Raf-1 activates MAP kinase-kinase.
The normal cellular homologue of the acutely transforming oncogene v-raf is c-raf-1, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases Erk1 and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K). Here we report that MAPK-K as well as Erk1 and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with M(r) 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-1 protooncogene product to be identified.
Kyriakis
J M
JM
Diabetes Unit, Medical Services, Massachusetts General Hospital, Charlestown 02129.
App
H
H
Zhang
X F
XF
Banerjee
P
P
Brautigan
D L
DL
Rapp
U R
UR
Avruch
J
J
D016475:3T3 Cells; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004789:Enzyme Activation; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction; D014162:Transfection
D008934:Mitogens; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases