MEKK3 interacts with the PA28 gamma regulatory subunit of the proteasome

The proteasome is a multisubunit proteolytic enzyme comprising activator complexes bound to the 20 S catalytic core. The functions of the proteasomal activator (PA) 700 in ubiquitin/ATP-dependent protein degradation and of the PA28 alpha/beta activators in antigen presentation are well defined. However, the function of a third PA, PA28 gamma, remains elusive. We now show that mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase kinase 3 (MEKK3), a MAPK kinase kinase (MAPKKK) involved in MAPK kinase 7 (MKK7)-c-Jun N-terminal kinase ('JNK') and MKK6-p38 signalling, can bind PA28 gamma but not PA28 alpha. In contrast, B-Raf, a MAPKKK specific for the MAPK/ERK kinase ('MEK')-ERK module, binds PA28 gamma and alpha. The PA28 gamma-binding domain of MEKK3 is located within its N-terminal regulatory domain (amino acids 1-178). Expression of MEKK3 in Cos-7 cells led to an increase in endogenous and co-expressed PA28 gamma protein levels, whereas kinase-deficient MEKK3 had no effect on PA28 gamma expression. Furthermore, in vitro assays indicated that PA28 gamma was a MEKK3 substrate. MEKK3 represents the first protein kinase capable of binding and phosphorylating a PA, and provides a potential mechanism to link stress-activated protein kinase signalling with the PA28 gamma-dependent proteasome.