At least 99% of protein phosphorylation in eukaryotic cells occurs on Ser and Thr residues. The greater than 300 protein Ser/Thr kinases in the human genome are opposed by less than 30 protein Ser/Thr phosphatases, of which protein phosphatase 1 and protein phosphatase 2A (PP2A) 2 contribute the bulk of activity in most cell types. The notion of Ser/Thr phosphatases as promiscuous and constitutively active enzymes that simply provide the substrates for regulated kinase signaling has been challenged by the discovery of batteries of catalytic subunit-interacting proteins, which impart substrate specificity, subcellular localization, and responsiveness to phosphorylation (1, 2). In the case of PP2A, the predominant holoenzyme is formed by association of a core dimer of catalytic and scaffold subunits with one of a least 12 regulatory subunits in vertebrates. Since vertebrate A and C subunits are each encoded by two genes and since many regulatory subunits are diversified by alternative splicing, several dozen different PP2A heterotrimers are likely to exist in any given cell type. The three unrelated regulatory subunit gene families (B, BЈ, BЉ) are almost certain to utilize different mechanisms to control enzymatic activity and cellular localization of PP2A. The B-family (PR55) of regulatory subunits consists of predicted -propellers with divergent N-terminal tails that act as subcellular targeting signals (3-5). In addition to interacting with phosphatase substrates (6 -12), BЈ-family (also referred to as B56, PR61) subunits are heavily phosphorylated, which may confer regulation by second messengers (13-16). Members of the BЉ-family of PP2A subunits (PR72/ 130, PR59, PR48) feature two calcium binding EF hands with presumed structural rather than regulatory functions (17). Striatin and SG2NA have been referred to as Bٞ regulatory subunits (18). It may be more appropriate to refer to them as PP2A dimer-associated proteins since their stability does not depend on association with the PP2A core enzyme (17, 19) and since they lack the A subunit binding consensus motif common to B, BЈ, and BЉ subunits (20).A growing body of evidence indicates that PP2A has complex inhibitory and stimulatory effects on hormone and growth factor signaling, in particular the extracellular-signal regulated (ERK)/mitogen-activated protein kinase (MAPK) cascade (21). PP2A substrates include G-protein-coupled receptors and receptor tyrosine kinases (22-24), receptorassociated proteins (25-28), and all three kinases of the ERK/MAPK cascade core module , MAPK/ERK kinase (MEK) (33)(34)(35) and ERK (36,37)).Here, we have begun to dissect the contribution of different PP2A holoenzymes to growth factor signaling in PC12 cells. The net effect of total PP2A silencing or inhibition was ERK and Akt hyperphosphorylation, most likely as a consequence of direct dephosphorylation of these kinases by PP2A. Cautioning against exclusive reliance on silencing approaches, protracted inhibition of PP2A by RNA interference (RNAi) resulted in a compensatory unco...
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 B␣/␦ regulatory subunits by ϳ3-fold or knock-down of B␣ 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-B␣/␦ 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 B␣ knock-down cells but elevated in B␣-and B␦-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 B␣ or B␦. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes AB␣C and AB␦C 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 AB␣C and AB␦C holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1. PP2A3 is a major serine/threonine phosphatase implicated in the control of numerous cellular processes including metabolism, transcription and translation, ion transport, development, inflammation, cell growth, differentiation, and apoptosis (for reviews, see Refs. 1 and 2). Heterotrimeric PP2A holoenzymes are composed of a scaffolding/ structural subunit (A), a variable regulatory subunit (B), and a catalytic subunit (C). Thus far, four distinct regulatory subunit families have been identified: B or PR55 (2-4), BЈ or PR61 (5, 6), BЉ or PR72 (7-9), and Bٞ or PR93/PR110 (10). Although the regulatory subunit families share little amino acid sequence homology, isoforms within each family exhibit significant sequence homology. The variable subunit plays a critical role in the control of PP2A by regulating substrate selectivity and/or directing the localization of the enzyme within the cell (11-15). Recent studies have also revealed a role for PP2A regulatory subunits in cell growth and apoptosis (16 -18), assembly and function of cytoskeletal proteins (14,19,20), and various cell signal transduction pathways (for a review, see Ref. 2...
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
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.
MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3 (MAP2K), and a MAPK kinase kinase (MAP3K). Regulation of the MAP3K, presumably by phosphorylation, provides the impetus for activation of the three-kinase module. Once activated, the MAP3Ks activate MAP2Ks by phosphorylation of two residues within the activation loop. Phosphorylation of MAP2Ks activates these dual specificity kinases to phosphorylate MAPKs on a conserved threonine and tyrosine motif, TXY, also within the activation loop. Once phosphorylated, the MAPKs phosphorylate protein substrates and regulate cellular processes like growth, protein synthesis, gene expression, and nucleotide synthesis (2).Over the last decade, a large body of work has characterized events that occur downstream of the MAPKs. However, little is known regarding the regulatory mechanisms that modulate the MEKK proteins to ultimately regulate the MAPKs. For example, it is known that overexpression of MEKK3 activates the ERK (3, 4), JNK (3-5), p38 (5, 6), ERK5 (7), and NF-B pathways (8 -10). Typically, MEKK3-dependent regulation of these pathways is studied by using transfection studies, and activation of these pathways rarely requires an agonist. Therefore, it appears that some process intrinsic to MEKK3 is critical and sufficient for activation of these pathways.The activation loop of some protein kinase families, such as the arginine-aspartate family, is positioned between subdomains VII and VIII and is phosphorylated by other protein kinases or through autophosphorylation of the kinase itself (11). Phosphorylation within the activation loop of protein kinases results in conformational changes in the protein structure that (i) enhance substrate binding, (ii) correctly position amino acids involved in catalysis, and (iii) relieve steric hindrance within the catalytic domain. Regardless of how the activation loop is phosphorylated, regulation of catalytic activity frequently correlates with phosphorylation of the activation loop.Given that phosphorylation plays a critical role in regulating the MAPKs and the MAP2Ks, we investigated how phosphorylation of MEKK3 might affect its catalytic activity. Since phosphorylation sites within the activation loop of MEKK3 have not been reported, we systematically mutated serine and threonine residues within the activation loop to alanine and monitored MEKK3-dependent activities in HEK293 EBNA cells. Two key amino acids were identified at positions 526 and 530 using a luciferase-based reporter gene assay as well as assays that measure the ERK, JNK, and p38 MAP...
To 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.
Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.
Tryptophan decarboxylase (TDC) from Cafharanfhus 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 fabaci (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.The process by which insects select a host plant for feeding and reproduction begins with the sensing of structural and metabolic components of the plant. Alkaloids are one of several plant-produced substances known to greatly influence insect recognition, feeding, and oviposition. Levels of the alkaloid gramine are inversely related to the extent of aphid infestation (ZuAiga et al., 1988). Furthermore, polyphagous aphids colonize only low-alkaloid-producing plants, whereas aphids with restricted host specificity prefer high-alkaloid producers, using the ingested alkaloids in their own defense (Niemeyer, 1990).It has been difficult to examine how alkaloids affect insect feeding, because to do so host plant lines must differ only in alkaloid content. Testing alkaloids in an artificial insect diet provides some information, but not a11 behavioral aspects of plant-insect interactions in situ can be reproduced exactly.To directly assess insect feeding on alkaloid-containing plants, we transferred and expressed in Nicotiana tabacum (tobacco) the gene encoding the TDC enzyme (EC 4.2.1.27) of Catharantkus Yoseus (L.) (periwinkle). In several species with TDC activity (Robinson, 1979), tryptamine is thought to participate in the protection of young seedlings against insects, particularly in newly emergent seedling stems and
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