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The structure of complete catalytic domain of D-myoinositol 1,4,5-triphosphate 3-kinase isoform B (IP(sub 3)-3KB), including the CaM binding domain in complex with Mg(super 2+) and ATP is presented. It is suggested that the IP(sub 3)-3KB structure, a mechanism of enzyme activation raises the possibility that interaction between IP(sub 3)-3KB molecules occur as part of the catalytic or regulatory cycle.

D-Myoinositol 1,4,5-trisphophate 3-kinases (IP3-3Ks) play important roles in metazoan cellular signaling. It has been demonstrated that mice without a functional version of IP3-3K isoform B are deficient in peripheral T-cells, indicating that IP3-3KB is essential to the developing immune system. The recent apo IP3-3KA structure exhibited a helix at the catalytic domain N-terminus exhibited a helix at the N-terminus of the catalytic domain, with a tryptophan indole moiety mimicking the binding mode of the substrate ATP purine ring, suggesting a mechanism of autoinhibition. Here we present the structure of the complete catalytic domain of IP3-3KB, including the CaM binding domain in complex with Mg2+ and ATP. The crystal structure reveals a homodimeric arrangement of IP3-3KB catalytic domains, mediated via an intermolecular antiparallel β-sheet formed from part of the CaM binding region. Residues from the putative autoinhibitory helix are rearranged into a loop configuration, with extensive interactions with the bound ATP. Mutagenesis of residues from this region reveals that substitution of the putative autoinhibitory tryptophan generates a hyperactive enzyme which retains Ca2+/CaM sensitivity. The IP3-3KB structure suggests a mechanism of enzyme activation, and raises the possibility that an interaction between IP3-3KB molecules may occur as part of the catalytic or regulatory cycle. [ABSTRACT FROM AUTHOR]

LMP1 is an Epstein-Barr virus (EBV)-encoded membrane protein essential for the proliferation of EBV-infected lymphoblasts (E. Kilger, A. Kieser, M. Baumann, and W. Hammerschmidt, EMBO J. 17:1700-1709, 1998). LMP1 also inhibits gene expression and induces cytostasis in transfected cells when it is expressed at levels as little as twofold higher than the average for EBV-positive lymphoblasts (M. Sandberg, A. Kaykas, and B. Sugden, J. Virol. 74:9755-9761, 2000; A. Kaykas and B. Sugden, Oncogene 19:1400-1410, 2000). We have found that in three different clones of EBV-infected lymphoblasts the levels of expression of LMP1 in individual cells in each clone ranged over 100-fold. This difference is due to a difference in levels of the LMP1 transcript. In these clones, cells expressing high levels of LMP1 incorporated less BrdU. We also found that induction of expression of LMP1 or of a derivative of LMP1 with its transmembrane domain fused to green fluorescent protein instead of its carboxy-terminal signaling domain resulted in phosphorylation of eIF2 alpha in EBV-negative Burkitt's lymphoma cells. This induction of phosphorylation of eIF2 alpha was also detected in EBV-infected lymphoblasts, in which high levels of LMP1 correlated with high levels of phosphorylation of eIF2 alpha. Our results indicate that inhibition of gene expression and of cell proliferation by LMP1 occurs normally in EBV-infected cells.

The EBV latent membrane protein 1 (LMP1) is an integral membrane protein that acts like a constitutively activated receptor. LMP1 interacts with members of the tumor necrosis factor receptor-associated factor family, as well as with tumor necrosis factor receptor-associated death domain, resulting in induction of nuclear factor-kappaB, the p38 mitogen-activated protein kinase pathway, and the c-Jun NH(2)-terminal kinase activator protein 1-signaling cascade. The binding of Janus kinase 3 results in activation of signal transducers and activators of transcription. The domain structure of LMP1 has been mapped extensively, but the quantitative contribution of distinct LMP1 domains to the efficiency of B-cell proliferation by EBV has not been determined. On the basis of the maxi-EBV system, which allows us to introduce and study mutations in the context of the complete EBV genome, a panel of 10 EBV mutants with alterations in the LMP1 gene locus was established. The mutant EBVs were tested for their efficiency to induce and maintain proliferation of clonal B-cell lines in vitro. Surprisingly and with reduced frequency, EBV mutants which deleted LMP1's COOH terminus, transmembrane domains, or the entire open reading frame were able to generate proliferating B-cell clones that were dependent on the presence of human fibroblast feeder cells. A B-cell clone carrying the LMP1-null mutant EBV genome was also analyzed for oncogenicity in severe combined immunodeficiency mice. Our results demonstrate that LMP1 is critical but not mandatory for the generation of proliferating B cells in vitro. LMP1 functions greatly contribute to EBV's transformation potential and appear essential for its oncogenicity in severe combined immunodeficiency mice.