A Proteomic Approach to Screen and Identify Spleen Tyrosine Kinase (Syk)-Binding Partners and Substrates

Syk is a 72 kDa non-receptor tyrosine kinase found in hematopoietic cells, and is essential for B cell receptor (BCR) function. Signals sent from the BCR can result in clonal expansion, anergy, apoptosis, and differentiation, which are mediated by Syk coupling the BCR to multiple downstream signaling pathways. Because Syk can modulate various biological processes in response to BCR signaling, identifying its associated proteins and direct substrates is critical to understanding its role and corresponding signaling pathways. In this work, we describe a robust proteomic strategy to screen and identify novel Syk-binding proteins and substrates. We identified several known Syk binding-proteins and substrates, such as USP25, PI3K, PLCg, BTK, BLNK, CD79a, PHB1, and TUB. For potential and unknown Syk-interacting proteins, we have identified and validated Nek9, Csk, and MST1. We further verified Nek9, a serine/threonine kinase with little known functions, as a Syk-interacting protein in pervanadate and anti-IgM stimulated DG75 B cells and demonstrated its phosphorylation on tyrosine. Inhibition of Syk with piceatannol, a Syk inhibitor, resulted in loss of Nek9 tyrosine phosphorylation, which was also observed in DT40 Syk-negative chicken B cells. Nek9 tyrosine phosphorylation by Syk was further confirmed using an in vitro kinase assay, resulting in increased Nek9 activity and phosphorylation on tyrosine 52, 520, 521, and 845. Our results suggest that Syk could be the activating kinase for Nek9 in BCR signaling, leading to the activation of other downstream kinases.