The highly conserved and ubiquitously expressed 14‐3‐3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14‐3‐3, we identified a novel transcriptional co‐activator, TAZ (transcriptional co‐activator with PDZ‐binding motif) as a 14‐3‐3‐binding molecule. TAZ shares homology with Yes‐associated protein (YAP), contains a WW domain and functions as a transcriptional co‐activator by binding to the PPXY motif present on transcription factors. 14‐3‐3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co‐activation through 14‐3‐3‐mediated nuclear export. The C‐terminus of TAZ contains a highly conserved PDZ‐binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ‐stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain‐containing protein NHERF‐2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14‐3‐3.

The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3 phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.Protein kinase B (PKB), also termed Akt, has been the subject of intense study due to its role in transducing signals from phosphoinositide 3-kinase (PI3K) that regulate cell survival and intermediary metabolism. Several protooncogene products modulate the activation of PI3K and, as a consequence, PKB has been shown to play roles in many of the cellular functions that are altered during oncogenesis and other diseases (reviewed in reference 12). Interference with PKB activation may therefore have therapeutic value.Activation of PKB entails a complex series of events involving additional proteins. First, the PI3K-generated lipid products PI(3,4,5)P 3 and PI(3,4)P 2 recruit PKB to the plasma membrane through their affinity for the PH domain of PKB (14,20,21). Once membrane proximal, at least two residues of PKB are rapidly phosphorylated, including threonine 308 (T308) and serine 473 (S473) (1). T308 lies within the kinase T loop, and its phosphorylation is presumed to generate a conformational change that permits access to the substrates, analogous to T-loop phosphorylation in other protein kinases. In the case of PKB, this reaction is catalyzed by another 3Ј phosphoinositide-regulated kinase termed PDK1 (2, 33). S473 is located within a hydrophobic region close to the carboxyl terminus of PKB and is also phosphorylated during activation (1), but the mechanism of its phosphorylation and the role it serves in activating PKB are incompletely understood.Several lines of evidence suggest that S473 is autophosphorylated. For example, catalytically inactive mutants of PKB do not undergo S473 phosphorylation (34). There is also evidence for an autonomous S473 ...

SummaryLow-dose exposures to common environmental chemicals that are deemed safe individually may be combining to instigate carcinogenesis, thereby contributing to the incidence of cancer. This risk may be overlooked by current regulatory practices and needs to be vigorously investigated.

Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor– or epidermal growth factor–dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor–dependent activation of Rac leading to lamellipodia.

Inactivating mutations in the serine-threonine kinase LKB1 (STK11) are found in most patients with PeutzJeghers syndrome; however the function of LKB1 is unknown. We found that LKB1 binds to and regulates brahma-related gene 1 (Brg1), an essential component of chromatin remodeling complexes. The association requires the N terminus of LKB1 and the helicase domain of Brg1 and LKB1 stimulates the ATPase activity of Brg1. Brg1 expression in SW13 cells induces the formation of flat cells indicative of cell cycle arrest and senescence. Expression of a kinase-dead mutant of LKB1, SL26, in SW13 cells blocks the formation of Brg1-induced flat cells, indicating that LKB1 is required for Brg1-dependent growth arrest. The inability of mutants of LKB1 to mediate Brg1-dependent growth arrest may explain the manifestations of Peutz-Jeghers syndrome. Peutz-Jeghers syndrome (PJS)1 is an autosomal dominant disorder characterized by mucocutaneous pigmentation, hamartomatous polyps of the gastrointestinal tract, and an 18-fold increase in the risk of developing cancer (1-3). Inactivating mutations in the serine-threonine kinase LKB1 (STK11) are found in most patients with PJS (4, 5). Many of the polyps that develop in PJS show loss of heterozygosity (6, 7) indicating that LKB1 is a tumor suppressor, and mutations in LKB1 have also been found in a small number of sporadic cancers (8, 9). Hypermethylation of CpG islands in the promoters of genes is an alternative mechanism by which tumor suppressor genes are inactivated, and the LKB1 promoter appears to be hypermethylated in some polyps from PJS patients (10).Xenopus lavis XEEK1 (xenopus egg and embryonic kinase 1 (11), Par4 Caenorhabditis elegans partitioning-defective gene 4 (12), and a Drosophila melanogaster gene (accession number AAF54972) are LKB1 orthologs, but little is known about the function of any of the proteins. Par4 is required for asymmetric cell division of the C. elegans embryo, but its specific role is not understood (12). XEEK1 is expressed early in Xenopus development, phosphorylates a protein of 155-kDa, and is a substrate for cAMP-dependent protein kinase (11). LKB1 can also be phosphorylated by cAMP-dependent protein kinase, as well as p90 Rsk (13,14). XEEK1 localizes exclusively to the cytoplasm (11), but LKB1 is found in both the nucleus and the cytoplasm (15). The SL26 mutant of LKB1, isolated from a patient with PJS (16), is localized almost exclusively to the nucleus (15, 17). Expression of LKB1 in G361 melanoma cells resulted in significant inhibition of cell growth because of a G 1 arrest, suggesting that LKB1 may be involved in cell cycle regulation (14,18). To understand the function of LKB1, we carried out a screen to identify proteins to which it binds. We found that LKB1 associates with Brg1, a component of SWI⅐SNF chromatin remodeling complexes. LKB1 stimulates the ATPase activity of Brg1 and is required for Brg1-induced growth arrest. EXPERIMENTAL PROCEDURESIn Vitro Expression Cloning-A unidirectional HeLa cell-derived cDNA library in pcDNA3.1 (I...

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Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1−/− mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK’s effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment.

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