E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas.

The E2A gene products, E12 and E47, are critical for proper early B-cell development and commitment to the B-cell lineage. Here we reveal a new role for E2A in T-lymphocyte development. Loss of E2A activity results in a partial block at the earliest stage of T-lineage development. This early T-cell phenotype precedes the development of a T-cell lymphoma which occurs between 3 and 9 months of age. The thymomas are monoclonal and highly malignant and display a cell surface phenotype similar to that of immature thymocytes. In addition, the thymomas generally express high levels of c-myc. As assayed by comparative genomic hybridization, each of the tumor populations analyzed showed a nonrandom gain of chromosome 15, which contains the c-myc gene. Taken together, the data suggest that the E2A gene products play a role early in thymocyte development that is similar to their function in B-lineage determination. Furthermore, the lack of E2A results in development of T-cell malignancies, and we propose that E2A inactivation is a common feature of a wide variety of human T-cell proliferative disorders, including those involving the E2A heterodimeric partners tal-1 and lyl-1.

T-cell fate.

Recent studies suggest that lineage commitment steps, which occur during T-cell differentiation, follow principles in common with fate specification in simple invertebrates. Here we review T-cell development from the perspective of developmental biology. We present models for alpha beta vs gamma delta and CD4 vs CD8 lineage commitment that are consistent with previously published and newly presented experiments.

Commitment to the CD4 lineage mediated by extracellular signal-related kinase mitogen-activated protein kinase and lck signaling.

The development of T cells results in a concordance between the specificity of the TCR for MHC class I and class II molecules and the expression of CD8 and CD4 coreceptors. Based on analogy to simple metazoan models of organ development and lineage commitment, we sought to determine whether extracellular signal-related kinase (Erk) mitogen-activated protein (MAP) kinase pathway signaling acts as an inductive signal for the CD4 lineage. Here, we show that, by altering the intracellular signaling involving the Erk/MAP kinase pathway, T cells with specificity for MHC class I can be diverted to express CD4, and, conversely, T cells with specificity for MHC class II can be diverted to express CD8. Furthermore, we find that activation of the src-family tyrosine kinase, p56lck is an upstream mediator of lineage commitment. These results suggest a simple mechanism for lineage commitment in T cell development.

Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis.

The MotA protein of Escherichia coli is a component of the flagellar motors that functions in transmembrane proton conduction. Here, we report several features of MotA structure revealed by use of a mutagenesis-based approach. Single tryptophan residues were introduced at many positions within the four hydrophobic segments of MotA, and the effects on function were measured. Function was disrupted according to a periodic pattern that implies that the membrane-spanning segments are alpha-helices and that identifies the lipid-facing parts of each helix. The results support a hypothesis for MotA structure and mechanism in which water molecules form most of the proton-conducting pathway. The success of this approach in studying MotA suggests that it could be useful in structure-function studies of other integral membrane proteins.

Tryptophan-scanning mutagenesis of MotB, an integral membrane protein essential for flagellar rotation in Escherichia coli.

The MotB protein of Escherichia coli is an essential component of the flagella that functions together with the MotA protein in transmembrane proton conduction. MotB has a single hydrophobic segment that spans the membrane. In order to determine which parts of the membrane-spanning segment can tolerate the introduction of a large, hydrophobic side chain, single Trp residues were substituted into many consecutive positions in the segment and the effects on function were measured. Trp residues were tolerated at positions near the periplasmic end of the MotB segment but not at positions near the cytoplasmic end. These results are different from what was seen in a similar mutational study of MotA, in that protein Trp residues were tolerated at positions that would be clustered together on one face of each hydrophobic segment if they are alpha-helices [Sharp, L. L., Zhou, J., & Blair, D. F. (1995) Proc. Natl. Acad. Sci. U.S.A. (in press)]. Those results suggested that the membrane-spanning segments of MotA are alpha-helices arranged in a bundle so that each has a face directed toward the lipid. The contrasting results seen with MotB indicate that its relationship to neighboring protein segments is different. Double-Trp substitutions, one each in MotA and MotB, also were studied. Many double substitutions had strongly synergistic effects which imply that the membrane segments of these proteins interact. (ABSTRACT TRUNCATED AT 250 WORDS)

The influence of the MAPK pathway on T cell lineage commitment.

During development, progenitor thymocytes differentiate into either CD4 or CD8 T cells, and this fate decision depends on the specificity of the T cell antigen receptor (TCR) for MHC class II or class I molecules. Based on the mechanisms of fate specification known for simple metazoan organisms, we sought to determine whether the extracellular signal-related kinases (ERKs) play a role in T cell differentiation and lineage commitment. Using a dominant gain-of-function mutant of the erk2 gene, we show that differentiation into the CD4 lineage is favored. We also show that, conversely, the addition of a pharmacological inhibitor of the ERK pathway favors differentiation into the CD8 lineage. We present a quantitative selection model that incorporates these results as well as those of recent reports on the role of Notch in T cell lineage specification.

Function of protonatable residues in the flagellar motor of Escherichia coli: a critical role for Asp 32 of MotB.

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.