Deanna G. Adams

Signal Transduction from N-cadherin Increases Bcl-2 REGULATION OF THE PHOSPHATIDYLINOSITOL 3-KINASE/Akt PATHWAY BY HOMOPHILIC ADHESION AND ACTIN CYTOSKELETAL ORGANIZATION

Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin·catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin·catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.

Positive Regulation of Raf1-MEK1/2-ERK1/2 Signaling by Protein Serine/Threonine Phosphatase 2A Holoenzymes

Protein serine/threonine phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged Bα/δ regulatory subunits by ∼3-fold or knock-down of Bα by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-Bα/δ subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in Bα knock-down cells but elevated in Bα- and Bδ-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing Bα or Bδ. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes ABαC and ABδC act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A ABαC and ABδC holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1.

Protease inhibitors of Manduca sexta expressed in transgenic cotton

SummaryTo explore the effectiveness of insect derived protease inhibitors in protecting plants against insect feeding, anti-trypsin, anti-chymotrypsin and anti-elastase protease inhibitor (PI) genes from Manduca sexta L. were expressed in transgenic cotton (Gossypium hirsutum L.). From 198 independent transformants, 35 elite lines were further analyzed. Under the control of the 35S promoter of CaMV, PI accumulated to approximately 0.1% of total protein, depending on the tissue analyzed. Using cell-flow cytometry, DNA content/ nuclei of transgenic and non-transformed cotton were identical. On cotton plants expressing PIs, fecundity of Bemisia tabaci (Genn.), the sweetpotato whitefly, was reduced compared to controls. Expression of these protease inhibitors may reduce the developmental rate of B. tabaci and other insects, and provide a strategy for cotton protection.

Tryptophan Decarboxylase, Tryptamine, and Reproduction of the Whitefly.

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine. When the TDC gene was expressed in transgenic tobacco, the 55-kD TDC enzyme and tryptamine accumulated. Bemisia tabaci (sweetpotato whitefly) reproduction on transgenic plants decreased up to 97% relative to controls. Production of tryptamine, its derivatives, or other products resulting from TDC activity may discourage whitefly reproduction and provide a single-gene-based plant protection strategy.

Phosphorylation of the stress-activated protein kinase, MEKK3, at serine 166

Much effort has focused on the identification of MAPK cascades that are activated by the MEKK family of protein kinases. However, direct phosphorylation and regulation of the MEKK proteins has not been shown. To address this question, we have expressed recombinant (His)6FLAG.MEKK3 in Sf9 insect cells and tethered the purified protein to Ni-Sepharose so that we could precipitate interacting proteins and then identify such proteins by liquid chromatography and mass spectrometry (LC-MS). We identified 14-3-3 proteins as interacting with MEKK3, which suggested that (His)6FLAG.MEKK3 was phosphorylated on serine since 14-3-3 proteins are known to associate with phosphorylated proteins. We identified two phosphorylated amino acids at Ser166 and Ser337 of tryptic peptides derived from (His)6FLAG.MEKK3 by using LC-MS. Antibodies were developed that recognize the specific phosphorylated amino acid and with these antibodies, we demonstrate that various stimuli (tumor necrosis factor, arsenite, forskolin, and serum) promote phosphorylation of Ser166 and Ser337. However, neither of these phosphorylated amino acids is required for association with 14-3-3 protein or regulation of MEKK3-dependent ERK and JNK activity. Nonetheless, these results suggest that MEKK3 is a convergence point of multiple upstream signaling pathways.

Isolation and Characterization of PP2A Holoenzymes Containing FLAG-Tagged B Subunits

Protein serine/threonine phosphatase 2A (PP2A) is a major cellular enzyme implicated in the control of a wide variety of biological processes. The predominant form of PP2A in cells is a heterotrimeric holoenzyme (ABC) consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although numerous signal transduction pathways are known to be regulated by PP2A, the identity of the PP2A holoenzymes controlling each pathway remains unclear. Studies aimed at elucidating substrates for individual PP2A holoenzymes have been hindered by the limited availability of purified endogenous holoenzymes and the inability to differentiate cellular roles of closely related PP2A holoenzymes. In this chapter, we describe a strategy for the functional expression of select FLAG-tagged regulatory B subunits in human embryonic kidney-293 cells and subsequent purification of PP2A holoenzymes containing the FLAG-tagged B subunit and endogenous A and C subunits (ABFLAGC). Biochemical analyses of the purified ABFLAGC holoenzymes reveal that they exhibit virtually indistinguishable specific activities and sensitivities to inhibitors as compared to the corresponding endogenous PP2A holoenzymes. The strategy described herein provides a straightforward method to purify individual PP2A holoenzymes from target mammalian cells for subsequent in vitro studies, as well as a powerful approach to identify cellular substrates and roles for each holoenzyme.

The actin cytoskeletal architecture of estrogen receptor positive breast cancer cells suppresses invasion

Estrogen promotes growth of estrogen receptor-positive (ER+) breast tumors. However, epidemiological studies examining the prognostic characteristics of breast cancer in postmenopausal women receiving hormone replacement therapy reveal a significant decrease in tumor dissemination, suggesting that estrogen has potential protective effects against cancer cell invasion. Here, we show that estrogen suppresses invasion of ER+ breast cancer cells by increasing transcription of the Ena/VASP protein, EVL, which promotes the generation of suppressive cortical actin bundles that inhibit motility dynamics, and is crucial for the ER-mediated suppression of invasion in vitro and in vivo. Interestingly, despite its benefits in suppressing tumor growth, anti-estrogenic endocrine therapy decreases EVL expression and increases local invasion in patients. Our results highlight the dichotomous effects of estrogen on tumor progression and suggest that, in contrast to its established role in promoting growth of ER+ tumors, estrogen has a significant role in suppressing invasion through actin cytoskeletal remodeling.Whilst estrogen is known to be tumorigenic in some breast cancer, in some contexts it can be protective against invasion and dissemination. Here, the authors show estrogen can promote generation of Suppressive Cortical Actin Bundles that can inhibit motility dynamics through EVL-mediated actin cytoskeletal remodeling.