MAP4K Family Kinases in Immunity and Inflammation

CHAPTER SEVEN

MAP4K Family Kinases in Immunity and Inflammation Huai-Chia Chuang*,†,1, Xiaohong Wang{,1, Tse-Hua Tan*,†,{,2 *Immunology Research Center, National Health Research Institutes, Zhunan, Taiwan, ROC † Research and Development Center for Immunology, China Medical University, Taichung, Taiwan, ROC { Department of Pathology & Immunology, Baylor College of Medicine, Houston, Texas, USA 2 Corresponding author: e-mail address: [email protected]

Contents 1. Overview of MAP4K Family Kinases 2. MAP4K1 (HPK1) 2.1 Regulation of HPK1 Kinase Activation 2.2 Activation of the JNK Pathway by HPK1 2.3 Regulation of NF-κB Signaling by HPK1 2.4 Negative Regulation of T-Cell Receptor Signaling by HPK1 2.5 Negative Regulation of B-Cell Receptor Signaling by HPK1 2.6 Regulation of Immune Cell Adhesion by HPK1 2.7 In Vivo Roles of HPK1 in Immune Responses and Autoimmune Diseases 2.8 In Vivo Roles of HPK1 in Tumor Immunity 3. MAP4K2 (GCK) 3.1 Activation of the JNK Pathway by GCK 3.2 Regulation of GCK Protein Stability 4. MAP4K3 (GLK) 4.1 Activation of the JNK Pathway by GLK 4.2 Regulation of GLK Activation in T Cells 4.3 Regulation of T-Cell Receptor Signaling by GLK 4.4 In Vivo Roles of GLK in Immune Responses and Autoimmunity 4.5 The Involvement of GLK in Cancer 5. MAP4K4 (HGK) 5.1 Activation of the JNK Pathway by HGK 5.2 HGK Function In Vitro 5.3 HGK Negatively Regulates IL-6 in T Cells 5.4 In Vivo Roles of HGK in Type 2 Diabetes and Immunometabolism 5.5 In Vivo Roles of HGK in Cell Migration and Cancer 6. MAP4K5 (KHS, GCKR) 6.1 Activation of the JNK Pathway by KHS 6.2 Regulation of KHS Activation in TNFα Signaling 1

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These authors contributed equally.

Advances in Immunology, Volume 129 ISSN 0065-2776 http://dx.doi.org/10.1016/bs.ai.2015.09.006

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2016 Elsevier Inc. All rights reserved.

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6.3 Activation of Wnt Signaling by KHS in B Cells 6.4 In Vivo Roles of KHS 7. MAP4K6 (MINK) 7.1 Activation of the JNK Pathway by MINK 7.2 MINK Function In Vitro 7.3 In Vivo Roles of MINK in T-Cell Development 8. Perspectives Acknowledgments References

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Abstract MAP kinase kinase kinase kinases (MAP4Ks) belong to the mammalian Ste20-like family of serine/threonine kinases. MAP4Ks including MAP4K1/HPK1, MAP4K2/GCK, MAP4K3/ GLK, MAP4K4/HGK, MAP4K5/KHS, and MAP4K6/MINK have been reported to induce JNK activation through activating the MAP3K-MAP2K cascade. The physiological roles of MAP4Ks in immunity and inflammation are largely unknown until recent studies using biochemical approaches and knockout mice. Surprisingly, JNK is not the major target of MAP4Ks in immune cells; MAP4Ks regulate immune responses through novel targets. HPK1 inhibits T-cell receptor (TCR) signaling and B-cell receptor signaling via inducing phosphorylation/ubiquitination of SLP-76 and BLNK, respectively. GLK activates TCR signaling through phosphorylating/activating PKCθ. T-cell-mediated immune responses and Th17-mediated experimental autoimmune diseases are enhanced in HPK1 knockout mice but ameliorated in GLK knockout mice. Consistently, HPK1 levels are decreased in peripheral blood mononuclear cells and T cells from patients with psoriatic arthritis and systemic lupus erythematosus (SLE), respectively. Moreover, GLK levels are increased in T cells from patients with SLE, rheumatoid arthritis, or adult-onset Still's disease; the percentages of GLK-overexpression T cells are correlated with the disease activity. In addition, HGK phosphorylates and induces TRAF2 protein degradation, leading to negative regulation of IL-6 production in resting T cells. Loss of HGK in T cells results in spontaneous systemic inflammation and type 2 diabetes in mice. HGK is also involved in cancer cell migration. To date, the phenotypes of knockout mice for GCK, KHS, and MINK have not been reported; the roles of these three MAP4Ks in immune cell signaling are discussed in this review. Taken together, MAP4K family kinases play diverse roles in immune cell signaling, immune responses, and inflammation.

1. OVERVIEW OF MAP4K FAMILY KINASES Mitogen-activated protein (MAP) kinases including ERK, JNK, and p38 play essential roles in regulating cell activation, proliferation, differentiation, and apoptosis (Chen & Tan, 2000; Kyriakis & Avruch, 2012). In the classical three-tiered MAPK pathways, MAP kinase kinase kinase (MAP3K) activates a MAP2K, which in turn induces dual threonine and tyrosine

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phosphorylation and subsequent activation of a MAPK (Chen & Tan, 2000; Kyriakis & Avruch, 2012). The phosphorylation of MAPKs can be dephosphorylated and inactivated by dual-specificity phosphatases (Huang & Tan, 2012; Li et al., 2014; Yang et al., 2014). While the upstream activators of MAP3Ks are often small GTP-binding proteins, but in some pathways, MAP3K can be activated by another kinase, which therefore is named as a MAP kinase kinase kinase kinase (MAP4K). To date, the nomenclature for six MAP4Ks has been approved by HUGO Gene Nomenclature Committee. This includes HPK1 (MAP4K1) (Hu, Qiu, Wang, Meyer, & Tan, 1996; Kiefer et al., 1996), GCK (MAP4K2) (Pombo et al., 1995), GLK (MAP4K3) (Diener et al., 1997), HGK/NIK (MAP4K4) (Su, Han, Xu, Cobb, & Skolnik, 1997; Yao et al., 1999), KHS/GCKR (MAP4K5) (Shi & Kehrl, 1997; Tung & Blenis, 1997), and MINK (MAP4K6) (Dan et al., 2000; Hu et al., 2004). All of these MAP4Ks belong to the mammalian Ste20-like serine/threonine kinase family, which are homologs of the yeast sterile20 protein (Ste20p), a putative MAP4K that activates a MAP3K in the yeast pheromone signaling pathway (Wu, Whiteway, Thomas, & Leberer, 1995). To date, a total of 26 mammalian Ste20-like kinases have been identified (Chen & Tan, 1999, 2000; Dan, Watanabe, & Kusumi, 2001; Delpire, 2009). These mammalian Ste20-like kinases are divided into two subfamilies based on the domain structures: the p21activated kinases (PAKs) and the germinal center kinases (GCKs) (Strange, Denton, & Nehrke, 2006). Among the GCK subfamily, several of them can activate the MAP3K kinase cascade, leading to JNK activation. The proposed four-tiered MAP4K-induced JNK kinase cascade is shown in Fig. 1. The structures of MAP4Ks share a high degree of similarity. They are all composed of an N-terminal kinase domain, proline-rich motifs, and a C-terminal citron-homology domain (Fig. 2). Studies in recent years have highlighted important roles of MAP4Ks in the regulation of cellular signaling, immune cell activation, cell transformation, and cell migration. Here, we will review the studies of MAP4Ks in the immune system, focusing on their roles in lymphocytes. Surprisingly, the regulatory functions of MAP4Ks in immune cells are largely mediated by JNK-independent mechanisms (Table 1).

2. MAP4K1 (HPK1) As its name indicates, hematopoietic progenitor kinase 1 (HPK1) is originally cloned from hematopoietic progenitor cells (Hu et al., 1996;

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Figure 1 JNK activation by MAP4K-mediated cascade. MAP4Ks induce activation of the MAPK JNK through sequential activation of MAP3Ks and MAP2Ks.

Figure 2 The structural domains of the MAP4K family kinases. MAP4Ks are composed of an N-terminal kinase domain, proline-rich motifs, and a citron-homology domain in the C-terminal region. The identified conserved phosphorylation sites and the caspase cleavage site (DDVD) are indicated. (The numbers designate human MAP4K protein sequences).

