New Genes

Kdelr1   Traf3   Jak3

Nek7 is an essential component of the NLRP3 inflammasome

IL-1β and IL-18 are inflammatory cytokines that are synthesized and released after pathogen invasion. Cytokines have several functions associated with infection, inflammation, and autoimmune processes by activating the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. IL-1β and IL-18 production is regulated by inflammasomes, which are multiprotein complexes that assemble upon detection of pathogenic or other danger signals in the cytoplasm. The composition of the inflammasome is dependent of the type of pathogen that initiates inflammasome assembly. For example, NLRP3 inflammasome formation is initiated by pathogens, DNA, single-stranded (ss) RNA, double-stranded (ds) RNA, bacterial toxins, environmental irritants, and endogenous danger signals (1, 2, 3). Formation of the NRLP3 inflammasome is initiated by aggregation of the NLRP3 protein and subsequent recruitment of the adaptor protein ASC and the cysteine protease pro-caspase-1. One of the goals of the Beutler laboratory is to identify components and factors associated with inflammasome function. The Cuties mouse strain was identified in the course of this study. The Cuties mice exhibit reduced IL-18 and IL-1β secretion, impaired NLRP3-dependent immune cell recruitment to sites of infection, and diminished association of NLRP3 with other proteins of the NLRP3 inflammasome after stimulation with nigericin and lipolysaccharide. DNA sequence analysis of the Cuties mice identified the causative mutation in the Nek7 gene. Nek7 encodes NEK7 (NIMA (never in mitosis gene a)-related expressed kinase 7), a member of the NEK family of serine/threonine kinases. NEK7 has known functions in several aspects of mitosis, the process of cell division (4, 5); the function of NEK7 in inflammasome function was unknown. Characterization of the Cuties mice determined that NEK7 binds NLRP3 to form a complex upon inflammasome stimulation, and that NEK7 functions as a switch between mitosis and NLRP3 inflammasome activation (6).

Figure 1. NLRP3 inflammasome pathway. NLRP3 is present in the cytosol in an inactive conformation state. The NLRP3 inflammasome is activated by a wide variety of agents. Extracellular ATP mediates inflammasome activation, triggering rapid K+ efflux from the cell (decreased potassium (K+) concentration may favor inflammasome assembly). Rupture of lysosomes containing certain particulates releases Cathepsin B into the cytoplasm, activating the inflammasome. NLRP3 activators may also trigger inflammasome assembly by generating reactive oxygen species (ROS), often produced under situations of cellular stress. Oligomerization of NLRP3 and the recruitment of caspase-1, ASC, and CARDINAL leads to the autoproteolytic maturation of caspase-1. Caspase-1 is capable of cleaving pro-1L-1β, pro-IL-18, and pro-IL-33. These mature cytokines mediate a variety of biological effects by activating key processes such as the nuclear factor κB and mitogen-activated protein kinase (MAPK) pathways. NEK7 was uncovered to be a component of the inflammasome.


  1. Quarmby LM & Mahjoub MR (2005) Caught nek-ing: Cilia and centrioles. J Cell Sci 118(Pt 22): 5161-5169.
  2. Mahjoub MR, Trapp ML & Quarmby LM (2005) NIMA-related kinases defective in murine models of polycystic kidney diseases localize to primary cilia and centrosomes. J Am Soc Nephrol 16(12): 3485-3489.
  3. Fry AM (2002) The Nek2 protein kinase: A novel regulator of centrosome structure. Oncogene 21(40): 6184-6194.
  4. Roig J, Mikhailov A, Belham C & Avruch J (2002) Nercc1, a mammalian NIMA-family kinase, binds the ran GTPase and regulates mitotic progression. Genes Dev 16(13): 1640-1658.
  5. Yissachar N, Salem H, Tennenbaum T & Motro B (2006) Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression. FEBS Lett 580(27): 6489-6495.
  6. Yin MJ, Shao L, Voehringer D, Smeal T & Jallal B (2003) The serine/threonine kinase Nek6 is required for cell cycle progression through mitosis. J Biol Chem 278(52): 52454-52460.
  7. Shi H, et al (2015) NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component. Nature Immunology 17(3): 250-258.
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Kdelr1 is essential for control of T-cell development, T-cell survival, and viral infection

