Preparation of a Cell-Free Protein-Synthesizing System from Wheat Germ

Anderson, Carl W.; Straus, J. William; Dudock, Bernard S.

doi:10.1016/b978-0-12-765560-4.50044-7

Cited by 38 publications

(41 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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“…Wheatgerm lysate was prepared and used according to Anderson et al (1983) Expression of and purification of protein A-glycolate oxidase…”

Section: Methodsmentioning

confidence: 99%

NADPH is a specific inhibitor of protein import into glyoxysomes

Pool¹,

López‐Huertas²,

Horng³

et al. 1998

The Plant Journal

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SummaryWe have studied the import of proteins into glyoxysomes in vitro and show that this process is specifically inhibited by NADPH. NADPH affects both binding and translocation of proteins into glyoxysomes, and inhibition is determined by the ratio of NADP ⍣ to NADPH. The site of action of NADPH is most likely within the glyoxysome because (1) pretreatment of glyoxysomes with NADPH, followed by re-isolation of the organelles prior to the import assay, resulted in inhibition of import that could be restored by the addition of NADP ⍣ ; (2) low concentrations of NADPH inhibited binding of proteins to broken glyoxysome membranes. The sensitivity of protein import to inhibition by NADPH declines as glyoxysomes are converted to leaftype peroxisomes. A model is proposed that speculates on a possible role for NADPH in regulating protein import into plant peroxisomes.

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“…The technique was again successful using the template of exogenous mRNA molecules (Schreier and Staehelin 1973). During approximately the same timeframe, investigators applied this capacity to extracts of wheat germs and, of great interest, found that the endogenous as opposed to exogenous expression of mRNA molecules manifested naturally low levels of protein (Marcus, Efron, and Weeks 1974;Roberts and Paterson 1973;Anderson, Straus, and Dudock 1983). Other techniques of eliminating endogenous mRNA were based on application of calcium iondependent bacterial RNAse, used to augment the efficiency of protein expression system www.intechopen.com in lysates of erythrocytes (Jackson and Hunt 1983;Merrick 1983;Pelham and Jackson 1976) as well as in other lysates originating from animal cells (Henshaw and Panniers 1983).…”

Section: The Beginning Of Cell-free Protein Synthesismentioning

confidence: 99%