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Table 1 Phenotypes for the Genetic Animal Models of MAP4K Family Kinases Phenotypes of Knockout MAP4Ks Alias (KO) Mice References

MAP4K1 HPK1

HPK1 KO mice show enhanced T-cell activation, increased cytokine production, and increased antibody production after KLH immunization. HPK1 KO mice are more susceptible to EAE induction. HPK1 KO T cells and B cells show enhanced cell activation and antigen receptor signaling. HPK1 KO dendritic cells show higher levels of costimulating molecules and proinflammatory cytokines.

Alzabin, Bhardwaj, Kiefer, Sawasdikosol, and Burakoff (2009); Shui et al. (2007)

MAP4K2 GCK, RAB8IP

GCK KO mice have been generated and are viable. The in vivo roles of GCK remain unclear.

Gavrilescu et al. (2012); Zhong et al. (2009)

MAP4K3 GLK, RAB8IPL1

GLK KO mice show impaired T-cell activation, reduced cytokine production, and decreased antibody production after KLH immunization. GLK KO mice are resistant to EAE induction.

Chuang et al. (2011)

Chuang et al. (2014); MAP4K4 HGK, NIK Whole-body HGK Vitorino et al. (2015); Xue knockout mice show embryonic lethality; HGK is et al. (2001) required for mesodermal and somite development. T cell-specific HGK conditional KO mice spontaneously develop systemic inflammation and type 2 diabetes. Endothelial cell-specific HGK Continued

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Table 1 Phenotypes for the Genetic Animal Models of MAP4K Family Kinases—cont'd Phenotypes of Knockout MAP4Ks Alias (KO) Mice References

conditional KO mice are embryonic lethal; the migration activity of endothelial cells are impaired by HGK knockout. MAP4K5 KHS, GCKR, KHS1

Shi, Huang, Harrison, No obvious abnormal Han, and Kehrl (2006) phenotypes of KHS KO mice have been observed; however, immune responses in KHS KO mice have not been characterized.

MAP4K6 MINK, hMINK, MINK1, MINK-1

MINK KO mice have not McCarty et al. (2005) been generated. Nonetheless, mice reconstituted with MINK siRNA-silenced bone marrow show normal development of B cells but defective negative selection of thymocytes.

HPK1, hematopoietic progenitor kinase 1; GCK, germinal center kinase; RAB8IP, rab8-interacting protein; GLK, GCK-like kinase; RAB8IPL1, RAB8-interacting protein-like 1; HGK, HPK1/GCKlike kinase; NIK, Nck-interacting kinase; KHS, kinase homologous to SPS1/STE20; GCKR, germinal center kinase related; MINK, misshapen/Nck-related kinase; hMINK, human MINK.

Kiefer et al., 1996). Northern blot analysis of tissue distribution of HPK1 shows that HPK1 is predominantly expressed in lymphoid organs/tissues including the bone marrow, fetal liver, lymph node, placenta, spleen, and thymus (Hu et al., 1996; Kiefer et al., 1996). In the hematopoietic compartment, HPK1 is expressed in all cell types, such as hematopoietic progenitor cells, T cells, B cells, macrophages, dendritic cells, neutrophils, and mast cells (Hu et al., 1996; Kiefer et al., 1996).

2.1 Regulation of HPK1 Kinase Activation Mutation of Lys-46 to Met (designated HPK1-M46) in the ATP-binding site of the kinase domain abolishes catalytic activation of HPK1 (Hu et al., 1996). Outside of the kinase domain, there are three proline-rich motifs in the middle region and a citron-homology domain in the

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C-terminal region. Proline-rich motifs in the middle of HPK1 mediate the interaction of HPK1 with many SH3 domain-containing proteins (Boomer & Tan, 2005). The citron-homology domain of HPK1 may be involved in HPK1-mediated lymphocyte adhesion because the citronhomology domain in another Ste20-like kinase TNIK binds to Rap2 and regulates actin cytoskeleton (Taira et al., 2004). Full-length HPK1 can be cleaved by caspase-3 at a caspase cleavage site (DDVD) in apoptotic cells, resulting in an enhanced catalytic activity of the N-terminal HPK1 fragment (Chen, Meyer, Ahmed, Yao, & Tan, 1999). The deficiency of the tyrosine kinases LCK or ZAP70 abolishes Tyr-379 phosphorylation and kinase activity of HPK1 in Jurkat T cells upon anti-CD3 stimulation, indicating that LCK and ZAP70 induce HPK1 Tyr-379 phosphorylation and kinase activation (Ling et al., 2001; Liou et al., 2000; Sauer et al., 2001). Protein phosphatase 4 (PP4) interacts with and dephosphorylates HPK1, leading to stabilization and activation of HPK1 (Zhou, Boomer, & Tan, 2004). Thr-355 autophosphorylation of HPK1 regulates ubiquitination and degradation of HPK1. Thr-355 is a PP4-targeted dephosphorylation site; this dephosphorylation prevents CUL7/Fbxw8-mediated ubiquitination and proteasomal degradation of activated HPK1 (Wang et al., 2009, 2014).

2.2 Activation of the JNK Pathway by HPK1 The initial characterization of the HPK1 function in JNK activation is conducted by overexpressing HPK1 in cell lines. Overexpression of HPK1 wild-type but not kinase-dead mutant (M-46) in human embryonic kidney 293 (HEK293) cells leads to catalytic activation of MEKK1 (a MAP3K), which in turn activates JNK (Chen & Tan, 2000; Hu et al., 1996). Kinase-dead mutation of MKK4, a MAP2K downstream of MEKK1 but upstream of JNK, abrogates HPK1-mediated JNK activation (Hu et al., 1996). These data suggest that HPK1 functions as a MAP4K that activates JNK through the MEKK1–MKK–JNK cascade. HPK1 overexpression in HEK293 or African green monkey kidney COS-1 cells can also activate JNK via other MAP3Ks including MLK3 and TAK1, which in turn activate two MAP2Ks, MKK4 and MKK7, leading to JNK activation (Kiefer et al., 1996; Wang, Zhou, Hu, Yao, & Tan, 1997). Direct phosphorylation of Ser281 in the activation loop of MLK3 by HPK1 in vitro further supports that HPK1 directly activates MAP3Ks (Leung & Lassam, 2001). Interestingly, HPK1 does not activate ERK or p38 in these studies (Hu et al., 1996; Kiefer et al., 1996).

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The studies using different hematopoietic cell lines or primary cells suggest that HPK1 also regulates JNK activation in hematopoietic cells. Overexpression of HPK1 activates JNK in human Jurkat T cells in response to anti-TCR or PMA stimulation (Liou et al., 2000). HPK1 overexpression also leads to increased JNK1/2 activation in human leukemia (HL)-60 cells upon anti-CD150 stimulation; furthermore, HPK1 is associated with CD150 receptor in HL-60 cells (Yurchenko et al., 2010). The HPK1 N-terminal kinase domain causes sustained JNK activation, Bad phosphorylation, and cell survival in myeloid cells independent of IL-3 stimulation (Arnold et al., 2007). Surprisingly, although HPK1 overexpression can lead to JNK activation in Jurkat T cells upon TCR stimulation (Liou et al., 2000), HPK1 knockout in mouse primary T cells does not compromise JNK activation upon anti-TCR stimulation (Shui et al., 2007). A possible explanation could be due to the dual roles of HPK1 in TCR signaling (see Section 2.4.2 for details) (Shui et al., 2007). Nevertheless, the strong evidence of activating the JNK cascade by HPK1 in different experimental systems has placed as HPK1 as a MAP4K.

