Characterization of the Polypeptide Composition of Human Factor VIII:C and the Nucleotide Sequence and Expression of the Human Kidney cDNA

Truett, Martha; Blacher, Russell; Burke, Rae Lyn; Caput, Daniel; Chu, C.; Dina, Dino; Hartog, K.; Kuo, Che‐Hui; Masiarz, Frank R.; Merryweather, James P.; Najarian, Richard C.; Pachl, Carol; Potter, S. J.; Puma, J. P.; Quiroga, Margarita; Rall, Leslie B.; Randolph, Anne; Urdea, Mickey S.; Valenzuela, Pablo; Dahl, Hans Henrik M.; Favalaro, J.; Hansen, J.S.; Nordfang, Ole; Ezban, Mirella

doi:10.1089/dna.1985.4.333

Cited by 102 publications

(47 citation statements)

References 44 publications

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“…Colonies were screened for mutations by direct sequencing (Sequenase version 2.0 DNA dideoxynucleotide sequencing kit; US Biochemical Corporation). The mutated fragment was then subcloned into the full-length c-kit cDNA in the mammalian expression vector pSV7d (Truett et al, 1985). Finally, the mutated c-kit cDNA was cloned into pcDNA1/Neo, a cytomegalovirus-based expression vector.…”

Section: Site-directed Mutagenesis and Plasmid Constructsmentioning

confidence: 99%

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction

et al. 1999

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In this report we show that Tyr568 and Tyr570 are phosphorylated in vivo in the Kit/stem cell factor receptor (Kit/SCFR) following ligand-stimulation. By mutation of Tyr568 and Tyr570 to phenylalanine residues and expression of the mutated receptors in porcine aortic endothelial (PAE) cells, we could demonstrate a loss of activation of members of the Src family of tyrosine kinases when Tyr568 was mutated, while mutation of Tyr570 only led to a minor decrease in activation of Src family members. Mutation of both tyrosine residues led to a complete loss of Src family kinase activation. Phosphorylation of the adapter protein Shc by growth factor receptors provides association sites for Grb2-Sos, thereby activating the Ras/MAP kinase pathway. A much lowered degree of Shc phosphorylation, Ras and Erk2 activation and c-fos induction was seen in the Y568F mutant, while in the Y570F mutant these responses were less a ected. In contrast, the mitogenic response was only slightly reduced. In a mutant receptor with both Tyr568 and Tyr570 mutated to phenylalanine residues, no phosphorylation of Shc and no activation of Ras and Erk2 was seen in response to stem cell factor stimulation, very weak induction of c-fos was seen and the mitogenic response was severely depressed. These data show that Ras/MAP kinase activation and c-fos induction by Kit/SCFR are mediated by members of the Src family kinases. However, the mitogenic response is only to a minor extent dependent on Src kinase activity.

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Section: Site-directed Mutagenesis and Plasmid Constructsmentioning

confidence: 99%

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction

et al. 1999

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“…The MscI site was used for ligation with a fragment encoding three consecutive HA-epitopes followed by a stop codon. The cDNA encoding HA-tagged DEP-1 was subsequently inserted as an Eco RI/Eco RI insert into the pSV7d expression vector (Truett et al, 1985). The DECD-DEP-1 pcDNA3 plasmid encodes a protein composed of the IgG heavy chain signal sequence followed by amino acid residues 895 ± 1336 of DEP-1 and carboxy-terminal HAepitopes.…”

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confidence: 99%

An extracellular ligand increases the specific activity of the receptor-like protein tyrosine phosphatase DEP-1

2001

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Cellular growth, di erentiation and migration is regulated by protein tyrosine phosphorylation. Receptor-like protein tyrosine phosphatases are thus likely to be key regulators of vital cellular processes. The regulation of these enzymes is in general poorly understood. Ligands have been identi®ed only for a small subset of the receptor-like protein tyrosine phosphatases and in no case has upregulation of the speci®c activity by extracellular ligands been demonstrated. Prompted by earlier ®ndings of ligands for receptor-like protein tyrosine phosphatases in extracellular matrix we investigated if Matrigel TM , a preparation of extracellular matrix proteins, contained modulators of the speci®c activity of the receptor-like protein tyrosine phosphatase DEP-1. Matrigel TM stimulation of cells increased the speci®c activity of immunoprecipitated DEP-1. Also, incubation of immunoprecipitated DEP-1 with Matrigel TM led to an increase in DEP-1 activity, which was blocked by soluble DEP-1 extracellular domain. Finally, immunoprecipitated DECD-DEP-1, a mutant form of DEP-1 lacking most of the extracellular domain, failed to respond to Matrigel TM stimulation. These experiments identify Matrigel TM as a source of DEP-1 agonist(s) and provide the ®rst evidence for upregulation of the speci®c activity of receptor-like protein tyrosine phosphatases by extracellular ligands. Oncogene (2001) 20, 5219 ± 5224.

