Mutation of the Two Asp (D) Residues in the MAPK CD Domain Does Not Enhance the Activation of SIPK or WIPK.
(A) The CD domain is evolutionarily conserved in plants, animals, and yeast.
(B) Mutation of the conserved Asp residues in the CD domain to Asn does not enhance SIPK activation. D353 alone, or both D350 and D353, in the CD domain of SIPK were replaced with Asn (N). SIPKN, the SIPK(D353N) mutant, and SIPKNN, the SIPK(D350N/D353N) double mutant, were transformed into tobacco leaves. Transgene expression was monitored by immunoblot (IB) analysis with anti-Flag antibody (top), and MAPK activity was determined by in-gel kinase assay with MBP as a substrate (bottom).
(C) Substitution of the conserved Asp residues in the CD domain of WIPK fails to result in its activation. D337 alone, or both D334 and D337, in the CD domain of WIPK were replaced with Asn (N). WIPKN, the WIPK(D337N) mutant, and WIPKNN, the WIPK(D334N/D337N) double mutant, were transformed into tobacco leaves. Transgene expression was monitored by immunoblot analysis with anti-Flag antibody (top), and MAPK activity was determined by in-gel kinase assay with MBP as a substrate (bottom).