MEKK2 and MEKK3 are two closely related mitogen-activated protein kinase (M
APK) kinase kinases, The kinase domains of MEKK2 and MEKK3 are nearly ident
ical, although their N-terminal regulatory domains are significantly diverg
ent. By yeast two-hybrid library screening, we have identified MEK5, the MA
PK kinase in the big mitogen-activated protein kinase 1 (BMK1)/ ERK5 pathwa
y, as a binding partner for MEKK2. MEKK2 expression stimulates BMK1/ERK5 ac
tivity, the downstream substrate for MEK5. Compared with MEKK3, MEKK2 activ
ated BMK1/ERK5 to a greater extent, which might correlate with a higher aff
inity MEKK2-MEK5 interaction. A dominant negative form of MEK5 blocked the
activation of BMK1/ERK5 by MEKK2, whereas activation of c-Jun N-terminal ki
nase (JNK) was unaffected, showing that MEK5 is a specific downstream effec
tor of MEKK2 in the BMK1/ERK5 pathway. Activation of BMK1/ERK5 by epidermal
growth factor and H2O2 in Cos7 and HEK293 cells was completely blocked by
a kinase-inactive MEKK3 (MEKK3kin(-)), whereas MEKK2kin(-) had no effect. H
owever, in D10 T cells, expression of MEKK2kin(-) but not MEKK3kin(-) inhib
ited BMK1/ERK5 activity. Two-hybrid screening also identified Lck-associate
d adapter/Rlk- and Itk-binding protein (Lad/RIBP), a T cell adapter protein
, as a binding partner for MEKK2. MEKK2 and Lad/RIBP colocalize at the T ce
ll contact site with antigen-loaded presenting cells, demonstrating cotrans
location of MEKK2 and Lad/RIBP during T cell activation. MEKK3 neither bind
s Lad/RIBP nor is recruited to the T cell contact with antigen presenting c
ell. MEKK2 and MEKK3 are differentially associated with signaling from spec
ific upstream receptor systems, whereas both activate the MEK5-BMK1/ERK5 pa
thway.