HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway.

Abstract

In mammalian cells, a specific stress‐activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. We have developed and applied a PCR‐based subtraction strategy to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. We show that one such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen‐activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3‐containing mixed lineage kinase MLK‐3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type‐specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation.

Sustained JNK signaling by proteolytically processed HPK1 mediates IL-3 independent survival during monocytic differentiation

“…Sustained JNK activation by constitutive kinase activity of HPK1-N. To better understand the role of endogenous HPK1 and HPK1-N as upstream activators of the JNK pathway, 7,8 we precipitated both HPK1 proteins from IL-3 stimulated or non-stimulated cells and determined their activity by an in vitro kinase assay. As expected, the kinase activity of endogenous full-length HPK1 was found to be IL-3 dependent (Figure 4d, top panel).…”

“…HPK1 and JNK kinase assays were performed as described before. 7 Briefly, 10 7 cell were lysed in 1 ml of buffer A (50 mM Tris/HCl, pH ¼ 8.0, 120 mM NaCl, 1% (v/v) NP-40, 200 mM Na 3 VO 4 , 5 mM DTT, 25 mM NaF, 1 mM PMSF) and antibodies directed against JNK1/2 (Santa Cruz and Abcam, 10 ml each) or HPK1 (rabbit sera no. 5/6, reacting with native full-length HPK1, 20 ml) or HPK1-N (rabbit sera no.9/10, reacting with the native HPK1 N-terminus, 20 ml) were used to immunoprecipitate kinase active proteins.…”

“…7 raised against HPK1 C-terminus) have been described previously. 7,8 The antibodies used were: anti-caspase-3 (R&D Systems), anti-JNK (Santa Cruz and Abcam), anti-phospho-JNK (Cell Signaling Technology), anti-Bad (Santa Cruz), anti-phospho-Bad (Cell Signaling Technology).…”

“…6 Ectopic expression renders full-length HPK1 active in epithelial cells resulting in selective activation of the JNK and the NFkB pathways. 7,8 Upon caspase-3 cleavage the N-terminal kinase domain of HPK1 (HPK1-N) is separated from the C-terminus (HPK1-C) resulting in suppression of NFkB. 8 In non-stimulated lymphocytes, HPK1 kinase activity is hardly detectable, but antigen receptor crosslinking leads to profound HPK1 activation.…”

From receptors to stress-activated MAP kinases

“…In addition, genetic evidence from yeast and¯ies suggests that a group of kinases which appear to function upstream of MAPKKKs may exist in certain MAPK modules (Widmann et al, 1999;Kyriakis, 1999), despite the lack of direct biochemical evidence that these kinases activate MAPKKK by phosphorylation. Thus, at least 14 di erent but structurally related kinases in this group are suggested to function as mammalian MAPKKKKs, including PAK1, 2, 3 and 4 (Manser et al, 1994;Knaus et al, 1995;Abo, 1998), GCK (Katz et al, 1994), GCKR/KHS (Shi and Kehrl, 1997;Tung and Blenis, 1997), GLK , HPK1 (Kiefer et al, 1996;Hu et al, 1996), NIK (Nck-interacting kinase)/HGK (Su et al 1997;Yao et al 1999), SOK1 (previously called UK-1; Pombo et al, 1996), Krs-1 (Taylor et al, 1996), MST1/Krs-2(Creasy and Cherno , 1995; Taylor et al, 1996), MST3 (Schinkmann and Blenis, 1997) and LOK (Kuramochi et al, 1997). Most of the MAPKKKKs, but not SOK1, Krs-1, MST3 and LOK, can activate JNK by overexpression.…”