Nckbeta adapter controls neuritogenesis by maintaining the cellular paxillin level.
The SH2/SH3 adapter Nck has an evolutionarily conserved role in neurons, linking the cell surface signals to actin cytoskeleton-mediated responses. The mechanism, however, remains poorly understood. We have investigated the role of Nck/Nckalpha/Nck1 versus Grb4/Nckbeta/Nck2 side-by-side in the process of mammalian neuritogenesis. Here we show that permanent genetic silencing of Nckbeta, but not Nckalpha, completely blocked nerve growth factor-induced neurite outgrowth in PC12 cells and dramatically disrupted the axon and dendrite tree in primary rat cortical neurons. By screening for changes among the components reportedly present in complex with Nck, we found that the steady-state level of paxillin was significantly reduced in Nckbeta knockdown, but not Nckalpha knockdown, neurons. Interestingly, Nckbeta knockdown did not affect the paxillin level in glial cells and several other cell types of various tissue origins. Genetic silencing of paxillin blocked neuritogenesis, just like Nckbeta knockdown. Reintroducing a nondegradable Nckbeta into Nckbeta short interfering RNA-expressing PC12 cells rescued paxillin from down-regulation and allowed the resumption of neuritogenesis. Forced expression of paxillin in Nckbeta knockdown PC12 also rescued its capacity for neuritogenesis. Finally, Nckbeta, but not Nckalpha, binds strongly to paxillin and treatment of the neurons with proteosome inhibitors prevented paxillin down-regulation in Nckbeta knockdown neurons. Thus, Nckbeta maintains paxillin stability during neuritogenesis.
Guan
Shengxi
S
Department of Dermatology and USC/Norris Cancer Center, University of Southern California Keck School of Medicine, 1303 North Mission Road, Los Angeles, CA 90033, USA.
Chen
Mei
M
Woodley
David
D
Li
Wei
W
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D020868:Gene Silencing; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D020932:Nerve Growth Factor / Q000378:metabolism; D016501:Neurites / Q000502:physiology* / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D051419:Paxillin / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; C078332:Nck protein; D015513:Oncogene Proteins; D051419:Paxillin; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D020932:Nerve Growth Factor