Nck Function in Tyrosine Kinase Signaling to the Actin Cytoskeleton

Abstract

Tyrosine kinase signaling leads to the post-translational modification of proteins and their binding partners. These modifications lead to the membrane recruitment of signaling proteins, promoting an increase in their local concentration, which results in a cellular response to the phosphorylation of tyrosine residues. Nck, an SH2/SH3 adaptor protein, functions in tyrosine kinase signaling by linking tyrosine phosphorylation on the membrane with binding partners, such as N-WASp, that function in facilitating actin nucleation and polymerization. Recent in vitro studies have demonstrated that N-WASp dimers bind to and activate N-WASp far more efficiently than individual N-WASp molecules. Because Nck has been shown to induce actin comet tail formation in an N-WASp dependent manner, we manipulate this molecular interaction to test the effect of N-WASp dimerization on actin polymerization. To manipulate the pathway, we vary the concentration of Nck within antibody-induced aggregates on the cellular surface and then use precise quantitative analysis of Nck density and induced actin polymerization to analyze the system. Using the Virtual Cell, we have built a comprehensive, quantitative actin cycle model. With this model we have tested 2 mathematical expressions for dimerized N-WASp activation of Arp2/3, which produce different experimentally testable predictions. These results demonstrate a nonlinear relationship between the density of Nck aggregates and actin polymerization and also demonstrate that a quadratic-based formula describing dimerized N-WASp activation of Arp2/3 most accurately predicts experimental observations. The combination of biochemical modeling and precise experimental manipulation and quantification provides unique insights into the relationship between the density of increased local concentrations of Nck and resulting localized actin polymerization. (Supported by NIH grants P41 RR013186 and U54 RR022232)