γ-Tocotrienol but Not γ-Tocopherol Blocks STAT3 Cell Signaling Pathway through Induction of Protein-tyrosine Phosphatase SHP-1 and Sensitizes Tumor Cells to Chemotherapeutic Agents

Although γ-tocotrienol (T3), a vitamin E isolated primarily from palm and rice bran oil, has been linked with anticancer activities, the mechanism of this action is poorly understood. In this study, we investigated whether γ-T3 can modulate the STAT3 cell signaling pathway, closely linked to inflammation and tumorigenesis. We found that γ-T3 but not γ-tocopherol, the most common saturated form of vitamin E, inhibited constitutive activation of STAT3 in a dose- and time-dependent manner, and this inhibition was not cell type-specific. γ-T3 also inhibited STAT3 DNA binding. This correlated with inhibition of Src kinase and JAK1 and JAK2 kinases. Pervanadate reversed the γ-T3-induced down-regulation of STAT3 activation, suggesting the involvement of a protein-tyrosine phosphatase. When examined further, we found that γ-T3 induced the expression of the tyrosine phosphatase SHP-1, and gene silencing of the SHP-1 by small interfering RNA abolished the ability of γ-T3 to inhibit STAT3 activation, suggesting a vital role for SHP-1 in the action of γ-T3. Also γ-T3 down-modulated activation of STAT3 and induced SHP-1 in vivo. Eventually, γ-T3 down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) gene products; and this correlated with suppression of proliferation, the accumulation of cells in sub-G(1) phase of the cell cycle, and induction of apoptosis. This vitamin also sensitized the tumor cells to the apoptotic effects of thalidomide and bortezomib. Overall, our results suggest that γ-T3 is a novel blocker of STAT3 activation pathway both in vitro and in vivo and thus may have potential in prevention and treatment of cancers.     

A Novel Cross-talk in Diacylglycerol Signaling: THE RAC-GAP β2-CHIMAERIN IS NEGATIVELY REGULATED BY PROTEIN KINASE Cδ-MEDIATED PHOSPHORYLATION*

Although the family of chimaerin Rac-GAPs has recently gained significant attention for their involvement in development, cancer, and neuritogenesis, little is known about their molecular regulation. Chimaerins are activated by the lipid second messenger diacylglycerol via their C1 domain upon activation of tyrosine kinase receptors, thereby restricting the magnitude of Rac signaling in a receptor-regulated manner. Here we identified a novel regulatory mechanism for β2-chimaerin via phosphorylation. Epidermal growth factor or the phorbol ester phorbol 12-myristate 13-acetate caused rapid phosphorylation of β2-chimaerin on Ser¹⁶⁹ located in the SH2-C1 domain linker region via protein kinase Cδ, which retained β2-chimaerin in the cytosol and prevented its C1 domain-mediated translocation to membranes. Furthermore, despite the fact that Ser¹⁶⁹ phosphorylation did not alter intrinsic Rac-GAP activity in vitro, a non-phosphorylatable β2-chimaerin mutant was highly sensitive to translocation, and displayed enhanced association with activated Rac, enhanced Rac-GAP activity, and anti-migratory properties when expressed in cells. Our results not only revealed a novel regulatory mechanism that facilitates Rac activation, but also identified a novel mechanism of cross-talk between diacylglycerol receptors that restricts β2-chimaerin relocalization and activation.     

Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signaling

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.     

Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway

GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.     

Novel interactions between NFATc1 (Nuclear Factor of Activated T cells c1) and STAT-3 (Signal Transducer and Activator of Transcription-3) mediate G protein-coupled receptor agonist, thrombin-induced biphasic expression of cyclin D1, with first phase influencing cell migration and second phase directing cell proliferation

Thrombin, a G protein-coupled receptor agonist, induced a biphasic expression of cyclin D1 in primary vascular smooth muscle cells. Although both phases of cyclin D1 expression require binding of the newly identified cooperative complex, NFATc1·STAT-3, to its promoter, the second phase, which is more robust, depends on NFATc1-mediated recruitment of p300 onto the complex and the subsequent acetylation of STAT-3. In addition, STAT-3 is tyrosine-phosphorylated in a biphasic manner, and the late phase requires NFATc1-mediated p300-dependent acetylation. Furthermore, interference with acetylation of STAT-3 by overexpression of acetylation null STAT-3 mutant led to the loss of the late phase of cyclin D1 expression. EMSA analysis and reporter gene assays revealed that NFATc1·STAT-3 complex binding to the cyclin D1 promoter led to an enhanceosome formation and facilitated cyclin D1 expression. In the early phase of its expression, cyclin D1 is localized mostly in the cytoplasm and influenced cell migration. However, during the late and robust phase of its expression, cyclin D1 is translocated to the nucleus and directed cell proliferation. Together, these results demonstrate for the first time that the dual function of cyclin D1 in cell migration and proliferation is temperospatially separated by its biphasic expression, which is mediated by cooperative interactions between NFATc1 and STAT-3.     

