Tyrosine phosphorylation of the proto-oncoprotein Raf-1 is regulated by Raf-1 itself and the phosphatase Cdc25A

Abstract

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.

FIG. 1

Significant accumulation of tyrosine phosphates on kinase-inactive Raf-1. (A) Sf21 insect cells were infected with recombinant baculoviruses encoding either the wild-type Raf-1 (Raf-1) or kinase-inactive Raf-1 (301), alone or in combination with baculoviruses encoding Jak2 or pp60^vsrc (Src). Anti-Raf-1 immunoprecipitates were analyzed by SDS-PAGE (8% gel), immunoblotted with antiphosphotyrosine (anti-P-tyr in all figures) antibody, and then reprobed with anti-Raf-1 antibody. The blots were developed by enhanced chemiluminescence. (B) NIH 3T3 cells were transfected with wild-type Raf-1 or with kinase-inactive mutant 301; 48 h posttransfection, cells were either unstimulated (−) or stimulated (+) with PDGF for 10 min. The expressed Raf-1 was immunoprecipitated and Western blotted with antiphosphotyrosine antibody 4G10 (top). Raf-1 expression was detected by probing the identical membrane with anti-Raf-1 antibody (bottom).

FIG. 2

Effects of a tyrosine phosphatase inhibitor on Raf-1 tyrosine phosphorylation. (A) Serum-starved NIH 3T3 cells were either stimulated (+) or not stimulated (−) with PDGF (20 ng/ml) for 10 min. The cells were immediately washed and lysed in the presence or absence of 1 mM sodium vanadate. Raf-1 immunoprecipitated (IP) complexes were prepared and analyzed by Western blotting. The blot was first probed with an antiphosphotyrosine antibody (top) and then stripped and reprobed with an anti-Raf-1 antibody (bottom). (B) Similar to panel A except that the properly starved NIH 3T3 cells were either stimulated (+) or not stimulated (−) with PDGF (20 ng/ml) in the presence (+) or absence (−) of sodium pervanadate for 10 min. (C) Sf21 insect cells were infected with recombinant baculoviruses encoding the vector itself (Mock), wild-type Raf-1 (Raf-1), or kinase-inactive Raf-1 (301) with or without coexpression of Jak2; 48 h postinfection, cells were untreated or treated with sodium pervanadate for 10 min. Raf-1 immune complexes were prepared and analyzed by Western blotting as described for panel A.

FIG. 3

(A) Dephosphorylation of tyrosine-phosphorylated Raf-1 by Cdc25A. Anti-Raf-1 immune complexes were prepared from Sf21 cells coinfected with various combinations of baculoviruses encoding proteins indicated above the lanes. Raf-1 immune complexes were analyzed on a Western blot probed first with antiphosphotyrosine antibody (top), and then with anti-Raf-1 antibody. (B) Augmented tyrosine phosphorylation of ectopic Raf-1 in cells coexpressing the phosphatase-inactive Cdc25A. Exponentially growing NIH 3T3 cells at 50 to 75% confluence were transfected with plasmid pLGP3, encoding HA-tagged wild-type Raf-1, alone or in combination with plasmid pcDNA3, encoding either the active Cdc25A or the phosphatase-inactive Cdc25A (Cdc25Ad). Transfected cells were serum starved and stimulated with PDGF (20 ng/ml) for 10 min prior to lysis. Raf-1 immune complexes were prepared by using anti-HA monoclonal antibody and analyzed on a Western blot probed first with antiphosphotyrosine antibody (left) and then with anti-Raf-1 antibody (right). (C) Effect of dephosphorylation by Cdc25A on Raf-1 kinase activity. Anti-Raf-1 immunoprecipitates were prepared from Sf21 cells infected with the indicated baculoviruses. 22W is an N-terminally truncated and constitutively active form of Raf-1. Extensively washed immune complexes were subjected to an in vitro kinase assay using purified Mek-1 as a substrate. The resulting phosphorylated products were analyzed with a Molecular Dynamics PhosphorImager.

FIG. 4

Enhanced tyrosine phosphorylation of Raf-1 during a kinase assay. (A) Anti-Raf-1 immunoprecipitates (IP) were prepared from either Sf21 cells infected with baculoviruses encoding Raf-1 alone or in combination with Jak2 (top left) or NIH 3T3 cells in the presence or absence of PDGF stimulation (top right). Half of the immunoprecipitates were subjected to an in vitro kinase assay in the absence of an exogenous substrate. Samples before or after the kinase assay were immunoprecipitated with anti-Raf-1 antibody and analyzed by Western blotting with an antiphosphotyrosine antibody. The blot was stripped and reprobed with anti-Raf-1 antibody to indicate Raf-1 protein levels (bottom). (B) The same as panel A except that anti-Raf-1 immunoprecipitates were prepared from Sf21 cells infected with baculoviruses encoding Raf-1 and in combination with p21^ras, Jak2, or pp60^vsrc as indicated. (C and D) The same as panel A except that anti-Raf-1 immune complexes were prepared in the presence (+) or absence (−) of the synthetic Raf-1 peptide (10 nM).