Decoding the signaling profile of hematopoietic progenitor kinase 1 (HPK1) in innate immunity: A proteomic approach

Signaling via beta(2) integrins (CD11/CD18) as well as TCRs and BCRs involves similar pathways. However, the activation of the same signaling molecule can result in opposing effects. One such example is the hematopoietic progenitor kinase 1 (HPK1), which negatively regulates T and B cell activation but enforces neutrophil adhesion via beta(2) integrins. This difference may be defined by specific HPK1 interacting networks in different leukocyte subsets which have already been described in the adaptive immune system. Here, we set out to identify interacting proteins of HPK1 in neutrophil-like differentiated HL-60 cells exposed to immobilized fibrinogen and left nonactivated or Mn2+-activated to allow beta(2) integrin-dependent adhesion. Co-IP experiments followed by mass spectrometry led to the identification of 115 HPK1-interacting proteins. A total of 58 proteins were found only in nonactivated cells and 39 proteins only in Mn2+-activated adherent cells. From these results, we decoded a pre-existing signaling cluster of HPK1 in nonactivated cells encompassing proteins essential for beta(2) integrin-mediated signaling during neutrophil trafficking, namely DNAX-activation protein 12 (DAP12), spleen tyrosine kinase (Syk), and Rac1. Thus, our study provides novel insights into the complex architecture of the signaling processes during neutrophil activation and the complex signaling profile of HPK1 in leukocytes.