Role of SH2/SH3 adapter Nck in regulation of actin cytoskeleton and cell motility

UnrestrictedNck family, including Nckalpha and Nckbeta, is involved in signaling pathways that regulate the actin cytoskeleton rearrangement. Although Nckalpha; and Nckbeta were initially considered functionally redundant, the specific role for each Nck has been revealed. To study the functions of Nckalpha and Nckbetain the regulation of actin cytoskeleton, we studied two cellular events involving the actin cytoskeleton rearrangements: neuritogenesis in neuronal cells and cell migration in fibroblasts.; First, we investigated the role of Nckalpha versus Nckbeta in the process of mammalian neuritogenesis. We found that knockdown of Nckbeta, but not of Nckalpha, completely blocked nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells and dramatically disrupted the axon and dendrite tree in primary rat cortical neurons. After screening the proteins that are reportedly in a complex with Nck, we found that the steady-state level of paxillin was significantly reduced in Nckbeta-knockdown neurons. Interestingly, Nckbeta knockdown did not affect the paxillin level in glial cells and several other cell types. Genetic silencing of paxillin blocked neuritogenesis, just as Nckbeta knockdown did. Reintroducing a non-degradable Nckbeta into Nckbeta-knockdown PC12 cells rescued paxillin from down-regulation and resumed neuritogenesis. Forced expression of paxillin in Nckbeta-knockdown PC12 also rescued neuritogenesis. Finally, Nckbeta, but not Nckalpha, binds strongly to paxillin and treatment of the neurons with proteosome inhibitors prevented paxillin from degradation. Thus, Nckbeta controls neuritogenesis by maintaining cellular paxillin stability.; Second, we have investigated the role of Nckalpha and Nckbeta in the PDGF-BB stimulated human dermal fibroblast (HDF) migration, a rate-limiting event during skin wound healing. Knockdown of either Nckalpha or Nckbeta completely blocked the PDGF-BBstimulated HDF migration. Consistently, mouse dermal fibroblasts isolated from either Nckalpha-/- or Nckbeta-/- mice did not migrate in response to PDGF-BB. Overexpression of middle SH3 domain of either Nckalpha or Nckbeta completely blocked the PDGF-BB induced HDF migration, suggesting that motility signaling from PDGF goes through the middle SH3 domain of both Ncks. Finally, downregulation of Nckalpha inhibited the Cdc42-induced filopodia formation, whereas downregualtion of Nckbeta inhibited the RhoA-induced stress fiber formation. Thus, Nckalpha and Nckbeta play non-compensating roles in the PDGFstimulated HDF migration and mediate different signaling pathways