PHOSPHORYLATION-SENSITIVE SECONDARY STRUCTURE IN A SYNTHETIC PEPTIDE CORRESPONDING TO THE ACTIVATION LOOP OF MAP KINASE

A 26-amino acid long synthetic peptide corresponding to the activation loop of Xenopus MAP kinase (MAPK), termed IDA (Inter-DFG-APE) MAPK pe ptide, was found to efficiently inhibit the immunoprecipitation of the enzyme with anti-IDA MAPK serum. The value of half-inhibition concent ration (100 nM) indicates that the IDA peptide and native MAPK activat ion loop are virtually indistinguishable in terms of antibody recognit ion. On the other hand, the Tyr-phosphorylated form of the peptide exe rted its inhibitory action at around one order higher concentration Sh orter nonapeptides covering the epitope sequence of anti-IDA MAPK anti body could also affect the immunoprecipitation but at much higher conc entrations (half-inhibition concentration similar to 100 mu M) and ind ependently of their phosphorylation state. Circular dichroic study rev ealed that a secondary structure could be readily induced with the aid of trifluoroethanol in the unphosphorylated and, to a less extent, in the Tyr-phosphorylated IDA MAPK peptide but not in the shorter nonape ptides. These results suggest that the secondary structure similar to that of the unphosphorylated activation loop of MAPK can be formed in the IDA MAPK peptide and may be lost upon its Tyr-phosphorylation. (C) 1997 Academic Press.