A PROLINE-RICH SEQUENCE UNIQUE TO MEK1 AND MEK2 IS REQUIRED FOR RAF BINDING AND REGULATES MEK FUNCTION

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is a bsent both from the yeast homologs Ste7 and Byr1 and from a recently c loned activator of the JNK/stress-activated protein kinases, SEK1/MKK4 . Since this PR sequence occurs in MEKs that are regulated by Raf fami ly enzymes but is missing from MEKs and SEKs activated independently o f Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 bl ocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following gr owth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for act ivation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosph orylation site within the PR sequence of MEK1 was required for sustain ed MEK1 activity in response to serum stimulation of quiescent fibrobl asts. Consistent with this observation, we observed that MEK2, which l acks a phosphorylation site at the corresponding position, was activat ed only transiently following serum stimulation. Finally, we found tha t deletion of the PR sequence from a constitutively activated MEK1 mut ant rendered the protein nontransforming in Rat1 fibroblasts. These ob servations indicate a critical role for the PR sequence in directing s pecific protein-protein interactions important for the activation, ina ctivation, and downstream functioning of the MEKs.