Regulation of many immune response genes depend on a 10 bp DNA sequence termed kappaB. This sequence is bound by a family of protein factors related to the Rel oncogene. The prototype transcription complex binding to the sequence, termed NF-kappaB, has been conventionally defined as a heterodimer between a P50 DNA binding protein and a P65 (RelA) activation protein that is typically sequestered in the cytoplasm by a protein called I-kappaB. Following certain types of stimulation to the cell, a specific protein kinase complex called I-kappaB kinase causes the phosphorylation of I-kappaB followed by its ubiquitination and degradation. Among the stimuli that can release NF-kappaB is the triggering of the T cell receptor (TCR) or B cell receptor (BCR) by antigen during an immune response. However, this transcription factor plays a role in the induction of diverse sets of genes throughout the body in response to hundreds of different inducers. While studying a rare clinical condition of immunodeficiency, we discovered that NF-kappaB has a more complex subunit composition than previously suspected. Specifically, we found that genes such as IL-2 and I-kappaB harbor kappaB sites of a particular sequence that bind a trimeric complex containing p50, p65, and ribosomal protein small subunit 3 (RPS3). RPS3 is a K-homology (KH) domain-containing protein that binds single-stranded nucleic acids and may enhance the affinity of the p50 and p65 Rel-homology proteins to these select kappaB sites. A subset of all NF-kappaB genes are dependent on RPS3 as well as NF-kappaB including crucial physiological functions such as expression of the immunoglobulin kappa light chain gene in B cells and the interleukin-2 gene in T cells. Rapid transcription of specific NF-kappaB target genes is vital for an effective immune response. We recently discovered that in response to stimuli that induce NF-kappaB, RPS3 also independently translocates to the nucleus in parallel to p65, where it facilitates high affinity binding of NF-kappaB to certain kappaB sites. for example, in T lymphocytes stimulation of the T cell receptor causes translocation of RPS3 as well as NF-kappaB into the nucleus. We went on to show that specific activating signals cause the stimulation-induced phosphorylation of RPS3 which is critical for its nuclear translocation. We found that the Inhibitor of kappaB (IkappaB) kinase beta (IKKbeta), when activated, specifically phosphorylates RPS3 at serine 209 (S209). Serine phosphorylation of RPS3, which also depends on contemporaneous IKKbeta-mediated phosphorylation and degradation of IkBalpha, augments its association with importin-alpha in the classic cytoplasmic karyopherin pathway which governs subsequent nuclear translocation. Moreover, the Escherichia coli O157:H7 type III secretion system effector protein NleH1 specifically inhibits RPS3 S209 phosphorylation to block subsequent RPS3 nuclear translocation. By selectively attenuating transcription of certain RPS3-dependent NF-kappaB target genes, E. coli O157:H7 may alter the NF-kappaB response to suppress host inflammation in the gut, which may improve bacterial dissemination. In summary, our results indicate that the IKKbeta-dependent modification of a single amino acid in RPS3 promotes the specific recognition of certain kappaB sites by NF-kappaB, unveiling a novel mechanism for RPS3 that underpins its regulatory role in transcription versus translation. This mechanism also illuminates novel therapeutic targets for the control of foodborne pathogens, especially during early E. coli O157:H7 infection. We have also discovered the first germline mutation in CARD11, a protein that forms a vital signaling link between the antigen receptor in both B and T lymphocytes, in one family with congenital lymphoid hyperplasia first reported in The New England Journal of Medicine in 1971 as well as in a child adopted from China in a second family. The affected family members exhibit excessive accumulation and defective differentiation of B lymphocytes but not T lymphocytes. The dominant missense mutations identified will constitutively activate NF-kappaB in both B and T cells contributing to downstream proliferation in B cells. However,it causes apparent non-responsivenes or anergy in T cells resulting in poor IL-2 production and proliferation. Thus, we have identified the underlying genetic cause of this hereditary B cell disorder and have uncovered a potential molecular explanation for why CARD11 mutations may predispose to B but not T lymphoid malignancies. This can be understood in terms of the 2 signal model in which T lymphocytes require antigen receptor(signal 1) as well as costimulatory (signal 2)both required for T cell proliferation, whereas the provision solely of signal 1 leads to poor responsiveness or anergy. We observed this phenomenon in our patients, since E127G CARD11 causes internal constitutive activation of NF-kappaB, an important feature component of signal 1, in the absence of a concomitant signal 2. In contrast, B cell proliferation can be triggered by BCR crosslinking alone, which is mimicked by mutant CARD11-driven NF-kappaB activity. We posit that a chronic TCR-like signal 1 provided through mutant CARD11 can be converted to a proliferative signal for the patients T cells in vivo when proper costimulation (signal 2) is provided by professional antigen presenting cells. Defects in T cell help to B cells, related to T cell hyporesponsiveness, may partly explain the paucity of germinal centers and autoimmune manifestations in these patients. On the other hand, deficiencies in T cell-independent humoral responses to polysaccharide antigens also point to intrinsic defects in B cell signaling and effector function with E127G CARD11 present. Our discovery of a germline gain-of-function mutation in CARD11 illuminates how antigen receptor signaling is regulated differently in B and T cells, even though the proximal signaling machinery is nearly identical. This surprising difference has not been revealed by somatic CARD11 mutations in diffuse large cell B cell lymphoma, in which only B cells harbor the mutation and can potentially explain the preponderance of B cell rather than T-cell lymphomas associated with activating mutations in this gene. Our molecular analysis of this autosomal dominant lymphoproliferative disorder, which may represent a novel precursor state for B cell malignancies like B-chronic lymphocytic leukemia, reveals how selective dysregulation of NF-kappaB via CARD11 may predispose to selective proliferation and differentiation arrest in B cells, but defective proliferation and function of T cells.

Project Start
Project End
Budget Start
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Support Year
16
Fiscal Year
2012
Total Cost
$452,258
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