Newly synthesized proteins enter the endoplasmic reticulum (ER) and move to the Golgi, where they can undergo post-translational modification prior to being distributed to their final location in the cell. In the ER, proteins are folded into their proper conformation with the aid of ER-resident proteins known as chaperones. Sorting of proteins in the ER is dependent on a Lys-Asp-Glu-Leu (KDEL) sequence at the C-terminus of the protein. The proper folding of proteins in the ER can be disrupted by various stresses such as ischemia, oxidative stress, and genetic mutations. These stresses can lead to the accumulation of misfolded proteins in the ER, which triggers the unfolded protein response (UPR). The UPR reduces the amount of misfolded proteins in the ER by stopping protein translation, degrading the misfolded proteins, and stimulating the production of additional chaperones. During protein translocation from the ER to the Golgi, chaperones often accompany their substrates. Retrieval of chaperones back to the ER is essential to prevent protein accumulation in the ER. KDELR1, a KDEL receptor, mediates the return of chaperones and other proteins from the Golgi to the ER by binding the KDEL sequence in the target protein. Once bound by KDELR1, the proteins are packaged into coat protein complex vesicles for retrograde transport to the ER. KDELRs also function in trafficking of proteins through the Golgi and regulate ER quality control (1). Mutations in Kdelr1 have been linked to the development of dilated cardiomyopathy in transgenic mice (2). The mutant mice exhibited accumulation of misfolded proteins in the ER due to impaired quality control in the ER. We recently identified a new strain of mice called daniel_gray that exhibited compromised antiviral immunity, reduced expression of the T cell receptor, and low numbers of lymphocytes in the blood (3). All of the phenotypes were linked to a mutation in the Kdelr1 gene. Mutation in Kdelr1 is likely to affect the retrieval of ER-resident proteins back to the ER, trafficking from the Golgi, and the regulation of ER stress. The reduced numbers of T cells were proposed to be due a sensitization of these cells to ER stress and subsequent apoptosis.

Figure 2. KDELR functions in vesicle trafficking. Newly synthesized proteins enter the ER and move on to the Golgi, where they are modified prior to being distributed through the secretory pathway to their final location. These newly synthesized proteins are folded into their proper conformation with the aid of ER-resident proteins known as chaperones. Chaperones often leave the ER and enter the Golgi along with their substrates via COPII vesicles. As COPII vesicles travel towards the Golgi, they are thought to become uncoated. Once inside the Golgi, the retrieval of chaperones back to the ER is dependent upon the recognition of C-terminal KDEL-like motifs by KDEL receptors, and subsequent transportation in COPI vesicles. During COPI vesicle formation, coat proteins are recruited from the cytosol to the Golgi membrane. Retrograde transport of KDELR is also dependent on the motor protein, kinesin-2, and may be modulated by Src activity as overexpression of activated Src relocated KDELR1 from the Golgi to the ER. Under the more neutral conditions in the ER, the chaperones are released from the receptor.


  1. Yamamoto K, et al (2001) The KDEL receptor mediates a retrieval mechanism that contributes to quality control at the endoplasmic reticulum. Embo j 20(12): 3082-3091.
  2. Hamada H, et al (2004) Dilated cardiomyopathy caused by aberrant endoplasmic reticulum quality control in mutant KDEL receptor transgenic mice. Mol Cell Biol 24(18): 8007-8017.
  3. Siggs OM, et al (2015) Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control viral infection. Proc Natl Acad Sci ,USA

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Traf3, an adaptor protein essential for T-dependent and T-independent antigen responses

Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) is an adaptor protein that directly binds the cytoplasmic regions of several immune response receptors, including, lymphotoxin-β receptor, CD40, TACI (transmembrane activator and CAML interactor), BCMA (B-cell maturation antigen), LMP1 (latent membrane protein 1), BAFFR (B-cell-activating factor receptor), RANK (receptor activator of NF-κB), HVEM (herpesvirus entry mediator), EDAR (ectodysplasin A receptor), XEDAR (X-linked ectodermal dysplasia receptor), CD137, and OX40 (1-4). TRAF3 also indirectly interacts with other receptors including TNFR1 and IL-1R as well as TLR3, TLR4, TLR7, and TLR9 by associating with adaptor proteins [(5); reviewed in (6)]. After receptor activation, TRAF3 promotes signaling by communicating the receptor signal to downstream signaling proteins. Downstream of the lymphotoxin-β receptor, BAFFR, and RANK receptors, TRAF3 is part of the non-canonical NF-κB (NF-κB2) signaling pathway (7, 8). The NF-κB2 signaling pathway regulates secondary lymphoid organogenesis, and thymic epithelial cell development as well as B cell development, maintenance, and antibody production by initiating the transcription of NF-κB2 target genes. Mutations in human TRAF3 are often observed in B cell malignancies including multiple myeloma, Waldenström's macroglobulinemia, non-Hodgkin lymphoma, splenic marginal zone lymphoma, B-cell chronic lymphocytic leukemia, and mantle cell lymphoma [(9-11); reviewed in (12)]. Changes in TRAF3 expression in these malignancies are linked to elevated NF-κB2 activity, which leads to enhanced B cell survival and an increased propensity for malignant transformation [reviewed in (13)]. We identified two mutations in Traf3 that caused immune deficiencies: hulk and banasplit. Both mouse strains exhibited deficient T-dependent and T-independent antibody responses to antigens as well as a reduced frequency of peripheral blood B cells. The phenotype was attributed to a loss in the ability of TRAF3 to mediate T helper cell function in antigen-specific IgG responses to T cell-dependent antigens (14, 15).

Figure 3. TRAF3 is an adaptor in immune signaling. TLR3,7, and 9 are localized in the endosome. Once TLR complexes recognize their ligands, they recruit combinations of adaptor proteins (MyD88, TICAM, TRAM, TIRAP) via homophilic TIR domain interactions.  In pDCs, activation of TLR7 and 9 in endosomes recruits MyD88 and IRAK4, which then interact with TRAF6, TRAF3, IRAK1, IKKα, and IRF7. IRAK-1 and IKKα phosphorylate and activate IRF7, leading to transcription of interferon-inducible genes and production of large amounts of type I IFN. In the TICAM-dependent pathway stimulated by TLR3 or 4 activation, TICAM recruits polyubiquitinated RIP1, which interacts with the TRAF6/TAK1 complex and leads to NF-κB activation and proinflammatory cytokine induction. TICAM signaling also leads to type I IFN production through phosphorylation and activation of IRF3 by a complex containing TRAF3, TBK1 and IKKe. In the NF-κB pathway, a subset of TNFR superfamily members such as CD40 can activate the canonical or non-canonical NF-κB signaling pathways. In the canonical pathway, membrane receptors isignal through kinases and TRAFs, subsequently resulting in IKK activation. This activation occurs after the K63 ubiquitination of TRAFs. TAK1 and its adaptor proteins TAB1 and TAB2 bind ubiquitin chains to TRAF and NEMO (IKKγ) resulting in the activation of the IKK complex (NEMO, IKKα and IKKβ). The IKK complex phosphorylates p105, resulting in IκB and p105 ubiquitination and degradation. Degradation of IκB releases activated NF-κB dimers for translocation to the nucleus. In the non-canonical pathway, the receptors bind to TRAFs to regulate NIK activity. TRAF3 and TRAF2 are recruited to the receptor along with cIAP1/2. TRAF2 undergoes K63 self-ubiquitination and is responsible for the K63 ubiquitination of cIAP1/2. TRAF3 is degraded by K48 ubiquitination, enhanced by the K63 ubiquitination of TRAF2 and cIAP1/2. As TRAF levels decrease, NIK is released and phosphorylates IKKα which phosphorylates p100. Phosphorylation and ubiquitination of p100 leads to the 26S proteasomal degradation of p100 and the processing of p52. P52 and RelB are released for translocation to the nucleus.


