14-3-3-PROTEIN INTERACT WITH SPECIFIC MEK KINASES

MEK (mitogen-activated protein kinase/extracellular signal-regulated k inase kinase) kinases (MEKKs) regulate c-Jun N-terminal kinase and ext racellular response kinase pathways. The 14-3-3 zeta and 14-3-3 epsilo n isoforms were isolated in a two-hybrid screen for proteins interacti ng with the N-terminal regulatory domain of MEKK3. 14-3-3 proteins bou nd both the N-terminal regulatory and C-terminal kinase domains of MEK K3. The binding affinity of 14-3-3 for the MEKK3 N terminus was 90 nM, demonstrating a high affinity interaction. 14-3-3 proteins also inter acted with MEKK1 and MEKK2, but not MEKK4. Endogenous 14-3-3 protein a nd MEKK1 and MEKK2 were similarly distributed in the cell, consistent with their in vitro interactions. MEKK1 and 14-3-3 proteins colocalize d using two color digital confocal immunofluorescence. Binding of 14-3 -3 proteins mapped to the N-terminal 393 residues of 196-kDa MEKK1. Un like MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1 demonstrat ed little or no ability to interact with 14-3-3 proteins. MEKK1, but n ot MEKK2, -3 or -4, is a caspase-3 substrate that when cleaved release s the kinase domain from the N-terminal regulatory domain. Functionall y, caspase-3 cleavage of MEKK1 releases the kinase domain from the N-t erminal 14-3-3-binding region, demonstrating that caspases can selecti vely alter protein kinase interactions with regulatory proteins. With regard to MEKK1, -2 and -3, 14-3-3 proteins do not appear to directly influence activity, but rather function as ''scaffolds'' for protein-p rotein interactions.