Tyrosine phosphorylation of the proto-oncoprotein Raf-1 is regulated by Raf-1 itself and the phosphatase Cdc25A.
There is a growing body of evidence demonstrating that Raf-1 is phosphorylated on tyrosines upon stimulation of a variety of receptors. Although detection of Raf-1 tyrosine phosphorylation has remained elusive, genetic analyses have demonstrated it to be important for Raf-1 activation. Here we report new findings which indicate that Raf-1 tyrosine phosphorylation is regulated in vivo. In both a mammalian and baculovirus expression system, a kinase-inactive allele of Raf-1 was found to be tyrosine phosphorylated at levels much greater than that of wild-type Raf-1. The level of tyrosine phosphate on Raf-1 was markedly increased upon treatment with phosphatase inhibitors either before or after cell lysis. Cdc25A was found to dephosphorylate Raf-1 on tyrosines that resulted in a significant decrease in Raf-1 kinase activity. In NIH 3T3 cells, coexpression of wild-type Raf-1 and phosphatase-inactive Cdc25A led to a marked increase in Raf-1 tyrosine phosphorylation in response to platelet-derived growth factor. These data suggest that the tyrosine phosphorylation of Raf-1 is regulated not only by itself but also by Cdc25A.
Xia
K
K
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Lee
R S
RS
Narsimhan
R P
RP
Mukhopadhyay
N K
NK
Neel
B G
BG
Roberts
T M
TM
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053614:Janus Kinase 2; D051379:Mice; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D018411:Spodoptera / Q000166:cytology; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology; D020687:cdc25 Phosphatases
D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D019908:Proto-Oncogene Proteins c-raf; C517005:Cdc25a protein, mouse; D017027:Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases