Citation/Abstract

SH2/SH3 adaptor proteins are essential components of the signal transduction pathways initiated by cytoplasmic tyrosine kinases. Nck is a ubiquitously expressed adaptor protein whose function has been enigmatic. We have utilized confocal microscopy and subcellular fractionation to demonstrate that Nck is both a nuclear and a cytoplasmic protein in NIH3T3 and A431 cells and that this subcellular localization is independent of growth factor stimulation. A nuclear-specific binding partner of Nck was identified as SAM68, an RNA binding protein. Nck is constitutively associated with SAM68 in both asynchronous and mitotic cells via its amino-terminal SH3 domain as shown by immunoprecipitation and GST pulldown. To further aid in the subcellular localization of Nck throughout the cell cycle, we have transfected NIH3T3 fibroblasts with full length Nck and various deletion constructs tagged with hemagglutinin A and found that the subcellular location of the 47 kDa human form of Nck is regulated in a cell cycle dependent manner. Ha-Nck was exclusively cytoplasmic in $\rm G\sb0/G\sb1$ phase cells and became predominantly nuclear when cells were blocked in S phase. Mutational analysis revealed that the third SH3 domain of Nck is responsible for this regulation. The overexpression of Nck in NIH3T3 fibroblasts delayed G$\sb1$ progression of these cells and was nontransforming, indicating a role for Nck in cell proliferation, particularly in G$\sb1$ phase progression. Sequestering Nck to a particular subcellular location may regulate the interaction it has with substrates at different stages of the cell cycle.