Identification of RAF kinase isoform-specific partners

RAF kintise has at lrast 3 isofonn in the mammalian genome: A. H and C RAF Alignment of these isoforms show that the biggest differences are in their N -terminus, in this work HAK kina>e isofonn-specific functions were studud using a two-hybrid screen. The N terminal region of HAF kinase iso forms was fused to the GAL'1 DNA binding domain and a comprehensive two hybrid screen performed against different cDNA fusion libraries. The screen with C RAF yielded H different rDNAs. For A-RAF 13 different rDNAs were ideindied The following C-RAF interarting proteins were found: 14 33 (53 time; Ha-ras (GB# .100277): Chaperonin IriC (GB #X7-180I) and pyruvate-kinase (GB #M26252). An unknown liutnan psetidogene d)NA wa,s found in a Hela cell library wi'th both C-RAF and A HAF. The following A-KAh in teractino proteins were found: R-ra>, huinan r.irbamovl phosphate synthase (GH #D90282), human arginine sun-mate syrithase (GH#X01630), and hu mati extracellular protein with EGE repeats (GB#U03877). Four proteins were found that sperifirallv interacted only with the A-RAF isoform. C'unver sly 1.33 and two novel proteins interacted with both H-RAt and C-RAF bur not A RAF. The minimal region sufficient for all ihese interactions is the cypleine rich domain (CRI), aa 70-153 of A-RAF). This domain has a Znfinger structure. Similar structures are found in a number of serine/threonine kinses such as PKC. KSR kiruise and MFK kinase 1 (GB# P53349). Our data Miggesls that the CRU doma.n iunctiun.s an art important protein-prntein intraction domain for a variety of protein kinaes.