The Synthesis of High-Molecular-Weight Proteins in the Wheat Germ Translation System

Morch, Marie-Dominique; Drugeon, Gabrièle; Zagórski, W.; Haenni, Anne Lise

doi:10.1016/b978-0-12-743655-5.50011-7

Cited by 11 publications

(9 citation statements)

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“…Next, immunoblotting studies were performed using To confirm that the preadsorbed antibodies do not crossreact with phosphorylated PTEN, rabbit reticulocyte and wheat germ lysate transcription-translation systems were used to generate phosphorylated and unphosphorylated PTEN protein, respectively, in the presence of radioactively labeled methionine. Unlike the rabbit reticulocyte system, wheat germ lysate exhibits very low endogenous posttranslational modification activity (2,27,38,39). Thus, the translated PTEN protein produced by the wheat germ lysate system should show minimal if any phosphorylation.…”

Section: Resultsmentioning

confidence: 99%

p85 Associates with Unphosphorylated PTEN and the PTEN-Associated Complex

Rabinovsky

Pochanard

McNear

et al. 2009

Molecular and Cellular Biology

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The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/ AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110␤ isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway.The phosphoinositide 3-kinase (PI3K)/AKT signaling pathway regulates glucose/nutrient homeostasis and cell survival and plays a central role in both normal metabolism and cancer. The PTEN tumor suppressor gene (29, 30, 54) negatively regulates the PI3K/AKT pathway by dephosphorylating the D3 hydroxyl subunit of phosphoinositide-3,4,5-trisphosphate, a key membrane phosphatidylinositol generated by PI3K (34). PTEN undergoes genetic or epigenetic inactivation in many malignancies, including glioblastoma, melanoma, and endometrial, prostate, and breast cancers, among others (6, 13, 22, 23, 47, 49-51, 55, 68). Similarly, germ line mutations of PTEN are associated with the development of hamartomatous neoplasias such as Cowden disease and Bannayan-Zonana syndrome (17,21,41).The tumor suppressor function of PTEN undergoes dynamic regulation involving both C-terminal phosphorylation and protein-protein interactions. Phosphorylation of serine and threonine residues at the PTEN C-terminal tail, mediated by kinases such as CK2 and glycogen synthase kinase 3␤, alters its conformational structure and association with PDZ domain-containing proteins and attenuates PTEN enzymatic activity (1, 11, 20, 32, 45, 61-63, 66, 67, 71). Conversely, PTEN function is promoted in large part through its stabilization in unphosphorylated form by incorporation into a high-molecular-weight protein complex (the PTEN-associated complex [PAC]) (66). We first demonstrated the existence of the PAC through gel filtration studies of rat liver extracts, which identified PTEN within a high-molecular-mass peak (Ͼ600 kDa), as well as a lowmolecular-mass peak (40 to 100 kDa) in which PTEN is monomeric and phosphorylated (66). Subsequently, several PDZ domain-containing proteins were shown to interact with PTEN, including MAGI-1b, MAGI-2, MAGI-3, ghDLG, hMAST205, MSP58/MCRS1, NHERF1, and NHERF2, which mediate indirect binding with platelet-derived growth factor (PDGF) receptor ␤ (25,36,4...

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Section: Resultsmentioning

confidence: 99%

p85 Associates with Unphosphorylated PTEN and the PTEN-Associated Complex

Rabinovsky

Pochanard

McNear

et al. 2009

Molecular and Cellular Biology

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“…Hybrid-arrest translation of GmHSP22.5 and pFS2033 transcripts was done with total RNA as described by Galau et al [ 11 ] using the same hybridization conditions as for the hybrid-select experiments. Translations were done with a wheat germ cell free translation system supplemented with 35S-methionine [32].…”

Section: Rna Blot Analysismentioning

confidence: 99%

Molecular characterization of cDNAs encoding low-molecular-weight heat shock proteins of soybean

et al. 1996

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Three cDNA clones (GmHSP23.9, GmHSP22.3, and GmHSP22.5) representing three different members of the low-molecular-weight (LMW) heat shock protein (HSP) gene superfamily were isolated and characterized. A fourth cDNA clone, pFS2033, was partially characterized previously as a full-length genomic clone GmHSP22.0. The deduced amino acid sequences of all four cDNA clones have the conserved carboxyl-terminal LMW HSP domain. Sequence and hydropathy analyses of GmHSP22, GmHSP22.3, and GmHSP22.5, representing HSPs in the 20 to 24 kDa range, indicate they contain amino-terminal signal peptides. The mRNAs from GmHSP22, GmHSP22.3, and GmHSP22.5 were preferentially associated in vivo with endoplasmic reticulum (ER)-bound polysomes. GmHSP22 and GmHSP22.5 encode strikingly similar proteins; they are 78% identical and 90% conserved at the amino acid sequence level, and both possess the C-terminal tetrapeptide KDEL which is similar to the consensus ER retention motif KDEL; the encoded polypeptides can be clearly resolved from each other by two-dimensional gel analysis of their hybrid-arrest translation products. GmHSP22.3 is less closely related to GmHSP22 (48% identical and 70% conserved) and GmHSP22.5 (47% identical and 65% conserved). The fourth cDNA clone, GmHSP23.9, encodes a HSP of ca. 24 kDa with an amino terminus that has characteristics of some mitochondrial transit sequences, and in contrast to GmHSP22, GmHSP22.3, and GmHSP22.5, the corresponding mRNA is preferentially associated in vivo with free polysomes. It is proposed that the LMW HSP gene superfamily be expanded to at least six classes to include a mitochondrial class and an additional endomembrane class of LMW HSPs.

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“…Wheat germ cell-free extracts catalyze the incorporation of amino acids into proteins in the presence of supplied mRNAs from various sources (for reviews see [1,2]. However, wheat germ extracts cannot be considered as a pure eukaryotic translation system since they also contain proplastid tRNAs and aminoacyl-tRNA synthetases which have been released from broken organelles during grinding (our unpublished observations).…”

Section: Introductionmentioning

confidence: 99%

Preparation of a tRNA-dependent wheat germ protein-synthesizing system

et al. 1989

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A method is described for the preparation of a tRNA-dependent wheat germ protein-synthesizing system. This system can be supplied with exogenous tRNAs of eukaryotic or prokaryotic origin. In order to obtain maximal aminoacylation of the added tRNAs, the translation assays can be supplemented with homologous aminoacyl-tRNA synthetase preparations. Such a tRNA-dependent wheat germ protein-synthesizing system, which is easy to prepare, can be used not only to translate plant cytoplasmic mRNAs in the presence of added cytoplasmic tRNAs, but also to determine the translation activity of exogenous tRNAs from various sources in the presence of either natural or in vitro synthesized mRNAs.

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The Synthesis of High-Molecular-Weight Proteins in the Wheat Germ Translation System

Cited by 11 publications

References 0 publications

p85 Associates with Unphosphorylated PTEN and the PTEN-Associated Complex

p85 Associates with Unphosphorylated PTEN and the PTEN-Associated Complex

Molecular characterization of cDNAs encoding low-molecular-weight heat shock proteins of soybean

Preparation of a tRNA-dependent wheat germ protein-synthesizing system

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