2.3 Regulation of NF-κB Signaling by HPK1 In addition to activating JNK, overexpression of HPK1 with IKKα or IKKβ in HEK293T cells substantially increases the in vitro kinase activity of IKKα/β toward IκB; this effect is abolished by an HPK1 kinase-dead mutant (Hu et al., 1999). HPK1 overexpression also leads to NF-κB activation in Jurkat T and COS-1 cells (Arnold, Liou, Drexler, Weiss, & Kiefer, 2001), whereas HPK1 siRNA knockdown abrogates IKK kinase activity and NF-κB activation in Jurkat T cells upon anti-CD3 stimulation (Brenner et al., 2009; Brenner, Golks, Kiefer, Krammer, & Arnold, 2005). These studies demonstrate that HPK1 kinase activation is required for IKK-NF-κB activation. It is believed that HPK1 activates IKK-NF-κB via regulating CARMA1. HPK1 inducibly interacts with CARMA1, which is an adaptor protein facilitating IKKβ activation, in Jurkat T cells upon anti-CD3 stimulation (Brenner et al., 2009). It has been further demonstrated that HPK1 directly phosphorylates CARMA1 at Ser-551, which is required for NF-κB activation (Brenner et al., 2009). Interestingly, while full-length HPK1 activates IKK, the HPK1 C-terminal fragment that is generated by caspase-mediated cleavage partially suppresses NF-κB activation (Arnold et al., 2001; Chen et al., 1999). A possible explanation is that the HPK1 C-terminal region may compete for the CARMA1 binding with full-length HPK1, resulting in the suppression of NF-κB activation (Brenner et al., 2009).

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2.4 Negative Regulation of T-Cell Receptor Signaling by HPK1 2.4.1 Regulation of HPK1 Activation in T Cells Upon TCR stimulation, the adaptor SLP-76 forms a signalosome complex by binding to PLCγ1, GADS, VAV, NCK, ITK, ADAP, and HPK1, which transmit TCR signaling to different effector molecules (Boomer & Tan, 2005). HPK1 directly interacts with the SH2 domain of the adaptor SLP-76, which facilitates HPK1 activation during TCR signaling (Boomer & Tan, 2005; Ling et al., 2001). Like SLP-76, other adaptors CLNK, CRK, and GADS also inducibly interact with HPK1 upon TCR signaling (Boomer & Tan, 2005). The inducible interactions between HPK1 and the adaptors induce HPK1 activation and HPK1 downstream signaling in T cells (Ling et al., 1999; Liu, Smith, Arnold, Kiefer, & McGlade, 2000; Ma et al., 2001; Oehrl et al., 1998; Yu et al., 2001). In addition to TCRinducible interactions, the adaptor CRKL and GRB2 constitutively binds to HPK1 in Jurkat T cells and also increases HPK1 kinase activity (Anafi et al., 1997; Ling et al., 2001, 1999; Oehrl et al., 1998). It would be interesting to study whether these adaptors differentially bind to HPK1 in different times or locations. 2.4.2 Negative Regulation of Adaptors by HPK1 in T Cells The studies using Jurkat T cell line have reported contrasting roles of HPK1 in T-cell activation depending on the stimuli and HPK1 expression levels (Brenner et al., 2009; Ling et al., 1999; Liou et al., 2000; Ma et al., 2001). The data derived from HPK1 knockout primary T cells demonstrate that HPK1 is mainly a negative regulator of TCR signaling (Shui et al., 2007). TCR-induced phosphorylation of ZAP70, SLP-76, LAT, VAV1, PLCγ1, and ERK, as well as calcium flux, is increased in HPK1 knockout primary T cells (Shui et al., 2007). Consistent with the enhanced TCR signaling, HPK1 knockout T cells show hyperproliferation and IL-2 production upon TCR ligation in vitro (Shui et al., 2007). Interestingly, the activated HPK1 also feedback phosphorylates SLP-76 at Ser-376 residue, which mediates SLP-76/14-3-3 interaction in TCR signaling (Di Bartolo et al., 2007; Shui et al., 2007). Loss of 14-3-3 binding in the SLP-76 S376A mutant leads to increased PLCγ1 phosphorylation, calcium signaling, and ERK activation, indicating that HPK1 attenuates TCR signaling by inducing SLP-76 phosphorylation via a negative feedback mechanism (Di Bartolo et al., 2007; Shui et al., 2007). In fact, the binding of 14-3-3 to Ser-376 of SLP-76 reduces the stability of SLP-76 microclusters

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and the formation of SLP-76 signaling complexes (Lasserre et al., 2011). SLP-76 wild-type but not S376A mutant can be ubiquitinated in Jurkat T cells upon TCR stimulation (Wang, Li, Chiu, et al., 2012). Furthermore, Lys-30 is identified as a SLP-76 ubiquitination site that targets the activated SLP-76 for the proteasomal degradation. The regulation of SLP-76 ubiquitination and degradation by HPK1-mediated Ser-376 phosphorylation suggests that the disappearance of SLP-76 microclusters is due to the degradation of SLP-76. In addition to inducing phosphorylation of SLP-76, HPK1 also inducibly phosphorylates GADS at Thr-254 in Jurkat T cells upon TCR stimulation, which leads to GADS/14-3-3 interaction (Lasserre et al., 2011). GADS T254A mutant enhances TCR-induced NF-AT activation, indicating that HPK1 attenuates T-cell activation also by inducing GADS phosphorylation (Lasserre et al., 2011). Besides SLP-76 and GADS, HPK1 may also negatively feedback regulate other HPK1-interacting adaptors by phosphorylation. Although HPK1 activates the JNK pathway in different overexpression systems including HEK293T, COS-1, and Jurkat T cells (Ma et al., 2001), HPK1 knockout primary T cells show unaffected JNK activity (Shui et al., 2007). A possible explanation is that HPK1 has dual and opposite roles in the regulation of JNK activation in T cells upon TCR stimulation. HPK1mediated inhibition of SLP-76 activation would lead to the inhibition of JNK activity in TCR signaling. However, the activation of JNK by HPK1 may neutralize the suppression of JNK by HPK1-mediated SLP76 inhibition in TCR signaling. Similarly, HPK1 seems to regulate IKK activation in two different and contrasting mechanisms. HPK1 activates IKK by directly phosphorylating CARMA1, while HPK1 also negatively regulates IKK activation by inhibiting SLP-76 activation. It is likely that HPK1 facilitates JNK and IKK activation in the initial phase of TCR signaling but plays a critical role in dampening TCR signaling in the late phase.

2.5 Negative Regulation of B-Cell Receptor Signaling by HPK1 Similar to its role in T cells, HPK1 also negatively regulates BCR-induced cell activation and proliferation in B cells (Wang, Li, Kuo, et al., 2012). HPK1 knockout mouse primary B cells show increased activation of BLNK (an SLP-76-related adaptor), PLCγ2, calcium signaling, ERK, p38, JNK, and IKK upon BCR stimulation (Wang, Li, Kuo, et al., 2012). In B cells, HPK1 tyrosine phosphorylation is induced by the tyrosine kinases

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SYK and LYN and is required for its kinase activity (Liou et al., 2000; Tsuji et al., 2001). BCR signaling-induced HPK1 and BLNK interaction is mediated by the phospho-Y379 of HPK1 and the SH2 domain of BLNK (Sauer et al., 2001; Tsuji et al., 2001). BLNK shRNA knockdown blocks HPK1 kinase activation in DT40 chicken B cells upon BCR signaling, indicating that BLNK positively regulates HPK1 activation (Liou et al., 2000). Double deficiency of GRB2 and GRAP, but not single deficiency of either GRB2 or GRAP, abrogates HPK1 activation in these cells (Liou et al., 2000), suggesting a redundant role of GRB2 and GRAP in the regulation of HPK1 activation. The negative role of HPK1 in B cells is mediated by HPK1-induced Thr-152 phosphorylation of BLNK, which binds to 14-3-3 (Wang, Li, Kuo, et al., 2012). 14-3-3 binding induces BLNK ubiquitination at Lys37, Lys-38, and Lys-40, which targets the activated BLNK for proteasomal degradation, leading to attenuation of B-cell activation (Wang, Li, Kuo, et al., 2012). A recent finding of SLP-76 microclusters regulation by HPK1-induced phosphorylation and 14-3-3 binding (Lasserre et al., 2011) suggests that Thr-152 phosphorylation may also regulate BLNK microclusters formation and persistence in BCR signaling. The model of lymphocyte signaling attenuation by HPK1 is shown in Fig. 3.

2.6 Regulation of Immune Cell Adhesion by HPK1 2.6.1 Regulation of Lymphocyte Adhesion by HPK1 TCR stimulation not only activates the signaling leading to cell survival and cytokine production, but it also induces integrin activation resulting in T-cell adhesion and immunological synapse formation. The interaction between SLP-76 and degranulation-promoting adaptor protein (ADAP) is required for TCR-induced integrin activation (Wang et al., 2004). ADAP is constitutively associated with SKAP55, which targets the activated small GTPase Rap1 to the plasma membrane, leading to integrin activation (Kliche et al., 2006). Thus, the SLP-76/ADAP/SKAP55 ternary complex in turn relays the TCR signal to adhesion molecules of the integrin family, promoting T-cell adhesion (Baker et al., 2009). Because both ADAP and HPK1 bind to the same motif (the SH2 domain) of SLP-76, HPK1 competes with ADAP for SLP-76 binding, which in turn dampens the activity of ADAP downstream effector Rap1 (Patzak, Konigsberger, Suzuki, Mak, & Kiefer, 2010). HPK1-deficient T cells show increased Rap1 activity and cell adhesion to ICAM-1, which is a ligand for integrin LFA-1 on T cells (Patzak et al., 2010). The negative role of HPK1 on integrin activation and

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Figure 3 Lymphocyte signaling attenuation by HPK1/MAP4K1 in T cells and B cells. HPK1/MAP4K1 negatively regulates T-cell or B-cell activation though phosphorylating adaptors. T-cell receptor (TCR) signaling- and B-cell receptor (BCR) signaling-induced HPK1 kinase activation is mediated by the adaptors SLP-76 and BLNK, respectively. HPK1 feedback phosphorylates SLP-76 Ser-376 and BLNK Thr-152, leading to ubiquitination and proteasomal degradation of the activated SLP-76 and BLNK.

cell adhesion is also reported in B cells. WEHI-231 B cells with HPK1 shRNA knockdown show increased basal Rap1 activation and adhesion to ICAM-1 (Konigsberger et al., 2010). The negative regulation of B-cell adhesion by HPK1 is independent of PI3K or PLC activities; instead, HPK1 is associated with a SKAP55 homologue, SKAP-HOM (Konigsberger et al., 2010), which is required for B-cell adhesion (Togni et al., 2005). HPK1 may induce a negative phosphorylation site on SKAP-HOM, which in turn suppresses Rap1 activation. Altogether, these results reveal a novel function for HPK1 in suppressing T- and B-cell adhesion. 2.6.2 Regulation of Neutrophil Adhesion by HPK1 During acute inflammation, polymorphonuclear neutrophils infiltrate into lesion sites and mediate host defense (Fournier & Parkos, 2012). β2-Integrin activation in neutrophils controls neutrophil trafficking, including slow rolling, tight binding, cell spreading, and diapedesis (Fournier & Parkos, 2012). Outside-in signaling of β2-integrin induces the interaction between

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actin and HPK1-interacting protein of 55 kDa (HIP-55; also named mAbp1 and SH3P7) (Ensenat et al., 1999; Han et al., 2003; Onabajo et al., 2008; Seeley-Fallen et al., 2014); this signaling event subsequently reinforces the high-affinity conformation of β2-integrin, contributing to neutrophil adhesion (Hepper et al., 2012; Schymeinsky et al., 2009). HPK1 colocalizes with HIP-55 and actin at the lamellipodium of neutrophils upon β2integrin-mediated adhesion ( Jakob et al., 2013). Similar to the case of HIP-55 (Schymeinsky et al., 2009), HPK1 is required for CXCL1-induced high-affinity LFA-1 for binding to ICAM1 ( Jakob et al., 2013). CXCL1mediated neutrophil adhesion is abolished by either HPK1 deficiency or HIP-55 deficiency in vitro and in vivo ( Jakob et al., 2013; Schymeinsky et al., 2009). Taken together, in contrast to the role of HPK1 in lymphocyte adhesion, HPK1 positively regulates neutrophil adhesion.

2.7 In Vivo Roles of HPK1 in Immune Responses and Autoimmune Diseases Generation and characterization of HPK1 gene knockout mice reveal the physiological roles of HPK1 in the regulation of immune responses in vivo. HPK1 deficiency does not affect the development of T cells and B cells (Shui et al., 2007; Wang, Li, Kuo et al., 2012). However, HPK1-deficient T cells show dramatically increased activation of TCR proximal signaling and downstream ERK, leading to hyperproliferation of these cells in vitro upon anti-CD3 stimulation (Shui et al., 2007). Furthermore, T cells from immunized HPK1-deficient mice are hyper-responsive upon antigenic specific stimulation and produce significantly higher levels of cytokines including IL-2, IFN-γ, and IL-4. These immunized HPK1-deficienct mice also produce much higher levels of IgM and IgG isoforms, suggesting the enhanced functioning of HPK1 knockout B cells (Shui et al., 2007). These data indicate that HPK1 negatively regulates TCR signaling and adaptive immune responses. HPK1 knockout mice are more sensitive to the induction of experimental autoimmune encephalomyelitis (EAE) (Shui et al., 2007), suggesting that HPK1 negatively controls autoimmunity in mice. The evidence of HPK1 downregulation in peripheral blood mononuclear cells of psoriatic arthritis patients or T cells of systemic lupus erythematosus (SLE) patients suggests that HPK1 attenuation also contributes to the abnormal T- and B-cell activation and to autoimmunity in humans patients (Batliwalla et al., 2005; Stoeckman et al., 2006; Zhang et al., 2011). Thus, loss of HPK1-mediated regulation of T-cell activation and immune responses may be a crucial mechanism for autoimmune pathogenesis.

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2.8 In Vivo Roles of HPK1 in Tumor Immunity In addition to lymphocytes, HPK1 also acts as a negative regulator in dendritic cell maturation and activation (Alzabin et al., 2009). The bone marrow-derived dendritic cells (BMDCs) from HPK1 knockout mice display enhanced levels of co-stimulatory molecules CD80/CD86 and increased production of proinflammatory cytokines (Alzabin et al., 2009). Antigen presentation activity of dendritic cells for immunity is more efficient in HPK1 knockout mice (Alzabin et al., 2009). Moreover, the tumor eradication by HPK1 knockout BMDC-mediated CTL response is more effective than that by wild-type BMDCs (Alzabin et al., 2009). Although the molecular mechanism of HPK1-mediated negative regulation of dendritic cells is unclear, it is likely similar to the negative feedback mechanism observed in lymphocytes. Furthermore, as HPK1 plays critical roles in regulating lymphocyte receptor signaling and function, it is very likely that HPK1 can also control antitumor immunity via lymphocyte-dependent mechanisms. The restricted expression of HPK1 in hematopoietic cells and the critical roles of HPK1 in immune cells suggest that HPK1 could be an ideal drug target for enhancing antitumor immunity.

3. MAP4K2 (GCK) Germinal center kinase (GCK) is named as its expression in lymphoid follicles is restricted to germinal center B cells despite GCK being ubiquitously expressed in other tissues (Katz, Whalen, & Kehrl, 1994). GCK is also detected in macrophages where its expression level is further increased upon LPS stimulation (Himes et al., 2006). GCK kinase activity is strongly induced by the stimulation with TNF-α, different toll-like receptor ligands (lipid A/TLR2 ligand, poly(I:C)/TLR3 ligand, and LPS/TLR4 ligand), IL-1, or CD40 ligand (Zhong & Kyriakis, 2004), suggesting that GCK plays important roles in innate immune response and cell signaling. The in vivo roles of GCK have been characterized using GCK knockout mice (Zhong et al., 2009); however, the authors retracted the paper (Gavrilescu et al., 2012; Kyriakis & Avruch, 2012). Thus, the in vivo roles of GCK in immune regulation remain unclear.

3.1 Activation of the JNK Pathway by GCK GCK specifically activates the JNK but not the NF-κB, p38 or ERK signaling pathway in HEK293T/COS-1 overexpression systems (Pombo et al.,

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1995; Shi & Kehrl, 1997; Yuasa, Ohno, Kehrl, & Kyriakis, 1998; Zhong & Kyriakis, 2004). Similar results are observed in B- and T-cell lines upon the stimulation with various agonists, such as TNFα, IL-1, anti-CD40, lipid A, LPS, poly(I:C), and anti-CD3/CD28 (Pombo et al., 1995; Shi & Kehrl, 1997; Yuasa et al., 1998; Zhong & Kyriakis, 2004). GCK activates JNK by directly binding to and inducing the activation MEKK1, a MAP3K that preferentially activates the JNK pathway (Chadee, Yuasa, & Kyriakis, 2002; Yuasa et al., 1998). Interestingly, the kinase domain of GCK is not required for optimal activation of MEKK1 in vitro; instead, GCK promotes MEKK1 oligomerization, which leads to autophosphorylation and activation of MEKK1 (Chadee et al., 2002). GCK also activates another MAP3K, MLK3, which is a specific and potent activator of MAP2Ks, MKK4, and MKK7. Compared to MEKK1 activation, MLK3 activation requires GCK kinase activity (Chadee et al., 2002), suggesting that GCK directly phosphorylates and activates MLK3, which in turn induces JNK activation. In addition to directly regulating MAP3Ks, GCK also activates JNK through TNF receptor associated factor 2 (TRAF2), which mediates signal transduction from members of the TNF receptor superfamily to the JNK and NF-κB pathways (Au & Yeh, 2007). GCK can bind to TRAF2 in vitro and in transfected HEK293 cells through the C-terminal 141 amino acids of GCK and with the TRAF domain of TRAF2 (Chadee et al., 2002; Yuasa et al., 1998). The deletion of the Ring domain from TRAF2 abrogates GCK-mediated MEKK1 activation in vitro, suggesting that TRAF2 acts downstream of GCK and that the E3-ligase activity of TRAF2 is required for MEKK1 activation (Chadee et al., 2002). In agreement with this hypothesis, TRAF2 directly interacts with and activates MEKK1 in vitro and in B cells upon CD40 stimulation (Chadee et al., 2002; Gallagher et al., 2007; Matsuzawa et al., 2008). TRAF2-mediated MEKK1 activation requires an intact TRAF2 RING domain (Chadee et al., 2002), suggesting that TRAF2 activates MEKK1 by inducing MEKK1 K63-linked ubiquitination (Fig. 4). Although the inducible interaction between GCK and TRAF2 upon the stimulation with TNFα or CD40 ligand remains to be demonstrated in B cells, it is reasonable to speculate that GCK may phosphorylate TRAF2, which in turn induces TRAF2 E3-ligase activity and subsequent activation of MEKK1 and JNK (Fig. 4).

3.2 Regulation of GCK Protein Stability GCK protein level is significantly induced in Jurkat T cells upon poly(I:C) or LPS stimulation (Zhong & Kyriakis, 2004). The treatment with the

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Figure 4 GCK/MAP4K2-mediated JNK activation. TNFα, IL-1, CD40 ligand, or TLR ligands induce GCK/MAP4K2 kinase activation in B cells. Upon stimulation, TRAF6 binds to GCK and further stabilizes GCK proteins through occupying the binding site for another E3 ligase, which mediates K48-linked ubiquitination and subsequent proteasomal degradation of GCK. The activated GCK induces the activation of the MLK3–MKK7–JNK cascade and the MEKK1–MKK4–JNK cascade. GCK interacts with the E3 ligase TRAF2, which in turn induces MEKK1 activation. The interaction between GCK and TRAF2 may lead to the phosphorylation and activation of TRAF2.

proteasomal inhibitor lactacystin increases GCK protein levels in both resting and stimulated cells (Zhong & Kyriakis, 2004), suggesting that GCK is constitutively ubiquitinated and targeted for proteasomal degradation. It has been further demonstrated that GCK is ubiquitinated via K48 linkage but not K63 linkage (Zhong & Kyriakis, 2004). TRAF6 mediates signaling from the TNF receptor superfamily (e.g., TNFR, CD40) as well as the Toll/IL-1 receptor family (e.g., TLRs and IL-1R), leading to the activation of NF-κB and JNK signaling. TRAF6 interacts with GCK in Jurkat T cells upon the stimulation by poly(I:C) (Zhong & Kyriakis, 2004). This interaction is mediated by the TRAF domain of TRAF6 and the C-terminus of GCK (Zhong & Kyriakis, 2004). TRAF6 knockdown suppresses protein levels of GCK in resting and agonist-stimulated Jurkat T cells (Zhong & Kyriakis, 2004). Although a lower GCK K48 ubiquitination has also been observed in TRAF6 knockdown cells (Zhong & Kyriakis, 2004), it could be due to the relatively low GCK protein levels in these cells. Interestingly, the stabilization of GCK protein levels by TRAF6 does not require RING

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domain (E3-ligase activity) of TRAF6; TRAF domain-deleted TRAF6 mutant is unable to stabilize GCK protein levels (Zhong & Kyriakis, 2004). These studies suggest that TRAF6 stabilizes GCK protein levels by direct binding instead of inducing GCK ubiquitination (Zhong & Kyriakis, 2004; Fig. 4). As a consequence of TRAF6-mediated stabilization of GCK proteins, GCK kinase activity and downstream JNK activity are increased in these stimulated cells (Zhong & Kyriakis, 2004).

4. MAP4K3 (GLK) Tissue screening has shown that GCK-like kinase (GLK) is ubiquitously expressed (Diener et al., 1997). GLK can be activated by UV irradiation and TNF-α stimulation in HEK293 cells, suggesting that GLK is involved in cell responses to stress and inflammation (Diener et al., 1997). Earlier studies show that GLK activates JNK, and recent studies indicate that GLK also activates mTOR signaling in an epithelial cell line upon sensing cellular nutrient and energy levels (Findlay, Yan, Procter, Mieulet, & Lamb, 2007) and plays a crucial role in activating NF-κB signaling in T cells upon antigenic stimulation (Chuang et al., 2011).

4.1 Activation of the JNK Pathway by GLK Like HPK1, overexpression of GLK activates the JNK, but not the ERK or p38, signaling cascade in HEK293 cells; this activity is abrogated by a dominant-negative MEKK1 (a MAP3K) mutant, indicating that GLK activates JNK via acting on MEKK1 (Diener et al., 1997).

4.2 Regulation of GLK Activation in T Cells GLK kinase activation is rapidly induced in T cells upon TCR stimulation (Chuang et al., 2011). SLP-76 deficiency or knockdown suppresses GLK kinase activity in T cells, indicating that SLP-76 is required for GLK activation, which is similar to HPK1 activation (Chuang et al., 2011). Furthermore, TCR stimulation induces GLK/SLP-76 interaction, which precedes the SLP-76/PKCθ interaction in these cells (Chuang et al., 2011). GLK directly binds to SLP-76 in a tyrosine phosphorylationdependent manner (Chuang et al., 2011). The exact binding domains between SLP-76 and GLK remain unclear. The Tyr-379 phosphorylation of HPK1 is the prerequisite for the kinase activation of HPK1 in TCR signaling (Tsuji et al., 2001). Thus, GLK is also likely to be tyrosine

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phosphorylated during TCR signaling. Identification of the critical tyrosine phosphorylation sites on GLK should reveal the important mechanism of GLK regulation in TCR signaling. In addition to tyrosine phosphorylation, phosphorylation of the activation loop may be important for GLK activation in TCR signaling. Autophosphorylation of Ser-170 in the activation loop has been previously identified in human GLK by mass spectrometry using GLK proteins isolated from transfected HEK293T cells; Ser-170 phosphorylation is important for both GLK kinase activity and GLK-mediated mTOR activation upon amino acid treatment (Yan et al., 2010). It is conceivable that Ser-170 phosphorylation of GLK may be also induced by TCR signaling. Future identification and characterization of novel GLK phosphorylation sites and GLK-interacting proteins will provide more insights into the regulation of GLK kinase activation and GLK’s functions in T cells.

4.3 Regulation of T-Cell Receptor Signaling by GLK Thr-538 phosphorylation at the activation loop of PKCθ phosphorylation is required for PKCθ catalytic activation. GLK knockout T cells show abolished phosphorylation of Thr-538 upon anti-CD3 stimulation, which leads to catalytic inactivation of PKCθ and impaired downstream IKK/ NF-κB activation in T cells (Chuang et al., 2011). Earlier studies suggest that PDK1 is a kinase responsible for the phosphorylation and activation of PKCθ (Park et al., 2009). However, PDK1 activation is unaffected in GLK knockout T-cell upon anti-CD3/CD28 costimulation, while PKCθ and IKK phosphorylation remain defective in these cells (Chuang et al., 2011). These data suggest that GLK activates PKCθ independent or downstream of PDK1. GLK is inducibly and directly associated with PKCθ in T cells upon anti-CD3 stimulation (Chuang et al., 2011). Furthermore, GLK directly phosphorylates PKCθ at Thr-538 but not Ser-695 and Ser676 residues in vitro (Chuang et al., 2011). These data clearly demonstrate that GLK is the kinase that directly phosphorylates and activates PKCθ during TCR signaling (Fig. 5).

