Interaction of hematopoietic progenitor kinase 1 with adapter proteins Crkand CrkL leads to synergistic activation of c-Jun N-terminal kinase

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is an upstream activator of c-Jun N-terminal kinase (JNK). In orde r to further characterize the HPK1-mediated JNK signaling cascade, re searc hed for HPK1-interacting proteins that could regulate HPK1. We found that H PK1 interacted with Crk and CrkL adaptor proteins in vitro and in vivo and that the proline-rich motifs within HPK1 were involved in the differential interaction of HPK1 with the Crk proteins and Grb2. Crk and CrkL not only a ctivated HPK1 but also synergized with HPK1 in the activation of JNK. The H PK1 mutant (HPK1-PR), which encodes the proline rich region alone, blocked JNK activation by Crk and CrkL. Dominant-negative mutants of HPK1 downstrea m effecters, including MEKK1, TAK1, and SEK1, also inhibited Crk-induced JN K activation. These results suggest that the Crk proteins serve as upstream regulators of HPK1. We further observed that the HPK1 mutant HPK1-KD(M46), which encodes the kinase domain with a point mutation at lysine-46, and HP K1-PR blocked interleukin-2 (IL-2) induction in Jurkat T cells, suggesting that HPK1 signaling plays a critical role in IL-2 induction. Interestingly, HPK1 phosphorylated CrK and CrkL, mainly on serine and threonine residues in vitro. Taken together, our findings demonstrate the functional interacti on of HPK1 with Crk and CrkL, reveal the downstream pathways of Crk- and Cr kL-induced JNK activation, and highlight a potential role of HPK1 in T-cell activation.