Figure 3. Analysis of phosphorylation of 1024 peptide array by c-Raf.

A. c-Raf phosphorylation of 1024 peptide array. Phosphorylation of the custom made 1024 peptide array, spotted in triplicate, on incubation with c-Raf and ³³P-γ-ATP for one hour shows differential phosphorylation of the various substrate peptides. B. Consensus sequence of c-Raf substrates using 1024 array design. Consensus sequence obtained from the 1024 peptide array shows a strong preference for arginine at −1,−4 and the −5 position while the −2 position shows a strong preference for a hydrophobic residue and no distinctive preference is seen at the −3 position. Analysis of the C-terminal position relative to the centrally fixed phosphorylated residue shows a very high preference for methionine besides an equal preference for other basic and hydrophobic amino acids at the +1 position. Arginine is preferred at the +2 and +4 positions while methionine and proline along with arginine are preferred at +3 positions. Hydrophobic residues are preferred over basic residues at +5.