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 peptide, was found to efficiently inhibit the immunoprecipitation of the enzyme with anti-IDA MAPK serum. The value of half-inhibition concentration (100 nM) indicates that the IDA peptide and native MAPK activation loop are virtually indistinguishable in terms of antibody recognition. On the other hand, the Tyr-phosphorylated form of the peptide exerted its inhibitory action at around one order higher concentration. Shorter nonapeptides covering the epitope sequence of anti-IDA MAPK antibody could also affect the immunoprecipitation but at much higher concentrations (half-inhibition concentration approximately 100 microM) and independently of their phosphorylation state. Circular dichroic study revealed 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 nonapeptides. 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.
Tokmakov
A A
AA
Graduate School of Science and Technology, Biosignal Research Center, Kobe University, Nada, Japan. tokmak@inherit.biosig.kobe-u.ac.jp
Sato
K I
KI
Fukami
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism / Q000648:ultrastructure*; D002942:Circular Dichroism; D004789:Enzyme Activation; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000648:ultrastructure; D017433:Protein Structure, Secondary; D014982:Xenopus laevis
D010748:Phosphopeptides; D010750:Phosphoproteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases