The implications of allosteric sites on SH3-mediated signaling

A remarkable number of cellular processes are mediated by kinases. Many of these signaling pathways involve sarcoma homology 3 (SH3) domain interactions. Due to the high number and sequence conservation of SH3 domains in proteins, the interactions between SH3 domains and their known binding motifs (proline-rich regions), are poorly selective. The resulting promiscuity in signaling could potentially lead to dysfunction and disease. Therefore, cells have evolved several mechanisms to enhance the specificity of SH3 interactions. Herein, we focus on SH3 domain specificity achieved through supplementary (so-called “tertiary”) interaction sites. Such a tertiary SH3 interaction was first observed in the association between the HIV-1 negative factor (Nef) protein and Src family SH3 domains. In the first part of our study, we investigate the hypothesis that these tertiary interactions do not only enhance the SH3 binding specificity, but also allosterically influence the recognition of other ligands by Nef. In a second part, we build on these findings to understand how SH3 domains can activate the phosphoinositide 3-kinase (PI3K) by binding to its regulatory p85 subunit. Based on our comparative thermodynamic, structural, and functional analyses on Nef and PI3K we propose that SH3 binding to p85 involves tertiary interactions with the C-terminal region of p85 that allosterically activate the catalytic subunit of PI3K. Interestingly, this C-terminal region is also controlling p85 dimerization and thus the activation of the PI3K pathway. Collectively, our work reveals that tertiary SH3 interactions can create a functional selectivity in signaling.