Mitogen regulation of c-Raf-1 protein kinase activity toward mitogen-activated protein kinase-kinase.
The c-raf-1 protooncogene encodes a Ser/Thr protein kinase. A mitogen-activated protein kinase-kinase (MAPKK) purified from bovine brain is phosphorylated and activated 4-9-fold in vitro by c-Raf-1 from mitogen-treated cells. c-Raf-1 protein kinase activity, measured by the phosphorylation of brain MAPKK substrate, is detectably activated within 1 min after addition of platelet-derived growth factor (PDGF) to 3T3 cells, increasing more rapidly than the endogenous NIH 3T3 cell MAPKK activity. c-Raf-1 activation is also induced by insulin, phorbol ester, thrombin, and endothelin. PDGF-, epidermal groth factor-, and insulin-stimulated 32P-c-Raf-1 yield very similar, complex tryptic 32P-peptide maps, wherein only 2 of 10 32P-peptides appear entirely de novo after growth factor addition. Mitogen-activated protein kinase/extracellular signal-regulated kinase-2 can phosphorylate c-Raf-1 in vitro on 4-6 tryptic 32P-peptides, all of which comigrate with tryptic 32P-peptides derived from c-Raf-1 labeled in situ. Mitogen-activated protein kinase phosphorylation of c-Raf-1 in vitro, however, does not 1) generate 32P-peptides that comigrate with those that appear de novo after PDGF or insulin treatment in situ; 2) does not convert c-Raf-1 polypeptides to a slower mobility on SDS-polyacrylamide gel electrophoresis as is seen after PDGF or insulin; 3) does not alter c-Raf-1 kinase activity toward MAPKK. Thus, based on overlapping site specificity, Erk-2 is a viable candidate to be among the PDGF-stimulated c-Raf-1 kinases. Although PDGF/insulin-stimulated c-Raf-1 Ser/Thr phosphorylation may be necessary to sustain the active state, a role for mitogen-activated protein kinase/extracellular signal-regulated kinase-2 phosphorylation in the initiation of c-Raf-1 activation is unlikely.
Kyriakis
J M
JM
Diabetes Unit, Massachusetts General Hospital, Charlestown 02129.
Force
T L
TL
Rapp
U R
UR
Bonventre
J V
JV
Avruch
J
J
D016475:3T3 Cells; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D004789:Enzyme Activation; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D008934:Mitogens / Q000494:pharmacology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D013379:Substrate Specificity
D008934:Mitogens; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020929:Mitogen-Activated Protein Kinase Kinases