Synergistic Regulation of Immunoreceptor Signaling by SLP-76-Related Adaptor Clnk and Serine/Threonine Protein Kinase HPK-1

Abstract

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...

Proteomic Analysis of the SH2Domain-containing Leukocyte Protein of 76 kDa (SLP76) Interactome

“…IP3 triggers the efflux of Ca 2ϩ from endothelial reticulum, leading to the influx of extracellular Ca 2ϩ , whereas DAG activates Protein Kinase C. By phosphorylating PLC␥-1, the Bruton's Tyrosine Kinase of the Tec family BTK plays a critical role in the activation of this phospholipase. SLP76 also associates with Clnk, a cytosolic adapter of the SLP76 family which promotes TCR-dependent activation of T cells (36,37) and FcRI-dependent responses of mast cells (38). Clnk deficiency, however, impaired neither TCR signaling in T cells nor FcRI signaling in mast cells (39).…”

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

“…9,10 HPK1 activation involves binding of cell-specific adaptor proteins, relocation to the plasma membrane, autophosphorylation and transphosphorylation by protein kinase D1. [11][12][13] While a role for HPK1 in the regulation of T-cell apoptosis was already suggested, 14 our recent studies have shown proteolytic processing of HPK1 into HPK1-C in non-apoptotic preactivated primary T cells. 15 In this study, the cleavage product HPK1-C sensitizes towards T-cell receptor-mediated cell death, while full-length HPK1 enables activation and survival of T cells.…”

HPK1 competes with ADAP for SLP‐76 binding and via Rap1 negatively affects T‐cell adhesion

“…Following recruitment to the TCR, HPK1 is phosphorylated on tyrosine 379 and binds the SH2 domain of SLP-76, which itself is phosphorylated by HPK1 [15][16][17][18][19]. Subsequent transphosphorylation by PKD1 and autophosphorylation within the kinase domain result in full activation of HPK1 [15], which then regulates different cellular responses including apoptosis, activationinduced cell death and autoimmunity [20][21][22].…”