Cell Motility

Touched and Moved by STAT3

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Science's STKE  11 Jul 2006:
Vol. 2006, Issue 343, pp. pe30
DOI: 10.1126/stke.3432006pe30

Abstract

STAT3, a member of the signal transducer and activator of transcription (STAT) family of transcription factors, is a known regulator of cell motility through its transcriptional activating functions. However, new evidence suggests a novel role for non–tyrosine-phosphorylated and cytoplasmically localized STAT3 in mediating cell migration by disrupting an interaction between microtubules and one of its partners, stathmin. The association of STAT3 with stathmin potentiates microtubule polymerization and cell movement.

The signal transducer and activator of transcription (STAT) family of proteins are essential mediators of cytokine and growth factor signaling (1). STAT signaling is required for diverse biological processes as varied as embryonic development and adulthood homeostasis. The activation of cytoplasmic, latent STATs is dependent on tyrosine phosphorylation, which induces dimerization through reciprocal phosphotyrosine–Src homology domain 2 (SH2) interactions between two STAT molecules (1). Activated STATs are translocated to the nucleus, where they bind to consensus promoter sequences of target genes and activate their transcription. Many tyrosine kinases—including Janus kinases (JAKs), receptor tyrosine kinases (RTKs), and non-RTKs—can mediate phosphorylation of STAT proteins. In normal cells, STAT tyrosine phosphorylation is transient, lasting from 30 min to several hours, and the tyrosine-dephosphorylated STAT proteins are shuttled back to the cytosol from the nucleus (1).

Among these STATs, STAT3 stands out by its constitutive phosphorylation in the majority of human neoplasms and its capacity to induce cell transformation and tumorigenesis (1, 2). It is believed that STAT3 mediates its effects through the nuclear localized transcriptional activity. As evidenced by more than 3000 reports, STAT3 target genes are implicated in all processes of tumorigenesis, including proliferation, apoptosis, angiogenesis, invasion, and migration. The latter two issues are of particular importance, because the biggest clinical challenge in controlling malignancies is preventing the spread of cancer cells.

There is substantial evidence for the involvement of STAT3 in cell motility, migration, and invasiveness under normal and abnormal biological situations. Specifically, STAT3 controls cell migration during normal zebrafish gastrulation through stimulation of the gene encoding LIV-1 (3, 4), a zinc transporter required for Snail nuclear localization. Snail is a transcriptional repressor and is involved in invasive and migratory phenotypes in epithelial cells (5). STAT3-deficient murine keratinocytes are unable to migrate in a wound assay, in part because of abnormal p130CAS phosphorylation (6). p130CAS is a focal adhesion–associated adaptor protein that is tyrosine-phosphorylated by kinases such as focal adhesion kinase (FAK) or Src and is dephosphorylated by protein tyrosine phosphatases such as PTP-PEST. p130CAS contributes to the formation and stability of focal adhesion complexes and reorganization of cytoskeletal proteins. Tyrosine-phosphorylated STAT3 potentiated the metastatic spread of melanoma-derived cell lines through the transcriptional activation of the matrix metalloproteinase 2 gene (MMP-2) (7). STAT3 activation was also required for induction of the genes encoding MMP-1 and MMP-10 in bladder cancer cells, leading to both increased migration and invasion in vivo (8). The JAK-STAT3 pathway is involved in trophoblast invasion (9), and STAT3 plays an indispensable role in interleukin-6 (IL-6)–mediated T cell migration (10). Depletion of STAT3 inhibited migration of ovarian carcinoma cells (11). In MCF-7 breast cancer cells, activation of STAT3 in response to the cytokine oncostatin M increased migration and caused morphological changes (12). Activated STAT3 promoted collagen production and cell migration in keloid pathogenesis (13), which causes excessive scarring at sites of injury.

These observations unambiguously champion a critical role for tyrosine-phosphorylated STAT3 in cell motility, but the mechanisms by which STAT3 imparts its function have been attributed almost completely to its tyrosine phosphorylation–mediated transcriptional activity. Some evidence has also been presented for non–tyrosine-phosphorylated STAT3 as a transcriptional regulator (14), but the relevance of these genes to cell motility has not been established. Ng et al. (15) provided evidence for a novel mechanism for STAT3 in cell motility and were the first to clearly demonstrate, through a direct interaction with a tubulin-binding protein, that non–tyrosine-phosphorylated, nontranscriptional STAT3 can stabilize the polymerization of microtubules (MTs).

Ng et al. identified stathmin as a novel STAT3 interacting partner. Stathmin is a small tubulin-binding protein that acts as a MT depolarization factor and contributes to cell motility (16). They demonstrated that modulation of stathmin expression had no effect on the phosphorylation, localization, or transcriptional activity of STAT3. However, STAT3 did influence the MT regulatory functions of stathmin. STAT3-deficient mouse embryo fibroblast (MEF) cells clearly displayed a disarrayed MT network and impaired cell motility due to defective α-tubulin polymerization and stabilized MTs. Reintroduction of either wild-type STAT3 or a tyrosine phosphorylation–defective mutant into these cells equally restored the MT array and normalized the polymerization of α-tubulin. However, a transcriptional dominant-active STAT3 mutant was unable to correct MT disarray. These experiments clearly show that a cytoplasmic, non–tyrosine-phosphorylated form of STAT3 rescued the migratory defect through the stabilization of MT organization. STAT3 binding was mapped to the C-terminal, tubulin-binding region of stathmin. Therefore, although it has no direct interaction with tubulin, STAT3 may inhibit stathmin’s MT depolymerization activity by competing with tubulin for stathmin binding and thereby disrupting stathmin-tubulin interactions. Indeed, overexpression of stathmin enhanced the MT network disturbance in STAT3-null MEFs but had little effect in wild-type MEFs. Down-regulation of stathmin by small interfering RNA partially corrected the disruption to the MT array and cell migration in MEFs lacking STAT3. Finally, STAT3 counteracted the MT disruption capacity of stathmin in vitro.

