Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xL and Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xL dissociation from Bak occurred on exposure of Bak's NH2 terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death.

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP.

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP\ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (α) of AMPK (Thr"(#) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in itro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr"(# on AMPK activity. Mutation of Thr"(# to an aspartic acid residue (T172D) in either α1 or α2 resulted in a kinase complex with approx. 50 % the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90 % following treatment with protein phosphatases, whereas the activity of the T172D mutant complex

Fat-associated lymphoid clusters (FALCs) are a recently discovered type of lymphoid tissue associated with visceral fat. Here we show that distribution of FALCs was heterogeneous with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 B cells in the peritoneal cavity through high expression of the chemokine CXCL13 and supported B cell proliferation and germinal center differentiation during peritoneal immune challenges. FALC formation was induced by inflammation, which triggered recruitment of myeloid cells that express tumor necrosis factor (TNF) necessary for TNF receptor-signaling in stromal cells. CD1d-restricted Natural killer T (NKT) cells were likewise required for inducible formation of FALCs. Thus, FALCs support and coordinate innate B and T cell activation during serosal immune responses.