Mitogen-activated protein (MAP) kinase cascades represent one of the major signal systems used by eukaryotic cells to transduce extracellular signals into cellular responses. Four MAP kinase subgroups have been identified in humans: ERK, JNK (SAPK), ERK5 (BMK), and p38. Here we characterize a new MAP kinase, p38␤. p38␤ is a 372-amino acid protein most closely related to p38. It contains a TGY dual phosphorylation site, which is required for its kinase activity. Like p38, p38␤ is activated by proinflammatory cytokines and environmental stress. A comparison of events associated with the activation of p38␤ and p38 revealed differences, most notably in the preferred activation of p38␤ by MAP kinase kinase 6 (MKK6), whereas p38 was activated nearly equally by MKK3, MKK4, and MKK6. Moreover, in vitro and in vivo experiments showed a strong substrate preference by p38␤ for activating transcription factor 2 (ATF2). Enhancement of ATF2-dependent gene expression by p38␤ was ϳ20-fold greater than that of p38 and other MAP kinases tested. The data reported here suggest that while closely related, p38␤ and p38 may be regulated by differing mechanisms and may exert their actions on separate downstream targets.

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Endotoxin (lipopolysaccharide; LPS) activates a wide variety of host defense mechanisms. In mammals LPS binding protein (LBP) and CD14 interact with LPS to mediate cellular activation. Using sucrose density gradients and a fluorescent endotoxin derivative we have investigated the mechanism of LPS binding to LBP and the soluble form of CD14 (sCD14). LPS binds to LBP to form two types of complex; at low ratios of LPS to LBP complexes with one molecule of LBP and 1-2 molecules of LPS predominate, while at high ratios of LPS to LBP a large aggregate of LBP and LPS predominates. Complexes of LPS with sCD14 do not form large aggregates, consisting of only 1-2 LPS bound to a single sCD14 even at high multiples of LPS to sCD14. LBP catalyzes LPS binding to sCD14. Catalysis by LBP apparently occurs because LBP provides a pathway for LPS to bind to sCD14 which avoids the necessity for LPS monomers in aqueous solution. The dissociation constants for LPS.LBP and LPS.sCD14 complexes were determined to be 3.5 x 10(-9) and 29 x 10(-9) M, respectively. These numbers suggest that when LBP and sCD14 are present at roughly equal concentrations as they are in normal human plasma and compete for limited LPS, the LPS will predominantly associate with LBP.

An essential step in the signal transduction pathway of Escherichia coli is the control of the protein kinase activity of CheA by the chemotaxis receptor proteins. This control requires the participation of the CheW protein. Although the biochemical nature of the coupling between the receptors and the kinase is unknown, it is likely that CheW interacts with the receptors and with CheA. In this communication, we report direct measurement of a physical interaction between CheW and CheA. We utilized the equilibrium column chromatography method of Hummel and Dreyer to show that CheW and CheA exhibit reversible binding with the stoichiometry of two CheW monomers per CheA dimer. CheW was found to exist as monomers and CheA was found to exist as dimers by equilibrium analytical ultracentrifugation. The dissociation constant for the CheW-CheA interaction (in 160 mM KCI/5 mM MgCl2, pH 7.4 at 40C) was determined to be in the physiologically relevant range of 17 FAM. No evidence for cooperativity in the association of CheW with CheA was found. CheW also plays a role in coordinating the kinase activity of CheA in the adaptation response. The cell adapts to stimuli by adjusting the methylation state of its receptors (9). Adaptation to attractant results in an increase in the methylation state ofthe receptor, whereas adaptation to a repellent results in a decrease. In the repellent adaptation response, CheA phosphorylates CheB (the methylesterase), which is then activated to demethylate the receptor and restore the cell to its prestimulus swimming state of random tumbles and smooth swims (10, 11). Demethylation in response to repellent requires both CheA and CheW. During the adaptation response to attractant stimuli, the methylesterase is transiently inhibited. The mechanism responsible for methylesterase inhibition is not fully understood, but the adaptation response to positive stimuli appears to require only CheW (11). This observation suggests that parts of a CheWdependent signaling pathway may be independent of CheA.We have initiated studies of the physical interactions between CheW and the other components of the signal transduction and adaptation pathways of the chemotaxis system. Through such experiments, we hope to obtain a more detailed picture of the molecular pathway involved in chemotaxis signal transduction. It is not known how the kinase activity of CheA is coupled to the receptor by CheW, but the lack of any known CheW covalent modification suggests that complex formation between CheW and CheA may be involved. Here we report the results of our investigation that demonstrate an interaction between monomeric Mr 18,000 CheW and dimeric Mr 142,000 CheA. The ultimate goal ofthis work is to understand the molecular events that control swimming behavior in response to chemotactic stimuli. It appears that the formation of reversible complexes among various chemotaxis proteins may be an important feature of these molecular events. MATERIALS AND METHODSBacterial Strains and Plasmids. The cheW overexpression plas...

Under physiological conditions, lipopolysaccharide (LPS) activation of cells involves the LPS binding protein (LBP) and either membrane or soluble CD14. We find LPS forms a ternary complex with LBP and membrane CD14 (mCD14). Subsequent to complex formation and distinct from signal transduction, LBP and LPS internalize. Internalization can be separated from signal transduction with the anti-LBP antibody 18G4 and the anti-CD14 antibody 18E12. 18G4 inhibits LBP binding to mCD14 without blocking signal transduction or LPS transfer to soluble CD14; 18E12 inhibits signal transduction without affecting LPS binding and uptake. These data show that while LPS signal transduction and LPS clearance utilize both LBP and mCD14, the pathways bifurcate after LPS binding to mCD14.