Signal Transduction In B Lymphocytes: Identification Of

Niaid Extramural Activities

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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 stress- activated protein kinase (SAPK, also referred to as Jun kinase or 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 counter-receptor CD40, 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 SAPK activation, and that signaling through these receptors leads to the formation of a trimolecular complex between GCKR, TRAF2, and TANK. CD40 and TNFa-induced activation of germinal center kinases and the subsequent activation of stress activated protein kinases requires TRAF2. While the TRAF2 TRAF domain binds GCK and GCKR, the RING finger domain is needed for their activation. We have found that GCKR activates the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNFa- and TRAF2-mediated GCKR and SAPK activation. TNFa signaling leads to TRAF2 polyubiquitination, TRAF2 oligomerization, and the recruitment, ubiquitination, and activation of GCKR, all sensitive to disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine-63 linked polyubiquitin chain by Ubc13/Uev1A is required for TNFa-mediated GCKR and SAPK activation. To better understand the role of GCK and GCKR in vivo, the murine GCK and GCKR gene have been isolated. Both genes are complicated spanning nearly 100 kilobases with more than 30 exons. This information has been used to create gene targeting constructs. Both GCK-/- and GCKR -/- mice have been created. Neither mutation dramatically alters mouse development as 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 their B cells are hyper-responsive to several proliferative signals including engagement of CD40. The B cells from these mice aberrantly activate signaling pathways normally stimulated by TNFalpha, CD40, and Wnt ligands. GCK-/- GCKR-/- mice have identified in crosses from interbreeding of double heterozygotic mice. These mice exhibit a similar immune phenotype to that of the GCKR-/- mice, but in addition are the males are sterile and the females have infrequent and small litters when crossed with wild type males. As a follow up study to the role of Ubc13 and TRAF2 in GCKR activation we have created a panel of TRAF2 expression contructs that express TRAF2 proteins that have individual lysines converted to alanines. We hope to identify the lysine residues in TRAF2 that undergo lysine-63 and lysine-48 linked ubiquitination and their role in the activation of Nuclear factor-kappaB and GCKR. In a continuation of our studies of PYK2 we have identified a role for Pyk2 in the activation of Signal transducers and activators of transcription factor-3 (STAT3). STATs are activated by tyrosine and serine phosphorylation, which normally occurs as a tightly regulated process. Our results indicate that Pyk2 facilitates epidermal growth factor receptor (EGFR)- and c-Src-mediated Stat3 activation, thereby implicating Pyk2 activation as a potential co-mediator in triggering Stat3-induced oncogenesis.