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 NFB transcription factors by HPK1 that was independent of SAPK/JNK activation. Overexpression of a dominant-negative SEK1 significantly inhibited SAPK/JNK activation, whereas NFB stimulation by HPK1 remained unaffected. Furthermore, activation of NFB required the presence of fulllength, 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 IKK␤ blocks HPK1-mediated NFB activation suggesting that HPK1 acts upstream of the IB 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 NFB activation. This dominant-negative effect was not only restricted to HPK1-mediated but also to NIKand tumor necrosis factor ␣-mediated NFB activation, suggesting an impairment of the IB kinase complex. Thus HPK1 activates both the SAPK/JNK and NFB pathway in hematopoietic cells but is converted into an inhibitor of NFB activation in apoptotic cells.

Recently, the identification of Clnk, a third member of the SLP-76 family of adaptors expressed exclusively in cytokine-stimulated hemopoietic cells, has been reported by us and by others. Like SLP-76 and Blnk, Clnk was shown to act as a positive regulator of immunoreceptor signaling. Interestingly, however, it did not detectably associate with known binding partners of SLP-76, including Vav, Nck, and GADS. In contrast, it became complexed in activated T cells and myeloid cells with an as yet unknown tyrosine-phosphorylated polypeptide of ϳ92 kDa (p92). In order to understand better the function of Clnk, we sought to identify the Clnk-associated p92. Using a yeast two-hybrid screen and cotransfection experiments with Cos-1 cells, evidence was adduced that p92 is HPK-1, a serine/threonine-specific protein kinase expressed in hemopoietic cells. Further studies showed that Clnk and HPK-1 were also associated in hemopoietic cells and that their interaction was augmented by immunoreceptor stimulation. A much weaker association was detected between HPK-1 and SLP-76. Transient transfections in Jurkat T cells revealed that Clnk and HPK-1 cooperated to increase immunoreceptor-mediated activation of the interleukin 2 (IL-2) promoter. Moreover, the ability of Clnk to stimulate IL-2 promoter activity could be blocked by expression of a kinase-defective version of HPK-1. Lastly we found that in spite of the differential ability of Clnk and SLP-76 to bind cellular proteins, Clnk was apt at rescuing immunoreceptor signaling in a Jurkat T-cell variant lacking SLP-76. Taken together, these results show that Clnk physically and functionally interacts with HPK-1 in hemopoietic cells. Moreover, they suggest that Clnk is capable of functionally substituting for SLP-76 in immunoreceptor signaling, albeit by using a distinct set of intracellular effectors.The activation of immune cells via antigen receptors or receptors for the Fc portion of immunoglobulins (so-called immunoreceptors) is a critical element of the normal immune response (37,42). Previous studies have shown that immunoreceptor signaling is initiated by ligand-induced tyrosine phosphorylation of a short sequence present in these receptors, named the immunoreceptor tyrosine-based activation motif. This motif functions by orchestrating the recruitment and activation of members of the Src, Syk/Zap-70, and Btk families of cytoplasmic protein tyrosine kinases (PTKs) (4, 33). These various PTKs mediate the tyrosine phosphorylation of several cellular polypeptides in response to immunoreceptor stimulation, including adaptors, such as LAT and SLP-76-related molecules, and enzymatic effectors, such as phospholipase C gamma (PLC-␥) and the exchange factor Vav (5,36,41). In turn, these events trigger intracellular calcium fluxes, the Rasmitogen-activated protein kinase (MAPK) cascade, lipid metabolism, and cytoskeletal reorganization, thereby leading to activation of such transcription factors as NFAT and AP-1. Ultimately, immunoreceptor signaling culminates in the induction of eff...

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