[35] Epitope tagging and protein surveillance

Kolodziej, Peter A.; Young, Richard A.

doi:10.1016/0076-6879(91)94038-e

Cited by 510 publications

(331 citation statements)

References 18 publications

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“…The plasmid pMMTV-ErbB2DIC containing the rat ErbB2 cDNA with a carboxyl-terminal truncation at residue 694 and an HA epitope-tag (Kolodziej and Young, 1991) fused with the mouse mammary tumor virus (MMTV) LTR (Figure 1) was constructed in two steps. First, the intermediate plasmid, pN-DID-HA, was generated by trimolecular ligation of EcoRI digested pcDL-SRa296 (Takebe et al, 1988), the 2.4 kb EcoRI-NdeI fragment from the rat ErbB2 cDNA (Bargmann et al, 1986), and the 236 bp NdeI ± EcoRI digested PCR product generated from the rat ErbB2 cDNA using a forward oligonucleotide primer upstream of the unique NdeI site and the reverse primer 5' GAATGAAT TCAGGCGTAATCAGGCACATCGTATGGGTACAGCC-TACGCATCGTATAC.…”

Section: Plasmidsmentioning

confidence: 99%

Expression of dominant-negative ErbB2 in the mammary gland of transgenic mice reveals a role in lobuloalveolar development and lactation

1999

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Overexpression of the receptor tyrosine kinase ErbB2/ HER2/Neu (ErbB2) occurs in 15 ± 40% of human breast cancers. To determine the function of ErbB2 signaling during normal mouse mammary gland development, we expressed a carboxyl-terminal truncated dominant negative allele of ErbB2 (ErbB2DIC) in the developing mouse mammary gland. Despite ErbB2DIC expression within mammary glands of pubescent virgin and pregnant mice, a phenotype was not observed until late in pregnancy. At 1 day post-partum, lactationally active, distended lobuloalveoli failed to form. This phenotype was exaggerated in multiparous females expressing ErbB2DIC. Immunohistochemical staining for ErbB2-DIC revealed a concordance between high levels of ErbB2DIC protein expression and the absence of lactational products within the lumens of ErbB2DIC stained lobuloalveoli. These results demonstrate that ErbB2 signaling is required for proper mammary development and lactation at parturition.

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Section: Plasmidsmentioning

confidence: 99%

Expression of dominant-negative ErbB2 in the mammary gland of transgenic mice reveals a role in lobuloalveolar development and lactation

1999

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“…A 0+7-kb DNA fragment containing part of the coding region of yPRP17 was PCR amplified from the previously described plasmid, pG1-yPRP17 (Lindsey & Garcia-Blanco, 1998)+ The forward primer y17FHAtag (Table 2) introduces an influenza virus HA epitope tag (Kolodziej & Young, 1991) at the N-terminus and creates two restriction sites (BamHI and NcoI), which were used for subcloning+ The reverse primer y17R25 contained the unique restriction site, SacI, located at nt 650 of the yPRP17 coding sequence+ The BamHI/SacI PCR product was inserted between the same sites of pG1-yPRP17, and the resulting plasmid was named pG1-HAyPRP17+…”

Section: Plasmid Construction and Mutagenesismentioning

confidence: 99%

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

Lindsey-Boltz

Chawla

Srinivasan

et al. 2000

RNA

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In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160-455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the b-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between b strands in the WD repeats, we have identified four amino acids, 235 TETG 238 , that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.

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“…Full-length Raf-1 was tagged at the amino terminus with a myc epitope (EQKLISEEDL) as previously described (Kolodziej and Young, 1991). All mutations were made using the Altered Sites Mutagenesis kit (Promega Corp.) and were con®rmed by DNA sequencing with Thermosequenase (Amersham).…”

Section: Dna Manipulation and Mutagenesismentioning

confidence: 99%

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

et al. 1998

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We have uniformly examined the regulatory steps required by oncogenic Ras, Src, EGF and phorbol 12-myristate 13-acetate (PMA) to activate Raf-1. Speci®-cally, we determined the role of Ras binding and the phosphorylation of serines 338/339, tyrosines 340/341 and the activation loop (491 ± 508) in response to these stimuli in COS-7 cells. An intact Ras binding domain was found to be essential for Raf-1 kinase activation by each stimulus, including PMA. Brief treatment of COS-7 cells with PMA was found to rapidly promote accumulation of the active, GTP-bound form of Ras. Furthermore, loss of the serine 338/339 and tyrosine 340/341 phosphorylation sites also blocked Raf-1 activation by all stimuli tested. Loss of the serine 497 and serine 499 PKCa phosphorylation sites failed to signi®cantly reduce Raf-1 activation by any stimulus including PMA. Alanine substitution of all other potential phosphorylation sites within the Raf-1 activation loop had little or no e ect on kinase regulation by Ras[V12] or vSrc although some mutants were less responsive to PMA. These results suggest that in mammalian cells, Raf-1 can be regulated by a variety of di erent stimuli through a common mechanism involving association with Ras-GTP and multiple phosphorylations of the amino-terminal region of the catalytic domain. Phosphorylation of the activation loop does not appear to be a signi®cant mechanism of Raf-1 kinase regulation in COS-7 cells.

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[35] Epitope tagging and protein surveillance

Cited by 510 publications

References 18 publications

Expression of dominant-negative ErbB2 in the mammary gland of transgenic mice reveals a role in lobuloalveolar development and lactation

Expression of dominant-negative ErbB2 in the mammary gland of transgenic mice reveals a role in lobuloalveolar development and lactation

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

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