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“…The PCR DNA products were cut with the restriction enzyme, Sail, and ligated into pSVTd [13] cut with restriction enzymes, Sinai and Satl. The plasmid li~tion mixture was used to transform E, coil K12-MCI061 strain u~ing CaCI, and heat shock [14].…”

Section: Experimentalmentioning

confidence: 99%

“…Inhibitory factors to the catalytic subunit of protein kinase A were removed by gel filtration, The activities of the recombinant proteins were determined by measuring chemiluminescence counts per 10 s in a buffer containing 20 mM Tris-acatate, 12 mM magnesium acetate, 0,3 mM dithiothreitel, 0,1% w/v bovine serum albumin and 200/tM lueiferin, pH 7.8, Specific activities were determined by relating light emission to ng protein measured from [" S]methionine incorporation [10l. Chemiluminescence was measured in a home-built chemiluminometer [12].The PCR DNA products were cut with the restriction enzyme, Sail, and ligated into pSVTd [13] cut with restriction enzymes, Sinai and Satl. The plasmid li~tion mixture was used to transform E, coil K12-MCI061 strain u~ing CaCI, and heat shock [14].…”

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Engineering firefly luciferase as an indicator of cyclic AMP‐dependent protein kinase in living cells

Sala-Newby

Campbell

1992

FEBS Letters

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A bioluminescent indicator for protein kinase A ba~ been developed by mutating V217 in firefly (Pltotinuspyralis) laciferase to R, and the C-terminal peroxisomal signal removed by PCR. The eDNA for normal and the RRFS mutant lueiferase were inserted into pSVTd and expressed in COS-7 ceils, Transient expression i,~ approximately 5% of cells was confirmed by extraction of active luciferase, light emission from cells in the presence of luciferin, and immuno.localisation, The cyclic-AMP analogue, 8-(4-chlorophenylthio)-c)'clic AMP caused a 5-10% decrease in light emission within 4 rain in COS cells expressing the RRFS mutaiat, but not in cells expressing normal lueiferase, This provides for the fir,zt time an indicator for detecting and quantifying protein kinase A activation in living cells, Cyclic AMP; Protein kinasc; Lucifcrasc. Bioluminescence 1, INTRODUCTIONIt is well established that both cyclic AMP and cyclic GMP play a key role in signalling cell activation, division and development, and defence [1,2]. The kinases and phosphatases which are activated by these intracellular signals have also been well characterised [3]. cAMP binds to the regulatory subunit (R) of" C~.R,, releasing the catalytic subunit which then binds to particular sites on targeted proteins. A key recognition sequence in these proteins is RRXS, although residues on either side of this can affect the selectivity and affinity for the kinase [3,4]. Yet precisely how protein kinase A is involved in many types of cell activation, particularly in the control of gene expression required for cell division or transformation, is not well established [1].Understanding of the role of Ca -'~ in cell activation and ceil injury has been revolutionised by the technology for measuring and manipulating free Ca :+ , and for locating it, in live cells [5][6][7][8]. A major problem in establishing definitively the role of protein kinase A in a cellular event has been the lack of methods for measuring and locating protein phosphorylation in living cells. We have recently established a strategy using engineering of bioluminescent proteins to solve this problem [9,10]. eDNA coding for firefly luciferase was engineered using PCR to contain a protein kinase A recognition site, RRFS [10], and lacking the C-terminal peroxisomal signal in native luciferase [11]. Phosphorylation by protein kinase A caused an 80-90% decrease in 2, EXPERIMENTALThe T7 RNA polymerase promoter was added to the 5' end of frefly lueiferase eDNA, elon~l in pcDVI primer and Honjo linker a~ previously described [10], a gall site to the 3' end and the codons for the last three amino acids, SKL. the peroxisomal signal [1 l], removed by PCR using a 3' anti-sense primer Cl"GCTTGAGCTCGTCGACTTACTTTCCGCCCTTCrTG. The eodon for reline at posilion 217, GTC, was mutated to the ¢odon for arginin¢, CGC, by two stage PCR as previously described [10]. generating a site coding for RRFS. Recombinant normal and mutant (RRFS) firefly lueiferases were generated by first transcribing the PCR DNA product using 1"7 RN...

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Characterization of the Polypeptide Composition of Human Factor VIII:C and the Nucleotide Sequence and Expression of the Human Kidney cDNA

Cited by 102 publications

References 44 publications

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction

An extracellular ligand increases the specific activity of the receptor-like protein tyrosine phosphatase DEP-1

Engineering firefly luciferase as an indicator of cyclic AMP‐dependent protein kinase in living cells

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