Signal Transduction In B Lymphocytes: Identification Of

Niaid Extramural Activities

Investigators

Linked publications & trials

Abstract

This project focuses on two serine threonine protein kinases, GCK and GCKR and a tyrosine kinase PYK2. GCK and GCKR belong to a subfamily of protein kinases, which also includes GLK, HPK1, and NIK. They are characterized by an N-terminal kinase domain related to the yeast STE20 protein kinase, a large C-terminal regulatory domain, and the ability to activate the Jun kinase (JNK) pathway. This pathway is activated by many cellular stresses, the inflammatory cytokine tumor necrosis factor (TNF) and CD40 ligand (CD40L). CD40L is present on activated T lymphocytes and interacts with its receptor CD40, [unless there is some other meaning to !?counter-receptor!? I would just stick with !?receptor!?] which is expressed by many of the important cells in the immune system, including dendritic cells, monocytes, and B-lymphocytes. Previously, we showed that GCK and GCKR are major intermediaries in CD40 and TNF-mediated JNK activation, and that signaling through these receptors leads to the formation of a trimolecular complex between GCKR, TRAF2, and TANK [if TRAF2 and TANK are acronyms, they should be defined here]. CD40 and TNF??-induced activation of germinal center kinases and the subsequent activation of stress activated protein kinases requires TRAF2. We have found that GCKR activates the JNK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. TNF?? signaling leads to TRAF2 polyubiquitination, TRAF2 oligomerization, and the recruitment, ubiquitination, and activation of GCKR, all sensitive to disruption of Ubc13 function.[unreadable] GCKR is also an intermediary in Wnt signaling pathways. Wnts are secreted cysteine-rich glycoproteins that regulate many cellular functions including proliferation, differentiation, survival, cell polarity, and migration. Wnts have emerged as key regulators of hematopoietic stem cells and early hematopoietic differentiation. Wnt proteins bind Frizzled (Fzd) receptors. Implicating Fzd receptors in B cell function, Fzd9-/- mice have defective B cell development, splenomegaly, thymic atrophy, and an accumulation of plasma cells in lymph nodes. Wnts activate a well characterized canonical signaling pathway, which lead to the stabilization of b-catenin, as well as others referred to as non-canonical pathways. One of these non-canonical pathways involves the activation of small GTPases and JNK. We have shown that normal mouse progenitor B cells, a mouse pre-B cell line, and 2 human B cell lines of germinal center origin all respond to Wnt3a by activating JNK and the canonical signaling pathway. The exposure of B cells to Wnt3a activates Rac, which depended upon two other proteins, Asef and APC. Wnt-mediated Rac activation led to GCKR and subsequent JNK activation. Surprisingly, we also provide evidence that GCKR regulates the canonical Wnt signaling pathway. The canonical pathway is notably disturbed in many cancers and mutations of various components of the pathway can be frequently found. In addition, dramatically increased expression of components of the Wnt/b-catenin pathway is found in many hematological malignancies. We have shown that Wnt signaling induces an interaction between GCKR and GSK3#], a key kinase in the canonical pathway. Furthermore, Wnt signaling results in decreased levels of b-catenin in Gckr-/- progenitor B cells compared to normal progenitor B cells. Consistent with this data silencing, GCKR expression in cell lines reduces Wnt3a induced accumulation of cytosolic b-catenin and the activity of a reporter gene that is sensitive to b-catenin levels. [unreadable] To better understand the role of GCK and GCKR in vivo, the murine Gck and Gckr genes have been isolated. Both Gck-/- and Gckr -/- mice have been created and backcrossed on to a C57Bl/6 background. The mutations did not affect mouse development as the Gck and Gckr deficient mice are born with a normal Mendelian frequency. Evaluation of the immune tissues from the Gckr-/- mice revealed evidence of abnormal immune reactivity. These mice possess an expanded population of germinal center B cells and have modest splenomegaly. There is an expansion of the number of marginal zone B cells and a reduction in the number of transitional B cells. In the bone marrow there is a mild reduction in early B cell progenitors. B cells isolated from the spleen of Gckr -/- mice are hyper-responsive to several proliferative signals including engagement of CD40 and anti-IgM. As indicated above progenitor B cells from these mice also aberrantly activate signaling pathways stimulated by Wnt ligands. Surprisingly, B cells from the Gck-/- mice exhibit a significantly reduced proliferative response to anti-IgM stimulation and to anti-IgM combined with IL-4 or with CD40. CD40 and LPS [define] induced a proliferative response similar to that observed with wild type mice. In contrast to the B cells from Gckr-/- mice, the Gck -/- B cells activate JNK normally following CD40 stimulation. Implicating GCK in chemokine receptor signaling, B cells from the Gck -/- mice migrate better at low concentrations of chemokine than do B cells from wild type mice (the Gckr -/- B cells responded similar to wild type cells). Gck -/- Gckr -/- mice have been identified in crosses from interbreeding of double heterozygotic mice. Complicating the breeding scheme, double KO males are sterile and the females have infrequent and small litters when crossed with wild type males. To better understand the defects in these mice, we isolated embryonic fibroblast cells and developed cell lines from Gck and Gckr knock-out mice as well as from the double knock-outs. In contrast to our expectations, the cell lines exhibited relatively normal JNK activation following exposure to TNF??. Analysis of the immune systems of the double KO mice is in progress. Similar to the Gckr -/- mice we have noted a marked expansion in the number of marginal zone B cells. They are also defective in CD40 induced JNK activation. The proliferative responses of the lymphoid cells to immune stimulants have been variable. In some instances we have noted dramatic increases in both B and T cell proliferation with the double knock-out cells. In other experiments we have noted modest reductions. We are attempting to determine what accounts for the marked variability in responses.