Figure 6.
SIMKK Phosphorylates SIMK at the Threonine and Tyrosine Residues of the Activation Loop.
For phosphorylation analysis, the kinase-inactive MAPKs SIMK(K84R), MMK2(K66R), and MMK3(K69R) were incubated in the presence of ATP without (−) or with (+) SIMKK.
(A) Phosphorylation of the TEY motif of the MAPKs was subsequently analyzed by immunoblotting with a pTEpY phosphospecific antibody.
(B) to (D) Phosphorylation of the TEY motif of SIMK(K84R) was analyzed by immunoblotting with a phosphotyrosine-specific antibody (B), immunoblotting with a phosphothreonine-specific antibody (C), and thin-layer chromatography of the phosphorylated amino acids (D). P-Y, P-T, and P-S indicate the mobility of phospho-tyrosine, phospho-threonine, and phospho-serine, respectively.