implicating the N-terminal domain in promoting Stat1-Stat1 dimer interaction.     

Stat3 beta inhibits gamma-globin gene expression in erythroid cells

We demonstrated previously gamma-globin gene inhibition in K562 cells and primary erythroid progenitors treated with interleukin-6. Although several cis-acting elements have been identified in the globin promoters, the precise mechanism for cytokine-mediated globin gene regulation remains to be elucidated. In this report we demonstrate inhibitors of Stat3 phosphorylation abrogate interleukin-6-mediated gamma gene silencing in erythroid cells. DNA-protein binding studies established Stat3 interaction in the 5'-untranslated gamma-globin promoter region. Furthermore, co-transfection experiments with Stat3 beta demonstrate gamma promoter inhibition in a concentration-dependent manner, which was significantly reversed when the cognate Stat3-binding site in the 5'-untranslated region was mutated. These studies establish a novel mechanism for gamma gene silencing through the STAT signal transduction pathway.     

Locking the beta3 integrin I-like domain into high and low affinity conformations with disulfides

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.