Identification of the major phosphorylation sites of the Raf-1 kinase.
Treatment of cells with various growth factors and mitogens results in the rapid hyperphosphorylation and activation of the Raf-1 kinase. To determine if phosphorylation events affect Raf-1 activity, we have initiated experiments to identify the phosphorylation sites of Raf-1. In this report, we find that Ser43, Ser259, and Ser621 are the major sites of Raf-1 which are phosphorylated in mammalian cells and in Sf9 insect cells infected with a recombinant baculovirus encoding human Raf-1. Mutant Raf-1 proteins lacking kinase activity are also phosphorylated on these sites in vivo, indicating that these phosphorylation events are not a consequence of autophosphorylation. Furthermore, we find that Thr268 is the predominant Raf-1 residue phosphorylated in in vitro autokinase assays. In addition, we have examined the biochemical activity of baculovirus-expressed Raf-1 proteins containing mutations at these phosphorylation sites. In in vitro protein kinase assays Ser259 mutant proteins were 2-fold more active than wild-type Raf-1 and Ser621 mutant proteins were inactive as kinases. Analysis of the residues surrounding Ser259 and Ser621 indicates that RSXSXP may be a consensus sequence for the kinase responsible for phosphorylation of Raf-1 at these sites. Interestingly, these RSXSXP sequences are completely conserved throughout evolution in all Raf family members.
Morrison
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DK
ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702.
Heidecker
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G
Rapp
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Copeland
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TD
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D012694:Serine / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf