Degradation of GAP-43 via the ubiquitin-proteasome system

K. DE MOLINER; M. WOLFSON; N. PERRONE-BIZZOZERO; E. SOTO; A. ADAMO

Newport Beach, California, USA

Congreso; Thirty-Fourth Annual Meeting of American Society for Neurochemistry.; 2003

American Society for Neurochemistry

GAP43 is a neuronal specific phosphoprotein that is present in growth cones ofdeveloping neurons and presynaptic terminals of certain mature neurons.GAP43 is thought to contribute to the significant morphological changes thatthese structures undergo during neural development and plasticity. It is of greatinterest to define the mechanisms by which neurons degrade proteins involvedin synaptic remodeling such as GAP43. In this study, we examined whetherthe degradation of GAP43 was mediated by the ubiquitin (Ub)-proteasomesystem. To this end, NIH3T3 cells were transfected with pcDNA3 constructsor pEGFP vectors, either empty or containing GAP43 sequences in the senseorientation. Selected clones expressing GAP43 were treated for 18 h with theproteasome inhibitors MG132 (25,50,75 lM) and Lactacystin (5,10,15 lM).We found that both inhibitors were equally effective at increasing GAP43protein levels, suggesting the involvement of the 26S proteasome in thisprotein degradation. To determine whether GAP43 degradation was dependenton ubiquitination, subsequent studies examined the presence ofUb-GAP43 complexes in transfected cells. Cell lysates were immunoprecipitatedwith specific antibodies to GAP43 followed by Western blots usinganti-Ub antibodies. The presence of several immunoreactive bands ofincreasing sizes was consistent with the formation of GAP43 complexes withvarious amounts of Ub. Last, we set up an in vitro protein degradation assayincubating a membrane fraction enriched in GAP43 at 37C in the presence ofATP.We found that MG132 (100 lM), Lactacystin (20 lM) and the Ub mutantK48R (100 lM) inhibited GAP43 degradation. Based upon these findings, weconclude that GAP43 is a substrate of the Ub-proteasome system.