4.4 In Vivo Roles of GLK in Immune Responses and Autoimmunity Antigen-immunized GLK knockout mice show dramatically impaired antibody productions and decreased Th1/Th2/Th17 cytokine levels in the sera. There are also reduced secondary immune responses in these mice, suggesting that GLK is important for mounting a protective immune

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Figure 5 GLK/MAP4K3-mediated T-cell activation and immune responses. Upon T-cell receptor (TCR) signaling, the adaptor SLP-76 mediates GLK kinase activation. GLK/ MAP4K3 interacts with and phosphorylates the kinase PKCθ, leading to activation of the NF-κB pathway. GLK-overexpressing T cells are associated with the disease activity in multiple autoimmune diseases. GLK is a novel biomarker/therapeutic target for autoimmune diseases. SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; AOSD, adult-onset Still's disease. Adapted/modified from Chuang et al. (2011).

memory against pathogens. The requirement of GLK in adaptive immune responses reflects the critical role of GLK in controlling PKCθ and NF-κB activation in T cells. In addition to regulating immune responses, GLK knockout mice are almost completely resistant to EAE, an animal model for multiple sclerosis in human patients. More excitingly, GLK expression levels are highly increased in the peripheral blood T cells isolated from patients of several autoimmune diseases, including SLE (Chuang et al., 2011), adult-onset Still’s disease (AOSD) (Chen et al., 2012), and rheumatoid arthritis (RA) (Chen et al., 2013). Furthermore, the GLK downstream targets PKCθ and IKKβ are highly activated in the GLK-expressing T cells from SLE patients. The percentage of GLK-expressing T cells is well correlated with diseases severity in SLE, AOSD, and RA patients, suggesting that GLK overexpression and its activated downstream signaling in T cells contribute to pathogenesis of autoimmune diseases (Fig. 5).

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4.5 The Involvement of GLK in Cancer GLK was thought to be a tumor suppressor in osteosarcoma U2OS cells and pancreatic cancers (Lam et al., 2009); however, other recent publications contradict the above conclusion. GLK induces mTOR signaling (Findlay et al., 2007) or NF-κB activation (Chuang et al., 2011), both of which contribute to tumorigenesis. Moreover, the 30 -UTR of GLK is targeted by the microRNA let-7c, which plays a negative role in cell migration in vitro. Let7c levels are significantly decreased in human non-small cell lung carcinoma (NSCLC) (Zhao et al., 2014). The inverse correlation between let-7c and GLK in NSCLC samples suggests that GLK may be involved in lung cancer (Zhao et al., 2014). Taken together, whether GLK is an oncogene or a tumor suppressor still needs to be clarified.

5. MAP4K4 (HGK) 5.1 Activation of the JNK Pathway by HGK Human MAP4K4 is named HPK1/GCK-like kinase (HGK) (Diener et al., 1997), while the mouse ortholog is named Nck-interacting kinase (NIK) (Su et al., 1997). HGK/NIK is widely expressed in different tissues (Diener et al., 1997). Early studies indicate that HGK is activated by TNFα stimulation, leading to specific activation of JNK via TAK1 (a MAP3K) and MKK4/MKK7 (MAP2Ks) in HEK293T cells (Diener et al., 1997; Su et al., 1997; Taira et al., 2004). HGK interacts with a small GTP-binding protein Rap2 but not with Rap1 or Ras (Machida et al., 2004). This interaction is mediated through the HGK C-terminal citron-homology domain (Machida et al., 2004). Rap2 enhances HGK-mediated JNK activation, suggesting that HGK is the effector of Rap2 (Machida et al., 2004).

5.2 HGK Function In Vitro The in vivo physiological roles of HGK are largely unknown due to the early lethality of HGK whole-body knockout mice (Xue et al., 2001). Studies using a wide range of cancer cell lines and tumor tissues suggest that HGK promotes cell transformation or migration in cancers (Liang et al., 2008; Meller et al., 1996; Ramnarain et al., 2006; Tang et al., 2006; Wright et al., 2003). Additional studies using cultured adipocytes, skeletal muscle cells, and pancreatic β cells suggest that HGK is also involved in regulation of adipogenesis and TNFα-induced insulin

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resistance, which is partially mediated by HGK-induced JNK activation (Aouadi et al., 2009; Bouzakri, Ribaux, & Halban, 2009; Bouzakri & Zierath, 2007; Guntur, Guilherme, Xue, Chawla, & Czech, 2010; Tesz et al., 2007). HGK plays an important role in regulating innate immune responses. HGK siRNA knockdown in macrophages in vitro inhibits production of TNFα and IL-1β but not IFNγ upon LPS stimulation (Aouadi et al., 2009). The induction of TNFα and IL-1β expression by HGK is independent of the MAPKs and NF-κB pathways, as HGK siRNA knockdown does not affect JNK, p38, ERK, or IKK activation in macrophages upon LPS stimulation (Aouadi et al., 2009). The regulation of TNFα signaling by HGK suggests that HGK may regulate TNFα-mediated functions, such as inflammatory responses, obesity, and insulin resistance. The regulation of TNFα expression by HGK is also reported in Jurkat T cells upon stimulation with superantigen-loaded antigen-presenting cells (Mack et al., 2005). PMA plus ionomycin-induced T-cell proliferation, IFNγ production, and MAPK activation are also abolished by HGK deficiency (Huang et al., 2014), suggesting that HGK may positively regulate T-cell activation. However, the above study only utilized in vitro experimental systems. The in vivo roles of HGK in T cells are not discovered until recent studies using T-cell-specific HGK conditional knockout mice (Chuang et al., 2014).

5.3 HGK Negatively Regulates IL-6 in T Cells A recent publication reported that the basal levels of serum IL-6 are drastically increased in T-cell-specific HGK conditional knockout mice, which spontaneously develop type 2 diabetes and systemic inflammation (Chuang et al., 2014). These studies demonstrate a molecular mechanism that HGK directly interacts with TRAF2 and phosphorylates TRAF2 at Ser-35, leading to lysosomal degradation of TRAF2 (Chuang et al., 2014). Although a previous publication shows that HGK interacts with TRAFs in vitro (Liu, Su, Becker, Treisman, & Skolnik, 1999), HGK only targets TRAF2 but not other TRAFs in resting T cells (Chuang et al., 2014). TRAF2 protein levels are induced during TCR stimulation (Lin et al., 2011), whereas HGK kinase activity is decreased by anti-CD3 stimulation (Chuang et al., 2014). These findings suggest that HGK plays an important role in maintaining a resting state of T cells. Thus, loss of HGK in T cells results in overexpression of basal TRAF2 levels and subsequent IL-6 production.

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5.4 In Vivo Roles of HGK in Type 2 Diabetes and Immunometabolism The proinflammatory cytokine IL-6 is involved in insulin resistance and type 2 diabetes (Bastard et al., 2002); these diseases are thought to be mainly caused by inflammatory macrophages (Donath & Shoelson, 2011). A recent publication reveals a T-cell-mediated pathogenesis of type 2 diabetes (Chuang et al., 2014), which is initiated by IL-6-overproducing T cells. In T-cell-specific HGK conditional knockout mice, IL-6-overproducing T cells (CCR6+) are attracted to adipose tissues by the chemokine CCL20, which is secreted from adipocytes (Duffaut et al., 2009). IL-6 secreted from HGK knockout T cells in turn stimulates the adipokine leptin levels (Chuang et al., 2014) and likely also stimulates CCL20 production in adipocytes (Suzuki & Mihara, 2012). Leptin subsequently promotes IL-6-overproducing HGK knockout T cells to further differentiate into Th17 cells in adipose tissue by synergistic effect of IL-6 and leptin (Chuang et al., 2014). The increased CCL20 levels may further retain IL-6+ Th17 cells in adipose tissue (Fig. 6). The circulating IL-6 and IL-17 cytokines in turn impair insulin sensitivity of insulin target cells including adipocytes, hepatocytes, and muscle cells. These findings are consistent with a genetic study using 1769 DNA samples of prediabetic Europeans (Sartorius et al., 2012); two single nucleotide polymorphisms (SNPs) of HGK gene are associated with increased plasma glucose levels or with enhanced plasma IL-6 but not with TNFα levels in prediabetic patients (Sartorius et al., 2012). However, this group also reported a paradoxical result that HGK siRNA knockdown impairs LPS-induced TNFα production in macrophage in vivo (Aouadi et al., 2009). Thus, the positive regulation of TNFα production by HGK is not supported by HGK knockout T cells (Chuang et al., 2014) and clinical samples (Sartorius et al., 2012). These conflicting results may be due to the difference of cell types or the potential off-target effect of siRNAs. This issue can be addressed using macrophage-specific HGK conditional knockout mice. In summary, the reciprocal regulation between HGK-deficient T cells and adipocytes in adipose tissue microenvironment plays an important role in the pathogenesis of type 2 diabetes (Fig. 6).