These findings are the first to describe a direct effect of non–tyrosine-phosphorylated, cytoplasmic STAT3 on cell motility through a nontranscriptional protein-protein interaction mechanism. But what remains to be determined are the functional domain(s) of STAT3 that interact with stathmin, how their interaction is regulated, and, most important, what contribution this interaction imparts to cell motility. Moreover, it is not yet known what form of nonphosphorylated STAT3 (monomer or polymer, acetylated or methylated) interacts with stathmin, nor whether other STATs—such as STAT5B, which has also been implicated in cell motility—have similar effects (17).

In addition to the transcriptionally mediated effects of STAT3 on cell migration, some evidence for a cytoplasmic, nontranscriptional role for STAT3 in cell movement has accumulated. STAT3-deficient keratinocytes were defective in cell migration and contained hyperphosphorylated p130CAS (6). STAT3’s transcriptional activity was not invoked because there was no alteration of the abundance of tyrosine kinases or phosphatases that regulate the phosphorylation of p130CAS. Phosphorylated STAT3 was found localized to cytosolic pseudopodial protrusions of migrating cells, as well as in focal adhesions along with FAK and paxillin (11, 18). Members of the Montell laboratory envisaged an "adaptor" or "sensor" role for a nontranscriptional STAT3 in mediating cell motility, but they were unable to provide direct experimental evidence. The findings by Ng et al. now suggest that STAT3 can directly interact with a tubulin-binding protein to affect cytoskeleton reorganization and cell motility.

Taken together, these results enable us to posit multiple routes through which STAT3 affects cell motility (Fig. 1). One route is through a transcription-dependent pathway by which STAT3 modulates the gene expression of migration- or invasion-promoting factors such as LIV-1 and the matrix metalloproteinases MMP-1, MMP-2, and MMP-10 (4, 7, 8). The other route for STAT3 is through its direct interaction and engagement of the central machinery for cell motility—such as focal adhesion components, FAK, paxillin, p130CAS, or cytoskeletal MTs—by binding the MT destabilization factor stathmin. The STAT3 protein involved in this nontranscriptional pathway can be either tyrosine-phosphorylated or nonphosphorylated. The transcriptional pathway would be slower and potentially less readily reversible, whereas the direct, nontranscriptional pathway through protein-protein interactions would be predicted to be rapid, dynamic, and reversible. Nonetheless, STAT3’s influence on cell migration likely involves a balance of both pathways, depending on both the cellular and extracellular contexts. STAT3’s control of cell motility, in addition to its cell proliferation–promoting and antiapoptotic activities, directly contributes to its role in tumorigenesis and tumor progression. More intensive studies on the STAT3–cell motility connection in normal and cancer cells will guarantee further insight into the understanding of the mechanisms of aberrant JAK-STAT signaling in tumor progression and metastasis and will likely lead to the discovery of novel therapeutic targets.

Fig. 1.

Multiple roles of STAT3 in a migrating cell. Cell motility is highly controlled and consists of processes such as focal adhesion, cytoskeletal reorganization, and digestion of the extracellular matrix. STAT3 is purportedly involved in all of these processes through its nuclear, transcription-dependent functions, its cytoplasmic transcription-independent functions, or both. Tyrosine-phosphorylated STAT3 protein dimers transcriptionally activate the expression of LIV-1 (in normal zebrafish gastrulation) and MMP-1, MMP-2, and MMP-10 (in human cancer cells) to promote cell migration. Tyrosine-phosphorylated STAT3 can localize to focal adhesions and interact with phosphorylated focal adhesion kinase (FAK) and paxillin. STAT3 may also stimulate dephosphorylation of the adaptor protein p130CAS in focal adhesions. Cytoplasmic, non–tyrosine-phosphorylated STAT3 directly interacts with a microtubule-destabilizing factor, stathmin, which dislodges tubulin from stathmin and promotes microtubule organization.

STAT3 now joins the ranks of cellular regulators that exhibit functional versatility. There are several proteins whose later characterized functions differ markedly from their original, "canonical" ones—for example, the well-studied tumor suppressor p53. Beside its well-established role as a sequence-specific DNA-binding nuclear protein and transcription factor, p53 is also known to regulate apoptosis and DNA repair processes by cytoplasmic, transcription-independent mechanisms, such as a direct interaction with mitochondria and components of repair pathways (1922). STAT3 protein is abundantly expressed in normal and cancer cells, but less than one-third of all STAT3 protein is transiently localized in the nucleus for the purpose of mediating transcription. Normal cellular functions are efficient and tightly controlled; thus, imagining a function for the abundantly cytoplasmically localized STAT3 seems reasonable. In addition to diversity in localization, with nuclear, cytoplasmic, and possibly even membrane raft (23) localizations, STAT3 may exhibit molecular diversity, with STAT3 proteins existing as monomers, dimers, and possibly even polymers (24) and in phosphorylated and nonphosphorylated forms. How each of these characteristics contributes to the functional versatility of STAT3 remains to be determined. It seems likely that a greater number of nontranscriptional functions for STAT3 protein (as well as other STATs) will be discovered and their involvement in normal and abnormal biological processes revealed.

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