Linked Life Data

11134016

Source:http://linkedlifedata.com/resource/pubmed/id/11134016

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
13
pubmed:dateCreated
2001-3-27
pubmed:abstractText
We recently established a two-stage in vitro assay for KSR kinase activity in which KSR never comes in contact with any recombinant kinase other than c-Raf-1 and defined the epidermal growth factor (EGF) as a potent activator of KSR kinase activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000) J. Biol. Chem. 275, 17276-17280). That study, however, did not address the mechanism of c-Raf-1 stimulation by activated KSR. Here we show that phosphorylation of c-Raf-1 on Thr(269) by KSR is necessary for optimal activation in response to EGF stimulation. In vitro, KSR specifically phosphorylated c-Raf-1 on threonine residues during the first stage of the two-stage kinase assay. Using purified wild-type and mutant c-Raf-1 proteins, we demonstrate that Thr(269) is the major c-Raf-1 site phosphorylated by KSR in vitro and that phosphorylation of this site is essential for c-Raf-1 activation by KSR. KSR acts via transphosphorylation, not by increasing c-Raf-1 autophosphorylation, as kinase-inactive c-Raf-1(K375M) served as an equally effective KSR substrate. In vivo, low physiologic doses of EGF (0.001-0.1 ng/ml) stimulated KSR activation and induced Thr(269) phosphorylation and activation of c-Raf-1. Low dose EGF did not induce serine or tyrosine phosphorylation of c-Raf-1. High dose EGF (10-100 ng/ml) induced no additional Thr(269) phosphorylation, but rather increased c-Raf-1 phosphorylation on serine residues and Tyr(340)/Tyr(341). A Raf-1 mutant with valine substituted for Thr(269) was unresponsive to low dose EGF, but was serine- and Tyr(340)/Tyr(341)-phosphorylated and partially activated at high dose EGF. This study shows that Thr(269) is the major c-Raf-1 site phosphorylated by KSR. Furthermore, phosphorylation of this site is essential for c-Raf-1 activation by KSR in vitro and for optimal c-Raf-1 activation in response to physiologic EGF stimulation in vivo.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
30
pubmed:volume
276
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
9733-41
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed-meshheading:11134016-Animals, pubmed-meshheading:11134016-COS Cells, pubmed-meshheading:11134016-Dose-Response Relationship, Drug, pubmed-meshheading:11134016-Enzyme Activation, pubmed-meshheading:11134016-Epidermal Growth Factor, pubmed-meshheading:11134016-Humans, pubmed-meshheading:11134016-Mice, pubmed-meshheading:11134016-Mutation, pubmed-meshheading:11134016-Phosphoric Monoester Hydrolases, pubmed-meshheading:11134016-Phosphorylation, pubmed-meshheading:11134016-Precipitin Tests, pubmed-meshheading:11134016-Protein Binding, pubmed-meshheading:11134016-Protein Kinases, pubmed-meshheading:11134016-Protein Structure, Tertiary, pubmed-meshheading:11134016-Proto-Oncogene Proteins c-raf, pubmed-meshheading:11134016-Recombinant Proteins, pubmed-meshheading:11134016-Serine, pubmed-meshheading:11134016-Signal Transduction, pubmed-meshheading:11134016-Threonine, pubmed-meshheading:11134016-Transfection, pubmed-meshheading:11134016-Tyrosine, pubmed-meshheading:11134016-Valine, pubmed-meshheading:11134016-ras Proteins
pubmed:year
2001
pubmed:articleTitle
Kinase suppressor of Ras signals through Thr269 of c-Raf-1.
pubmed:affiliation
Laboratory of Signal Transduction, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't