5.5 In Vivo Roles of HGK in Cell Migration and Cancer Many publications using embryos (Zohn et al., 2006), hepatoma cells (Han et al., 2010), glioma cells (Loftus et al., 2013), ovarian carcinoma cells

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Figure 6 HGK/MAP4K4 knockout T cells-mediated insulin resistance. HGK/MAP4K4 induces lysosomal degradation of TRAF2 proteins; loss of HGK in T cells results in overproduction of IL-6. IL-6-overproducing HGK knockout T cells (CCR6+) are attracted to adipose tissue by the chemokine CCL20, which can be secreted from adipocytes. In adipose tissue microenvironment, IL-6 secreted from HGK knockout T cells further enhances production of the adipokine leptin and likely also CCL20. Leptin in turn cooperates with IL-6 to promote Th17 differentiation. The cytokines IL-6 and IL-17 from T cells impair insulin signaling of adipocytes, leading to insulin resistance. Black line denotes an activating effect; blue line denotes an inhibitory effect. Adapted/modified from Chuang et al. (2014).

(Dragone et al., 2006), pancreatic ductal adenocarcinoma (Liang et al., 2008), or medulloblastoma cells (Santhana Kumar et al., 2015) demonstrate that HGK plays critical roles in cell migration in vitro. Recently, in vivo roles of HGK in cell migration have been studied using conditional knockout mice (Vitorino et al., 2015). Endothelial cell-specific HGK conditional knockout embryos are lethal due to defective endothelial cell migration and impaired vascular development (Vitorino et al., 2015). HGK activates moesin through phosphorylating its activation site Thr-558, leading to inactivation of integrin signaling and promotion of focal adhesion disassembly (Vitorino et al., 2015). Moreover, in a mouse tumor model, tumor vascular perfusion and endothelial membrane retraction are reduced by HGK deficiency (Vitorino et al., 2015). This publication indicates that HGK regulates

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both normal and pathological angiogenesis through controlling endothelial cell migration. In vivo functions of HGK in tumor metastasis remain to be demonstrated.

6. MAP4K5 (KHS, GCKR) 6.1 Activation of the JNK Pathway by KHS Kinase homologous to SPS1/STE20 (KHS) (Tung & Blenis, 1997), also called GCK-related kinase (GCKR) (Shi & Kehrl, 1997), is ubiquitously expressed in different tissues and cells including T and B cells (Shi & Kehrl, 1997). Similar to the case with GCK, the kinase activity of KHS is potently induced by TNFα in HEK293 cells as well as lymphocyte cell lines (Jurkat T cells and HS-Sultan B cells) (Shi & Kehrl, 1997; Shi, Leonardi, Kyriakis, Siebenlist, & Kehrl, 1999). CD40 signaling also induces endogenous KHS activation in primary B cells, suggesting that KHS is involved in CD40-mediated B-cell functions (Chin et al., 1999). KHS wild-type but not T178A kinase-dead mutant activates JNK, indicating that the kinase activity is required for KHS-mediated JNK activation (Shi & Kehrl, 1997). Overexpression of a catalytically inactive MAP3K mutant, MEKK1 (K432M), abolishes KHS-mediated JNK activation in TNFα signaling (Shi & Kehrl, 1997). Overexpression of a dominant-negative mutant of SEK1, a MAP2K downstream of MEKK1, also abolishes KHS-mediated JNK activation (Chin et al., 1999). These studies indicate that KHS induces the MEKK1–SEK1–JNK cascade (Chin et al., 1999; Shi & Kehrl, 1997); however, whether KHS directly interacts with and phosphorylates MEKK1 has not been reported.

6.2 Regulation of KHS Activation in TNFα Signaling As KHS is involved in TNFα and CD40 signaling, the relationships between TRAF proteins and KHS have been examined. KHS kinase activity is enhanced by coexpression with TRAF2, suggesting that TRAF2 acts upstream of KHS (Shi et al., 1999). Similar to GCK, KHS interacts with TRAF2 via its C-terminal 150 amino acids with the TRAF domain of TRAF2 (Shi et al., 1999). TRAF2 RING domain does not mediate the interaction with KHS but is essential for TRAF2-mediated KHS kinase activation, suggesting that TRAF2 acts as an E3 ligase upstream of KHS (Shi & Kehrl, 1997; Shi et al., 1999). It has been further demonstrated that TRAF2 in complexes with an E3 enzyme Ubc13 induces KHS K63-linked

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ubiquitination, which in turn activates KHS possibly by facilitating KHS oligomerization (Shi & Kehrl, 2003). TRAF-associated NF-κB activator (TANK) binds all known TRAF proteins and enhances TRAF2/KHS interaction, which in turn leads to JNK activation in TNFα signaling (Chin et al., 1999). The potential effects of other TRAF proteins on KHS activation have not been reported. TANK can synergize with TRAF2, TRAF5, and TRAF6 but not with TRAF3 in the JNK activation (Chin et al., 1999), suggesting that TRAF5 and TRAF6 but not TRAF3 may also interact with KHS and regulate KHS kinase activation. As GCK can activate MEKK1 by directly inducing MEKK1 oligomerization or by phosphorylating/activating TRAF2, a similar mechanism may also occur during KHS-mediated MEKK1 activation. The model of TRAF2-mediated KHS and JNK activation is shown in Fig. 7.

Figure 7 KHS/MAPK5-mediated JNK activation in TNFα signaling. TNFα stimulation induces kinase activation of KHS/MAP4K5 in T cells and B cells, leading to activation of the MEKK1–MKK4–JNK cascade. TRAF2 binds to KHS and induces K63-linked ubiquitination of KHS, resulting in KHS kinase activation. TRAF2 may also induce MEKK1 activation through its E3-liase activity. Because TNF-associated NF-κB activator (TANK) interacts with TRAF2 and synergize with TRAF2, TANK may further enhance MEKK1 activation in a KHS-dependent or independent manner.

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6.3 Activation of Wnt Signaling by KHS in B Cells Wnt ligands are a large family of secreted cysteine-rich glycoproteins that bind receptors of the Frizzled family to control cell proliferation, differentiation, survival, polarity, and migration. There are three major intracellular signaling pathways induced by Wnt ligands. The well-characterized canonical signaling pathway involves the regulation of β-catenin accumulation and ultimately T-cell factor/LEF-1-mediated gene transcription (Buechling & Boutros, 2011). Two less well-defined noncanonical Wnt pathways include the Wnt/Ca2+ pathway and the planar cell polarity pathway (Buechling & Boutros, 2011). The Wnt/Ca2+ pathway triggers calcium flux perhaps via the activation of heterotrimeric G proteins, whereas the planar cell polarity pathway involves the activation of small GTPases and JNK. An important role of KHS in the regulation of Wnt signaling has been demonstrated in KHS siRNA-knockdown B lines and KHS-deficient primary B cells (Shi et al., 2006). Wnt3a-conditioned media induces KHS activation in different B-cell lines (Shi et al., 2006). Knockdown of Rac (a small GTPase) in Raji B cells suppresses KHS kinase activity and JNK activation in Wnt signaling (Shi et al., 2006). Furthermore, Rac is associated with KHS, suggesting that Rac/KHS interaction induces KHS activation in Wnt signaling (Shi et al., 2006). JNK phosphorylation is impaired in KHS siRNA-knockdown B-cell lines as well as KHS-deficient primary B cells in Wnt signaling, indicating that Rac activates JNK through KHS in Wnt signaling (Shi et al., 2006). In addition to mediating Wnt-induced Rac-JNK signaling, KHS also positively regulates β-catenin stabilization in Wnt signaling (Shi et al., 2006). KHS deficiency in primary progenitor B cells increases cytosolic β-catenin upon Wnt3a stimulation (Shi et al., 2006). Glycogen synthase kinase 3β (GSK3β) negatively regulates β-catenin stability by inducing phosphorylating and subsequent ubiquitination of β-catenin. KHS directly interacts with GSK3β and phosphorylates GSK3β at Ser-9 residue (Shi et al., 2006). Phosphorylation of GSK3β Ser-9 suppresses GSK3β activation and subsequently activates β-catenin (Cross, Alessi, Cohen, Andjelkovich, & Hemmings, 1995). Hence, KHS positively regulates the β-catenin pathway (canonical Wnt signaling) by inducing the negative phosphorylation of GSK3β, which in turn stabilizes β-catenin.

