CD45 recruits adapter protein DOK-1 and negatively regulates JAK–STAT signaling in hematopoietic cells
Introduction
Adequate immune cell expansion and differentiation require T cell receptor (TCR) activation, which mediates intracellular signaling events necessary for a proper immune response. One of most striking events during such a response is the inducible phosphorylation of tyrosine residues within the TCR cytoplasmic domain, thereby creating binding sites for SH2 domain containing proteins such as Syk family kinases (Syk kinase for B cells and Zap-70 kinase for T cells). This procedure is though to be mediated by Src-family kinases, p56 Lck and p59 Fyn (Kane et al., 2000, Koretzky, 2003). CD45 plays a key role in the activation and regulation of TCR-mediated signaling pathways. It has been suggested that CD45 predominantly functions as a positive regulator of TCR signaling through its ability to dephosphorylate negative regulatory sites of Src-family kinases (Alexander, 2000, Frearson and Alexander, 1997, Huntington and Tarlinton, 2004, Mustelin et al., 2004), and the activity of p56 Lck is differentially regulated by CD45 during T cell development (Falahati and Leitenberg, 2007). Previously we have observed that the immune-restricted adaptor Src kinase associated protein, SKAP55, mediates the interaction between CD45 and Fyn kinase (Wu et al., 2002a, Wu et al., 2002b), and we hypothesized that the CD45-mediated regulation of Src-family kinases might require other novel protein–protein interactions possibly through intermediate players, such as the adaptor SKAP55 (Wu et al., 2002a, Wu et al., 2002b).
While CD45 plays a role as a positive effector in TCR-mediated signaling, recent studies have shown that CD45 can negatively regulate T cell and macrophage function by dephosphorylating Janus-kinases (JAK)s, leading to the inactivation of the JAK/STAT pathway (Fujii et al., 2003, Irie-Sasaki et al., 2003, Penninger et al., 2001, Wu et al., 2002a, Wu et al., 2002b). JAK/STAT signaling has been shown to be activated in CD45 deficient mice (Irie-Sasaki et al., 2001). Moreover, JAKs, such as JAK-1 and JAK-2, and downstream substrates, such as STAT3 and STAT5, are hyperphosphorylated upon stimulation with IL-3 and INF-α in CD45−/− mice when compared to CD45+/+ mice. However, the underlying mechanism of CD45 regulation of the JAK/STAT signaling pathway is largely unknown.
Intracellular adaptor proteins play a pivotal role in linking one or more signaling components upon cell activation, capable of integrating multiple signaling pathways used to coordinate signals within and between cells (Jordan et al., 2003, Koretzky and Myung, 2001, Rudd, 1998, Wilkinson et al., 2004). For instance, a hyper-phosphorylated protein Downstream of Kinase 1 (DOK-1), with a molecular weight of 62 kDa, interacts with Abelson tyrosine kinase (ABL) in hematopoietic cells (Carpino et al., 1997, Yamanashi and Baltimore, 1997). Despite DOK-1 being ubiquitously expressed, the highest amount of DOK-1 expression is found in T cell and macrophages (Songyang et al., 2001, Tamir et al., 2000, Yamakawa et al., 2002, Yamanashi et al., 2000). DOK-1 contains several features consistent with its function in signal transduction. It has been well characterized that the DOK-1 gene contains a PH domain at the N-terminus for lipid membrane targeting, a PTB domain in the central region for binding of tyrosine-phosphorylated proteins, and a sequence at the C-terminus that is readily tyrosine phosphorylated. Functional data arising from DOK-1 overexpression and DOK-1 gene knockout mice show that DOK-1 is a negative regulator of the mitogen-activated protein kinase (MAPK) pathway in B cells and other hematopoietic cells (Ott et al., 2002, Tamir et al., 2000, Yamanashi et al., 2000, Zhao et al., 2006), although it has a positive effect in insulin signaling and tumor cell migration (Noguchi et al., 1999). Nonetheless, DOK-1 has consistently been shown to be negatively involved in TCR/BCR signaling (Tamir et al., 2000, Yamanashi et al., 2000, Yasuda et al., 2007). Furthermore, DOK-1 is tyrosine phosphorylated by Lck kinase (Nemorin and Duplay, 2000, Nemorin et al., 2001) in response to anti-CD2 and CD3 stimulation. It is thought that once DOK-1 is phosphorylated, it binds to a Ras GTPase-activating protein (Ras-GAP), thereby increasing intrinsic Ras hydrolysis activity and hence converting Ras-GTP to Ras-GDP.
In order to further investigate the negative regulatory function of CD45 in the JAK/STAT pathway, we employed a CD45 trapping mutant as bait in a yeast two-hybrid system to identify novel interacting partners of CD45. We report here that DOK-1 associates with CD45 in a tyrosine phosphorylation dependent manner. DOK-1 is dephosphorylated by CD45 in the presence of wild-type CD45. Moreover, site-directed mutagenesis led to the identification of tyrosine residue (Y296) in Dok-1 as a pivotal site for CD45/Dok-1 interaction. Upon anti-CD3/TCR stimulation, DOK-1 translocates from the cytoplasm to the plasma membrane, an event that requires CD45 expression. Stable expression of DOK-1 in K562 and Jurkat cells dramatically decreases phosphorylation of JAK and STAT3/5 upon IL-3 and IFN-α stimulation.
Section snippets
Antibody reagents and cell lines
Anti-CD3 and anti-CD45 were purchased from BD Pharmingen and rat anti-hemagglutinin (HA) monoclonal antibody (clone 3F10) was bought from Boehringer Mannheim (Mannheim, Germany). Anti-Myc (9E10) monoclonal and anti-DOK-1 as well as SLP-76 and ERK1/2 monoclonal antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Anti-phosphotyrosine antibody (4G10) was obtained from Upstate Biotechnology (New York, USA). Phospho-JAK-1 and JAK-2 were purchased from QCB. Anti-phospho-MAPK antibody
CD45 interacts with DOK-1 in vivo
In order to identify novel CD45-associated proteins involved in the TCR-signaling pathway, the cytoplasmic domain of the substrate-trapping mutant CD45-D819V was utilized as bait in a yeast two-hybrid screen against a cDNA library derived from Jurkat cells (Wu et al., 2002a). Positive clones were identified by DNA sequencing and two independent clones containing the full length encoding for DOK-1 protein, known as BCR-ABL oncogenic interacting protein, were isolated. Interaction between
Discussion
To date extensive studies have demonstrated that the hematopoietic-specific tyrosine phosphatase CD45 plays a critical role in positively regulating TCR-mediated activation. The manner in which CD45 negatively regulates the JAK/STAT pathway, however, is less well documented. Based on our results, we report an internal mechanism by which CD45 functions in the JAK/STAT pathway through a regulatory adaptor, DOK-1, that integrates the crosstalk among T-cell receptor (TCR)-mediated multiple
Acknowledgments
We thank Zhen Li for useful discussion and Denis L’Abbe for technical assistance. This work was supported in part by the Natural Science and Engineering Research Council of Canada Grant GP0183691 and the Canadian Institute of Health Research Grant MOP82807.
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