Crystal Structure of the Entire Ectodomain of gp130: INSIGHTS INTO THE MOLECULAR ASSEMBLY OF THE TALL CYTOKINE RECEPTOR COMPLEXES*

gp130 is the shared signal-transducing receptor subunit for the large and important family of interleukin 6-like cytokines. Previous x-ray structures of ligand-receptor complexes of this family lack the three membrane-proximal domains that are essential for signal transduction. Here we report the crystal structure of the entire extracellular portion of human gp130 (domains 1–6, D1–D6) at 3.6 Å resolution, in an unliganded form, as well as a higher resolution structure of the membrane-proximal fibronectin type III domains (D4–D6) at 1.9 Å. This represents the first atomic resolution structure of the complete ectodomain of any “tall” cytokine receptor. These structures show that other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. They also reveal that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire tall receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C termini of the membrane-proximal fibronectin type III domains of the tall cytokine receptors in close proximity within the transmembrane complex, favorable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.     

PTP1B Targets the Endosomal Sorting Machinery: DEPHOSPHORYLATION OF REGULATORY SITES ON THE ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT COMPONENT STAM2*

Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated STAM2 specifically suppressed Akt activation, and a phosphorylation-deficient STAM2 mutant displayed prolonged localization on endosomes following EGF stimulation. These results reveal a novel link between the dephosphorylation and endocytic machinery and suggest that PTP1B can affect RTK signaling in a previously unrecognized manner.     

Novel Role for Matricellular Proteins in the Regulation of Islet β Cell Survival: THE EFFECT OF SPARC ON SURVIVAL,PROLIFERATION, AND SIGNALING*

Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining β cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of β cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by Western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in β cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared with controls. Purified SPARC inhibits growth factor-induced signaling in both INS-1 β cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 β cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and β cell growth.     

FRAX597, a Small Molecule Inhibitor of the p21-activated Kinases,Inhibits Tumorigenesis of Neurofibromatosis Type 2 (NF2)-associated Schwannomas

The p21-activated kinases (PAKs) are immediate downstream effectors of the Rac/Cdc42 small G-proteins and implicated in promoting tumorigenesis in various types of cancer including breast and lung carcinomas. Recent studies have established a requirement for the PAKs in the pathogenesis of Neurofibromatosis type 2 (NF2), a dominantly inherited cancer disorder caused by mutations at the NF2 gene locus. Merlin, the protein product of the NF2 gene, has been shown to negatively regulate signaling through the PAKs and the tumor suppressive functions of Merlin are mediated, at least in part, through inhibition of the PAKs. Knockdown of PAK1 and PAK2 expression, through RNAi-based approaches, impairs the proliferation of NF2-null schwannoma cells in culture and inhibits their ability to form tumors in vivo. These data implicate the PAKs as potential therapeutic targets. High-throughput screening of a library of small molecules combined with a structure-activity relationship approach resulted in the identification of FRAX597, a small-molecule pyridopyrimidinone, as a potent inhibitor of the group I PAKs. Crystallographic characterization of the FRAX597/PAK1 complex identifies a phenyl ring that traverses the gatekeeper residue and positions the thiazole in the back cavity of the ATP binding site, a site rarely targeted by kinase inhibitors. FRAX597 inhibits the proliferation of NF2-deficient schwannoma cells in culture and displayed potent anti-tumor activity in vivo, impairing schwannoma development in an orthotopic model of NF2. These studies identify a novel class of orally available ATP-competitive Group I PAK inhibitors with significant potential for the treatment of NF2 and other cancers.     

Curcumin suppresses T cell activation by blocking Ca2+ mobilization and nuclear factor of activated T cells (NFAT) activation

Curcumin is the active ingredient of the spice turmeric and has been shown to have a number of pharmacologic and therapeutic activities including antioxidant, anti-microbial, anti-inflammatory, and anti-carcinogenic properties. The anti-inflammatory effects of curcumin have primarily been attributed to its inhibitory effect on NF-κB activity due to redox regulation. In this study, we show that curcumin is an immunosuppressive phytochemical that blocks T cell-activation-induced Ca(2+) mobilization with IC(50) = ～12.5 μM and thereby prevents NFAT activation and NFAT-regulated cytokine expression. This finding provides a new mechanism for curcumin-mediated anti-inflammatory and immunosuppressive function. We also show that curcumin can synergize with CsA to enhance immunosuppressive activity because of different inhibitory mechanisms. Furthermore, because Ca(2+) is also the secondary messenger crucial for the TCR-induced NF-κB signaling pathway, our finding also provides another mechanism by which curcumin suppresses NF-κB activation.