6.4 In Vivo Roles of KHS KHS-deficient mice have been generated by gene targeting the exons 6–8 (Shi et al., 2006). KHS-deficient mice are viable and show no obvious

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phenotypic abnormalities. The in vivo functions of KHS in the immune system remain unclear. As KHS regulates JNK activation in TNFα signaling, KHS is possibly involved in TNFα-associated the inflammatory responses, such as autoimmune inflammation, obesity, and type 2 diabetes. CD40 signaling plays important functions in regulating germinal center and memory B-cell formation, as well as DC maturation and activation. The regulation of CD40 signaling by KHS suggests that KHS may regulate adaptive immune responses. In addition, KHS also regulates Wnt signaling, which is important for B-cell and T-cell development and differentiation (Gattinoni et al., 2009; Liang et al., 2007; Reya et al., 2000). Furthermore, KHS may also regulate tumorigenesis. KHS is activated by an oncogenic form of Ras or Bcr-Abl, leading to JNK activation in the Philadelphia chromosome-positive myelogenous leukemia cell line (Shi, Tuscano, & Kehrl, 2000). Ras-dependent KHS activation is positively regulated by Crk family adaptor proteins (Crk I, Crk II, and CrkL), which interact with KHS and synergistically with KHS to activate JNK (Oehrl et al., 1998; Shi et al., 2000).

7. MAP4K6 (MINK) 7.1 Activation of the JNK Pathway by MINK Misshapen/Nck-related kinase (MINK) shares greater than 90% amino acid identity with Nck-interacting kinase (NIK, a mouse homolog of HGK/ MAP4K4) and Traf2- and Nck-interacting kinase (TNIK). MINK is ubiquitously expressed in most tissue in mice and humans, with its greatest abundance in the brain (Dan et al., 2000; Hu et al., 2004). MINK activates JNK and p38 when overexpressed in cell lines, although the downstream target of MINK in mediating JNK activation has not been elucidated. The activation of MINK may be regulated by TRAF proteins. Msn (a homolog of MINK in drosophila) interacts with the TRAF domain of drosophila TRAF1 (DTRAF1) but not DTRAF2 (Liu et al., 1999). Overexpression of the TRAF1 TRAF domain alone leads to the activation of Msn (Liu et al., 1999). DTRAF1 lacks the RING domain and E3 ligase activity, which suggests that DTRAF1-mediated Msn activation does not require ubiquitination of Msn.

7.2 MINK Function In Vitro MINK has been shown to regulate cytoskeleton organization and cell–cell adhesion, which is possibly mediated by its interaction with NCK (Dan

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et al., 2000; Hu et al., 2004), an adaptor protein involved in integrin signaling for cell adhesion and migration. MINK also regulates neuronal structure and function by binding to and activating Rap2 (Hussain, Hsin, Huganir, & Sheng, 2010).

7.3 In Vivo Roles of MINK in T-Cell Development The roles of MINK in the immune system have been characterized using Rag2-deficient mice reconstituted with MINK-deficient hematopoietic stem cells, which were generated by lentivirus delivery of MINK siRNA (McCarty et al., 2005). MINK-deficient mice show normal peripheral B-cell development but increased CD4 single-positive thymocytes (McCarty et al., 2005). It has been further demonstrated that negative selection of autoreactive thymocytes is impaired in MINK-deficient mice. Increased JNK but not ERK phosphorylation is observed in MINKdeficient thymocytes upon anti-TCR stimulation. Furthermore, proapoptotic BimEL protein is increased in MINK-deficient thymocytes upon TCR stimulation in vitro and in vivo (McCarty et al., 2005). These data suggest that MINK positively regulates negative selection of thymocytes by inducing JNK-mediated apoptosis (McCarty et al., 2005). In addition, drosophila MINK1 phosphorylates Prickle and positively regulates the β-catenin-independent Wnt signaling pathway (Daulat et al., 2012), which is required for T-cell development (Staal, Luis, & Tiemessen, 2008). These studies suggest that MINK may also positively regulate thymocyte development through Wnt signaling.

8. PERSPECTIVES MAP4Ks control many signaling pathways, which could regulate different aspects of immune cell functions. The roles of HPK1 and GLK in lymphocyte activation and adaptive immune responses have been well characterized. It is interesting that both HPK1 and GLK are activated by TCR signaling and interact with SLP-76 (Fig. 8); however, they play contrasting roles in modulating T-cell activation, immune responses, and autoimmunity. In contrast to HPK1 and GLK, HGK kinase activity is downregulated by TCR signaling. Loss of HGK in resting T cells can induce IL-6 production and subsequent Th17 cell differentiation. It is conceivable that other MAP4Ks (GCK, KHS, and MINK) are also involved in T-cell activation and immune regulation. Further characterization of MAP4Ks’ functions

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Figure 8 Regulation of T-cell activation by MAP4Ks. Both HPK1 and GLK bind to the adaptor SLP-76 but play opposite roles in T-cell receptor (TCR) signaling. SLP-76 mediates kinase activation of GLK. The activated GLK interacts with and activates PKCθ, leading to T-cell activation. HPK1 phosphorylates Ser-376 of SLP-76 and induces proteasomal degradation of the activated SLP-76, leading to attenuation of TCR signaling. HGK maintains a resting state of T cells through phosphorylating TRAF2 and inducing its lysosomal degradation. Other MAP4Ks (GCK, KHS, and MINK) may be also involved in T-cell activation. Black line denotes an activating effect; blue line denotes an inhibitory effect.

in different types of immune cells should provide significant insight into the regulation of the immune system. Both GCK and KHS regulate JNK activation in TNFα signaling. It is unclear whether GCK and KHS are differentially regulated and involved in TNFα-mediated JNK activation in different conditions. It is also unclear whether these two kinases are redundant in TNFα signaling. There is a close relationship between MAP4Ks and TRAF proteins. TRAF6 stabilizes GCK protein levels independent of the TRAF6 E3-ligase activity, whereas TRAF2 functions downstream of KHS to induce MEKK1 ubiquitination and activation. In contrast, TRAF2 acts in the upstream of KHS by inducing K63 ubiquitination and activation of KHS. We propose that there is a

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reciprocal regulation between GCK (or KHS) and TRAF proteins and that the mechanism for either GCK or KHS may be applicable to each other. In addition, HGK also interacts with and phosphorylates TRAF2, leading to TRAF2 protein degradation in lysosomes. Moreover, drosophila TRAF1 interacts with drosophila MINK, resulting in MINK activation. The interactions of TRAF proteins with other two MAP4Ks (HPK1 and GLK) have not been studied. Nevertheless, the close relationship between MAP4Ks and TRAF proteins strongly suggests that MAP4Ks play important roles in regulating the signaling pathways mediated by TRAF proteins or TNF receptor superfamily proteins. It is also likely that TRAF proteins may feedback regulate the function or expression of MAP4Ks by the E3-ligase activity of TRAF proteins. The importance of several MAP4Ks in human diseases has been revealed. HPK1 downregulation or GLK overexpression in T cells may cause autoimmune diseases. HGK downregulation in T cells could cause insulin resistance and type 2 diabetes. Moreover, HPK1, GLK, and HGK are also involved in tumorigenesis. These findings suggest that MAP4Ks are novel biomarkers and/or drug targets for treating these diseases. Studying the functions of MAP4Ks in different types of cells may help further understanding the pathogenesis of human diseases.

ACKNOWLEDGMENTS This work was supported by grants from the National Health Research Institutes, Taiwan (98A1-IMPP01-014, to T.-H.T.). T.-H.T is a Taiwan Bio-Development Foundation (TBF) Chair in Biotechnology.

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