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In this report, we find that Ser43, Ser259, and Ser621 are the key web-sites of Raf-one that are phosphorylated in mammalian cells As well as in Sf9 insect cells contaminated which has a recombinant baculovirus encoding human Raf-1. Mutant Raf-1 proteins lacking kinase action also are phosphorylated on these websites in vivo, indicating that these phosphorylation events aren't a consequence of autophosphorylation. Furthermore, we see that Thr268 would be the predominant Raf-one residue phosphorylated in in vitro autokinase assays. Also, We now have examined the biochemical exercise of baculovirus-expressed Raf-1 proteins that contains mutations at these phosphorylation web sites. In in vitro protein kinase assays Ser259 mutant proteins were 2-fold far more Energetic than wild-kind Raf-one and Ser621 mutant proteins had been inactive as kinases.
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