Inhibition of Raf-1 signaling by a monoclonal antibody, which interferes with Raf-1 activation and with Mek substrate binding.
Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.
Kolch
W
W
Department of Medicine, University of California at San Diego, La Jolla 92093, USA.
Philipp
A
A
Mischak
H
H
Dutil
E M
EM
Mullen
T M
TM
Feramisco
J R
JR
Meinkoth
J L
JL
Rose
D W
DW
D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology*; D017124:Conserved Sequence; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D000939:Epitopes / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology; D048728:MAP Kinase Kinase Kinase 1; D051379:Mice; D008845:Microinjections; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D018631:ras Proteins / Q000378:metabolism / Q000502:physiology
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D006133:Growth Substances; D011518:Proto-Oncogene Proteins; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048728:MAP Kinase Kinase Kinase 1; C482578:Map3k1 protein, mouse; D018631:ras Proteins