Dynamic changes in C-Raf phosphorylation and 14-3-3 protein binding in response to growth factor stimulation: differential roles of 14-3-3 protein binding sites.
Phosphorylation events play a crucial role in Raf activation. Phosphorylation of serines 259 and 621 in C-Raf and serines 364 and 728 in B-Raf has been suggested to be critical for association with 14-3-3 proteins. To study the functional consequences of Raf phosphorylations at these positions, we developed and characterized phosphospecific antibodies directed against 14-3-3 binding epitopes: a monoclonal phosphospecific antibody (6B4) directed against pS621 and a polyclonal antibody specific for B-Raf-pS364 epitope. Although 6B4 detected both C- and B-Raf in Western blots, it specifically recognizes the native form of C-Raf but not B-Raf. Contrary to B-Raf, a kinase-dead mutant of C-Raf was found to be only poorly phosphorylated in the Ser-621 position. Moreover, serine 259 to alanine mutation prevented the Ser-621 phosphorylation suggesting an interdependence between these two 14-3-3 binding domains. Direct C-Raf.14-3-3 binding studies with purified proteins combined with competition assays revealed that the 14-3-3 binding domain surrounding pS621 represents the high affinity binding site, whereas the pS259 epitope mediates lower affinity binding. Raf isozymes differ in their 14-3-3 association rates. The time course of endogenous C-Raf activation in mammalian cells by nerve growth factor (NGF) has been examined using both phosphospecific antibodies directed against 14-3-3 binding sites (6B4 and anti-pS259) as well as phosphospecific antibodies directed against the activation domain (anti-pS338 and anti-pY340/pY341). Time course of Ser-621 phosphorylation, in contrast to Ser-259 phosphorylation, exhibited unexpected pattern reaching maximal phosphorylation within 30 s of NGF stimulation. Phosphorylation of tyrosine 340/341 reached maximal levels subsequent to Ser-621 phosphorylation and was coincident with emergence of kinase activity. Taken together, we found substantial differences between C-Raf.14-3-3 binding epitopes pS259 and pS621 and visualized for the first time the sequence of the essential C-Raf phosphorylation events in mammalian cells in response to growth factor stimulation.
Hekman
Mirko
M
Institute for Medical Radiation and Cell Research, University of Wuerzburg, 97078 Wuerzburg.
Wiese
Stefan
S
Metz
Renate
R
Albert
Stefan
S
Troppmair
Jakob
J
Nickel
Joachim
J
Sendtner
Michael
M
Rapp
Ulf R
UR
D048948:14-3-3 Proteins; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D007313:Insecta; D051379:Mice; D008807:Mice, Inbred BALB C; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D048491:Proto-Oncogene Proteins A-raf; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D000911:Antibodies, Monoclonal; D010750:Phosphoproteins; D012694:Serine; D020932:Nerve Growth Factor; D014446:Tyrosine 3-Monooxygenase; C482120:Braf protein, mouse; C482123:Braf protein, rat; D048491:Proto-Oncogene Proteins A-raf; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf