MITOGEN-ACTIVATED PROTEIN (MAP) KINASE PHOSPHORYLATION OF MAP KINASE KINASE - DETERMINATION OF PHOSPHORYLATION SITES BY MASS-SPECTROMETRY AND SITE-DIRECTED MUTAGENESIS

Mitogen-activated protein kinase kinase (MKK) phosphorylates and activ ates mitogen-activated protein kinase (MAPK) in response to stimulatio n of various eukaryotic signaling pathways. Conversely, a recent repor t showed that MAPK phosphorylates MKK in vitro [Matsuda, S., Gotoh, Y. , and Nishida, E. (1993) J. Biol, Chem. 268, 3277-3281]. To gain insig ht into the function of this feedback phosphorylation, we identified t he major sites targeted for phosphorylation by MAPK and examined wheth er such a modification plays a role in regulating the basal and stimul ated MKK activities. Two phosphopeptides generated by tryptic digestio n of MAPK-phosphorylated MKK were identified by electrospray ionizatio n mass spectrometry. Cyanogen bromide cleavage also yielded two phosph opeptides whose sequence overlapped with the tryptic phosphopeptides. Both sets of phosphopeptides contained candidate MAPK target sites at Thr292 and Thr386 that fit the consensus sequence ProXThrPro. Replace ment of either Thr292 or Thr386 with alanine by site-directed mutagene sis reduced the phosphate incorporation respectively to 32 or 75% that of wild type MKK. Replacement of both threonine residues with alanine reduced phosphate incorporation to 2.5% that of wild type enzyme. Com parison of MAPK-phosphorylated vs. unphosphorylated MKK showed no sign ificant differences in basal or Raf-1-stimulated MKK activity. We conc lude that the phosphorylation of MKK at Thr292 and Thr386 does not int erfere with catalysis in vitro.