Interaction of Hematopoietic Progenitor Kinase 1 with Adapter Proteins Crk and CrkL Leads to Synergistic Activation of c-Jun N-Terminal Kinase

Abstract

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is an upstream activator of c-Jun N-terminal kinase (JNK). In order to further characterize the HPK1-mediated JNK signaling cascade, we searched for HPK1-interacting proteins that could regulate HPK1. We found that HPK1 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 activated HPK1 but also synergized with HPK1 in the activation of JNK. The HPK1 mutant (HPK1-PR), which encodes the proline-rich region alone, blocked JNK activation by Crk and CrkL. Dominant-negative mutants of HPK1 downstream effectors, including MEKK1, TAK1, and SEK1, also inhibited Crk-induced JNK 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 HPK1-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 interaction of HPK1 with Crk and CrkL, reveal the downstream pathways of Crk- and CrkL-induced JNK activation, and highlight a potential role of HPK1 in T-cell activation.

Caspase-mediated cleavage and functional changes of hematopoietic progenitor kinase 1 (HPK1)

“…JNK cascade (Hu et al, 1996;Wang et al, 1997;Zhou et al, 1999). HPK1 may mediate extracellular signals through interaction with Src-homology 3 (SH3) domaincontaining adaptor molecules (Ana® et al, 1997;Oehrl et al, 1998;Ling et al, 1999). Here we report that HPK1 was cleaved by caspase activity during apoptosis.…”

“…We and others found that HPK1 physically interacts with adaptor molecules including Crk, CrkL, and Grb2 via these proline-rich motifs (Ana® et al, 1997;Oehrl et al, 1998;Ling et al, 1999). Co-expression of Crk with CrkL enhances HPK1 activity through mechanisms yet unknown (Ling et al, 1999). It is possible that binding of adaptors to the proline-rich motifs of HPK1 also relieves the self-inhibitory e ect.…”

“…One interesting structural feature of HPK1 is its four proline-rich motifs (putative SH3 domain-binding sites) in its regulatory region (Hu et al, 1996). We and others found that HPK1 physically interacts with adaptor molecules including Crk, CrkL, and Grb2 via these proline-rich motifs (Ana® et al, 1997;Oehrl et al, 1998;Ling et al, 1999). Co-expression of Crk with CrkL enhances HPK1 activity through mechanisms yet unknown (Ling et al, 1999).…”

“…It has been shown that interaction between HPK1 and adaptor molecules may play a role in recruiting HPK1 to the surface receptor complexes (Ana® et al, 1997;Ling et al, 1999). The cleavage of HPK1 greatly reduced its ability to associate with Grb2 and Crk and, very likely, may change HPK1 localization and functions.…”

“…We and others found that HPK1 physically interacts with adaptor molecules including Crk, CrkL, and Grb2 via proline-rich motifs in the C-terminal regulatory region (Ana® et al, 1997;Oehrl et al, 1998;Ling et al, 1999). Cleavage of HPK1 by caspases separates the Nterminus of HPK1 (containing kinase domain and the ®rst proline-rich motif) from the C-terminus (containing the remaining three proline-rich motifs (Figure 2a).…”