A GTPase-activating protein binds STAT3 and is required for IL-6–induced STAT3 activation and for differentiation of a leukemic cell line

Abstract

IntroductionThe signal transducer and activator of transcription (STAT) family members STAT1-4, STAT5A, STAT5B, and STAT6 are activated through phosphorylation by the Janus kinase (JAK) family upon cytokine stimulation. The phosphorylated STATs form homodimers or heterodimers and translocate into the nucleus, where they regulate expression of their target genes. [1][2][3][4] Among them, STAT3 is activated mainly by the interleukin-6 (IL-6) family cytokines including IL-6, oncostatin M, and leukemia inhibitory factor (LIF), and is implicated in a wide range of biologic processes, including nephrogenesis, gliogenesis, hepatogenesis, T-cell proliferation, inflammation, and oncogenesis. [5][6][7][8][9][10][11] The critical role of STAT3 in myeloid differentiation was demonstrated by the use of dominant negative mutants. [12][13][14] In contrast, in embryonic stem cells, STAT3 is required for self-renewal. [15][16][17][18][19] In addition, STAT3 is activated in a broad spectrum of human hematologic malignancies. 20 STAT3 can also be negatively regulated. Among the known inhibitors of STAT proteins are the suppressor of cytokine signaling (SOCS) proteins, 21 also known as Janus kinase binding (JAB) proteins 22 or STAT-induced STAT inhibitors (SSIs). 23 While SOCS proteins interact with JAKs and reduced their tyrosine kinase activity, 21-23 a STAT3 inhibitor protein inhibitor of activated STAT3 (PIAS3) directly binds to STAT3 and inhibits its activity. 24 A zinc finger protein Gfi-1 enhances STAT3 signaling by preventing this binding of PIAS3 to STAT3. 25 Several other molecules have been found to interact with activated STAT3. Among them, cellular Jun (c-Jun) forms a complex with STAT3 and activates the ␣ 2 -macroglobulin promoter that contains both STAT3-and c-Jun-binding sites. 26,27 Like many other transcription factors, STAT3 associates with a transcriptional cofactor, cAMP response element binding proteinbinding protein/p300 (CBP/p300), to form a transcriptional complex. 28 A protein called gene-associated with retinoid interferoninduced mortality (GRIM)-19 suppresses STAT3 activity through cytoplasmic retention of STAT3, whereas an endothelial cellderived zinc finger protein (EZI) enhances STAT3 activity through nuclear retention of STAT3. 29,30 There may be more such regulators in various steps of STAT3 action, such as translocation to the nucleus, induction of chromatin remodeling, and proteolysis.In a search for key molecules that prevent IL-6-induced terminal differentiation of murine myeloid leukemia M1 cells, we identified an antisense DNA for the full-length form of male germ cell Rac guanosine triphosphatase-activating protein (MgcRac-GAP) through functional cloning. 31 An N-terminus-truncated form of MgcRacGAP had been isolated and named male germ cell Rac GAP, as its expression was highest in testis. 32 Rac, Cdc42, and RhoA, the Rho family of small guanosine triphosphates (GTPases), play pleiotropic roles in a variety of cell functions such as transformation, migration, cytokinesis, and transcriptional...

Implantation of the human embryo requires Rac1-dependent endometrial stromal cell migration

“…In contrast, some studies suggest that RacGAP1 is able to mediate some of its functions independently of its GAP activity (38). Recently, RacGAP1 has also been implicated in regulating the localization and transcriptional activity of signal transducer and activation of transcription (STAT) proteins (39,40), a family of proteins that have been strongly implicated in the stromal decidualization response and embryo implantation (41). Here, we have shown that hESC Rac-GAP1 levels are down-regulated in response to human embryos and that this correlates with an increase in Rac1 activity observed in hESCs.…”

PDGF-induced human airway smooth muscle cell proliferation requires STAT3 and the small GTPase Rac1

“…The function of the nuclear Rac1, however, is still not understood. While STAT3 requires facilitated import into the nucleus via importin ␣ 5 (27), molecules other than importins, such as Rac1, are specifically required for the nuclear translocation of STAT3 (46). Kawashima et al (21) show that activated Rac1 is specifically required for the interaction of phospho-STAT5A with importins.…”

“…Additionally, we have previously determined that STAT3 activation is directly regulated by the small GTPase Rac1 in fibroblasts (44). Recent studies by us and others have shown that Rac1 contributes to maximal STAT3 activation, transcriptional activity, and nuclear translocation and that the STAT3/Rac1 interaction is important in STAT3 function (21,36,37,44,46,47). However, the results regarding the direct regulation of STAT3 by Rac1 have been somewhat contradictory, likely due to the fact that the studies have used different cell types and stimuli, and have often relied on overexpression studies using dominant negative forms of Rac1 (DnRac1).…”

Disruption of Sept6, a Fusion Partner Gene of MLL, Does Not Affect Ontogeny, Leukemogenesis Induced by MLL-SEPT6, or Phenotype Induced by the Loss of Sept4

“…These siRNA duplexes were manufactured and supplied by Dharmacon. Transfection was done with Lipofectamine 2000 (Invitrogen) as described previously (55). At 72 h after transfection, the cells were counted and subjected to Western blot analysis.…”