This project focuses on two serine threoninc 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 on 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, that signaling through these receptors leads to the formation of a trimolecular complex between GCKR, TRAF2, and TANK. A screen for molecules that bind GCKR has resulted in the identification of a panel of strong interacting proteins that includes a number of adaptor proteins such as Crk and CrkL as well as several proteins that participate in the Ras- signaling pathway. 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. The ES cells in which one allele of the GCKR gene has been inactivated have been created and are being using used to develop mice that are GCKR deficient. The tyrosine kinase PYK2 links signals generated by g- protein coupled receptors to downstream signaling pathways. One type of G-protein, G13alpha, potently activates PYK2 kinase activity, which leads to the activation of a small GTPase called RhoA, which in turn stimulates morphological changes in cells and triggers a conserved signaling pathway important for cell growth. The activation of g-proteins in the cells leads to a transient association between PYK2 and G13alpha. A pathway that leads from Gqalpha to the activation of NF-kappaB has been found to depend upon activation of PYK2 as well as the lipid kinase PI-3 kinase and the serine threonine kinase Akt. Finally, in conjunction with J. Tuscano we have developed a panel of antibodies against B lymphocyte cell surface molecules.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000739-06
Application #
6508519
Study Section
(LIR)
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2001
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Gibbons, Deena L; Abeler-Dörner, Lucie; Raine, Tim et al. (2011) Cutting Edge: Regulator of G protein signaling-1 selectively regulates gut T cell trafficking and colitic potential. J Immunol 187:2067-71
Sinha, Rajesh K; Park, Chung; Hwang, Il-Young et al. (2009) B lymphocytes exit lymph nodes through cortical lymphatic sinusoids by a mechanism independent of sphingosine-1-phosphate-mediated chemotaxis. Immunity 30:434-46
Shi, Chong-Shan; Huang, Ning-Na; Harrison, Kathleen et al. (2006) The mitogen-activated protein kinase kinase kinase kinase GCKR positively regulates canonical and noncanonical Wnt signaling in B lymphocytes. Mol Cell Biol 26:6511-21
Shi, Chong-Shan; Kehrl, John H (2004) Pyk2 amplifies epidermal growth factor and c-Src-induced Stat3 activation. J Biol Chem 279:17224-31
Shi, Chong-Shan; Kehrl, John H (2003) Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2). J Biol Chem 278:15429-34
Shi, C S; Kehrl, J H (2001) PYK2 links G(q)alpha and G(13)alpha signaling to NF-kappa B activation. J Biol Chem 276:31845-50
Shi, C S; Sinnarajah, S; Cho, H et al. (2000) G13alpha-mediated PYK2 activation. PYK2 is a mediator of G13alpha -induced serum response element-dependent transcription. J Biol Chem 275:24470-6
Shi, C S; Tuscano, J; Kehrl, J H (2000) Adaptor proteins CRK and CRKL associate with the serine/threonine protein kinase GCKR promoting GCKR and SAPK activation. Blood 95:776-82
Ivanov, V N; Kehrl, J H; Ronai, Z (2000) Role of TRAF2/GCK in melanoma sensitivity to UV-induced apoptosis. Oncogene 19:933-42
Chin, A I; Shu, J; Shan Shi, C et al. (1999) TANK potentiates tumor necrosis factor receptor-associated factor-mediated c-Jun N-terminal kinase/stress-activated protein kinase activation through the germinal center kinase pathway. Mol Cell Biol 19:6665-72

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