  1. Sato T, Irie S & Reed JC (1995) A novel member of the TRAF family of putative signal transducing proteins binds to the cytosolic domain of CD40. FEBS Lett 358(2): 113-118.
  2. Xie P, Stunz LL, Larison KD, Yang B & Bishop GA (2007) Tumor necrosis factor receptor-associated factor 3 is a critical regulator of B cell homeostasis in secondary lymphoid organs. Immunity 27(2): 253-267.
  3. Ansieau S, et al (1996) Tumor necrosis factor receptor-associated factor (TRAF)-1, TRAF-2, and TRAF-3 interact in vivo with the CD30 cytoplasmic domain; TRAF-2 mediates CD30-induced nuclear factor kappa B activation. Proc Natl Acad Sci U S A 93(24): 14053-14058.
  4. Mosialos G, et al (1995) The epstein-barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 80(3): 389-399.
  5. Oganesyan G, et al (2006) Critical role of TRAF3 in the toll-like receptor-dependent and -independent antiviral response. Nature 439(7073): 208-211.
  6. Hacker H, Tseng PH & Karin M (2011) Expanding TRAF function: TRAF3 as a tri-faced immune regulator. Nat Rev Immunol 11(7): 457-468.
  7. He JQ, et al (2006) Rescue of TRAF3-null mice by p100 NF-kappa B deficiency. J Exp Med 203(11): 2413-2418.
  8. Hauer J, et al (2005) TNF receptor (TNFR)-associated factor (TRAF) 3 serves as an inhibitor of TRAF2/5-mediated activation of the noncanonical NF-kappaB pathway by TRAF-binding TNFRs. Proc Natl Acad Sci U S A 102(8): 2874-2879.
  9. Otto C, et al (2012) Genetic lesions of the TRAF3 and MAP3K14 genes in classical hodgkin lymphoma. Br J Haematol 157(6): 702-708.
  10. Braggio E, et al (2009) Identification of copy number abnormalities and inactivating mutations in two negative regulators of nuclear factor-kappaB signaling pathways in waldenstrom's macroglobulinemia. Cancer Res 69(8): 3579-3588.
  11. Keats JJ, et al (2007) Promiscuous mutations activate the noncanonical NF-kappaB pathway in multiple myeloma. Cancer Cell 12(2): 131-144.
  12. Yi Z, Lin WW, Stunz LL & Bishop GA (2014) Roles for TNF-receptor associated factor 3 (TRAF3) in lymphocyte functions. Cytokine Growth Factor Rev 25(2): 147-156.
  13. Hildebrand JM, et al (2011) Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions. Immunol Rev 244(1): 55-74.
  14. Zapata JM, et al (2009) Lymphocyte-specific TRAF3 transgenic mice have enhanced humoral responses and develop plasmacytosis, autoimmunity, inflammation, and cancer. Blood 113(19): 4595-4603.
  15. Xie P, Kraus ZJ, Stunz LL, Liu Y & Bishop GA (2011) TNF receptor-associated factor 3 is required for T cell-mediated immunity and TCR/CD28 signaling. J Immunol 186(1): 143-155.

Jak3 is essential for cytokine signaling that mediates immune cell survival and function

The JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway is activated upon binding of cytokines to their receptors on the immune cell surface. Cytokine binding to cytokine receptors stimulates the activation of the JAK tyrosine kinases, which are associated with the cytoplasmic tails of the receptors. The JAK proteins phosphorylate residues on the receptor, creating a ligand for the STAT proteins. After binding to the receptor and phosphorylation by the JAK proteins, the STAT proteins are activated, promoting their movement from the cytoplasm of the cell to the nucleus, where they promote transcription (1). In the course of our immunological screening pipeline, three independent mouse strains, thistle, citron, and mount_tai, were identified that exhibited various aberrant immunological phenotypes, including reduced numbers of T cells and natural killer cells as well as increased numbers of neutrophils and macrophages. The mice also exhibited increased susceptibility to mouse cytomegalovirus (MCMV) virus. The thistle mouse exhibited a diminished T-dependent antibody response to ovalbumin administered with aluminum hydroxide. All three strains were linked to mutations in Jak3. JAK3 is expressed in natural killer (NK) cells, T cells, B cells, and intestinal epithelial cells, and is downstream of several cytokine receptors including those for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The functions of these cytokines is known: IL-2 functions in peripheral T cell homeostasis and antigen-driven T-cell expansion; IL-4 functions in B-cell maturation and isotype switching; IL-7 is necessary for T and B cell development; IL-9 serves as a growth factor for T cells and mast cells as well as a regulator of hematopoiesis; IL-15 functions in NK cell differentiation; and IL-21 effects on several immune cell types, including the regulation of humoral immune responses, B cell apoptosis, and T cell differentiation [reviewed in (2)]. Mutations in JAK3 are linked to autosomal recessive T- and NK-cell negative/B-cell positive type of severe combined immunodeficiency, which is characterized by a loss of T and NK cells [TB+NK- SCID; (3-6)]. Patients with SCID have persistent, recurring infections due to loss of T cell-associated immunity. Gain-of-function mutations in JAK3 have also been linked to adult T cell leukemia/lymphoma, early T cell precursor acute lymphoblastic leukemia (TdT+:ALL), T-cell prolymphocytic leukemia (T-PLL), acute megakaryoblastic leukemia (AMKL), cutaneous T cell lymphoma, and extranodal nasal-type natural killer cell lymphoma (7-11).

Figure 4. Jak3 regulates cell apoptosis and proliferation. Cytokine receptors are associated with the normally dephosphorylated and inactive JAK tyrosine kinases. Latent STAT1 exists in the cytoplasm as a monomer. Upon receptor stimulation, JAK proteins phosphorylate the receptor cytoplasmic domains. STAT proteins are recruited to the receptor, tyrosine phosphorylated by JAKs, and dimerize for translocation to the nucleus with the assistance of importin-α5 (associated with importin-β). Once STAT1 binds to its DNA target, importin-α5 is recycled to the cytoplasm by the cellular apoptosis susceptibility protein (CAS) export receptor. Suppressors of cytokine signaling (SOCS) proteins can directly bind and suppress JAKs or can compete with STATs for receptor binding. The tyrosine phosphatases SHP1 and SHP2 inhibit signaling by dephosphorylating STAT proteins.


  1. Schindler C, Shuai K, Prezioso VR & Darnell JE,Jr. (1992) Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science 257(5071): 809-813.
  2. Wu W & Sun XH (2012) Janus kinase 3: The controller and the controlled. Acta Biochim Biophys Sin (Shanghai) 44(3): 187-196.
  3. Notarangelo LD, et al (2001) Mutations in severe combined immune deficiency (SCID) due to JAK3 deficiency. Hum Mutat 18(4): 255-263.
  4. Candotti F, et al (1997) Structural and functional basis for JAK3-deficient severe combined immunodeficiency. Blood 90(10): 3996-4003.
  5. Macchi P, et al (1995) Mutations of jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature 377(6544): 65-68.
  6. Russell SM, et al (1995) Mutation of Jak3 in a patient with SCID: Essential role of Jak3 in lymphoid development. Science 270(5237): 797-800.
  7. Walters DK, et al (2006) Activating alleles of JAK3 in acute megakaryoblastic leukemia. Cancer Cell 10(1): 65-75.
  8. Elliott NE, et al (2011) FERM domain mutations induce gain of function in JAK3 in adult T-cell leukemia/lymphoma. Blood 118(14): 3911-3921.
  9. Bellanger D, et al (2014) Recurrent JAK1 and JAK3 somatic mutations in T-cell prolymphocytic leukemia. Leukemia 28(2): 417-419.
  10. Bergmann AK, et al (2014) Recurrent mutation of JAK3 in T-cell prolymphocytic leukemia. Genes Chromosomes Cancer 53(4): 309-316.
  11. Bouchekioua A, et al (2014) JAK3 deregulation by activating mutations confers invasive growth advantage in extranodal nasal-type natural killer cell lymphoma. Leukemia 28(2): 338-348.

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