Caspase-mediated Cleavage of Hematopoietic Progenitor Kinase 1 (HPK1) Converts an Activator of NFκB into an Inhibitor of NFκB

Ruediger Arnold,Jen Liou,Hannes C.A Drexler,Arthur Weiss,Friedemann Kiefer

doi:10.1074/jbc.m008343200

Abstract

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related protein kinase, is a potent stimulator of the stress-activated protein kinases (SAPKs/JNKs). Here we report activation of NFkappaB transcription factors by HPK1 that was independent of SAPK/JNK activation. Overexpression of a dominant-negative SEK1 significantly inhibited SAPK/JNK activation, whereas NFkappaB stimulation by HPK1 remained unaffected. Furthermore, activation of NFkappaB required the presence of full-length, kinase-active HPK1, whereas the isolated kinase domain of HPK1 was sufficient for activation of SAPK/JNK. We also demonstrate that overexpression of a dominant-negative IKKbeta blocks HPK1-mediated NFkappaB activation suggesting that HPK1 acts upstream of the IkappaB kinase complex. In apoptotic myeloid progenitor cells HPK1 was cleaved at a DDVD motif resulting in the release of the kinase domain and a C-terminal part. Although expression of the isolated HPK1 kinase domain led to SAPK/JNK activation, the C-terminal part inhibited NFkappaB activation. This dominant-negative effect was not only restricted to HPK1-mediated but also to NIK- and tumor necrosis factor alpha-mediated NFkappaB activation, suggesting an impairment of the IkappaB kinase complex. Thus HPK1 activates both the SAPK/JNK and NFkappaB pathway in hematopoietic cells but is converted into an inhibitor of NFkappaB activation in apoptotic cells.

Highlights

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Sterile 20 (Ste20)-related protein kinase, is a potent stimulator of the stress-activated protein kinases (SAPKs/JNKs)
Inflammatory cytokines like tumor necrosis factor ␣ (TNF␣)1 and interleukin 1 trigger intracellular pathways resulting in the activation of the stress-activated protein kinases SAPKs/JNKs and p38s as well as of NF␬B family transcription factors
The mutations did not impair protein stability, as they did not decrease HPK1-associated kinase activity or the capacity of HPK1 to activate SAPK/JNK (Fig. 3D). These results demonstrate a strong dependence of NF␬B activation on the proline-rich SH3binding sites in HPK1, whereas those sites were dispensable for SAPK/JNK activation

Summary

The abbreviations used are

TNF␣, tumor necrosis factor ␣; HPK1, hematopoietic progenitor kinase 1; SAPK, stress-activated protein kinases; GCK, germinal center kinase; PAK, p21-activated kinase; CNH, citron homology domain; JNK, c-Jun N-terminal kinase; IKK, I␬B kinase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; IL, interleukin; TRAF2, TNF receptor-associated factor 2; MLK3, mixed lineage kinase 3; NIK, NF␬B-inducing kinase; GST, glutathione Stransferase; CHO, aldehyde. GCK, GCKR/KHS, GLK, and HPK1, which will be referred to as subfamily I, share a C-terminally located regulatory domain, called citron homology domain (CNH) [8, 9]. Four proline-rich stretches located between the kinase domain and CNH domain of HPK1 contain a PXXP motif, the minimal sequence requirement for SH3 domain ligands Three of these have been shown to interact with small adaptor proteins including Grb2 [22], Nck [22], HS1 [19], and Crk [23, 24], providing a possible link to activated transmembrane receptors. In apoptotic cells HPK1 was cleaved by a caspase 3-like activity, resulting in the generation of a dominant-negative C-terminal fragment that inhibited NF␬B stimulation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION