Role of tyrosine kinases in lymphocyte activation.

Interaction of T- and B-cell antigen receptors with cytoplasmic non-receptor tyrosine protein kinases is critical to the activation of lymphocytes by antigen. Both the src-family tyrosine protein kinases Lck, Fyn, Lyn and Blk and the syk-family tyrosine protein kinases Syk and ZAP-70 play a role in lymphocyte activation. The antigen receptors are coupled to this cluster of kinases by the cytoplasmic tails of the gamma, delta, epsilon, zeta, and eta subunits of the T-cell receptor, and the Ig-alpha and Ig-beta subunits of the B-cell receptor. Each of these proteins contains one or more 'tyrosine based activation motifs', with the amino acid sequence D/EX7D/EXXYXXL/IX7YXXL/I. This motif appears to allow binding of one or more src-like kinases, via their unique amino termini, before the onset of lymphocyte activation. Invariant tyrosines in the motif become phosphorylated following the triggering of lymphocyte activation, and this modification induces the binding of the src- and syk-family tyrosine protein kinases, and potentially other signalling molecules, through SH2 domains to the antigen receptors.

Activation of B- and T-cells by the cytoplasmic domains of the B-cell antigen receptor proteins Ig-alpha and Ig-beta.

In addition to membrane immunoglobulin (mIg), the B-cell antigen receptor contains Ig-alpha/Ig-beta heterodimers that link mIg to intracellular signaling molecules. To compare the ability of the cytoplasmic domains of Ig-alpha and Ig-beta to transduce signals in B- and T-cells, we constructed chimeric genes encoding the extracellular and transmembrane domains of human CD8 alpha and the cytoplasmic domain of murine Ig-alpha (CD8/Ig-alpha) or Ig-beta (CD8/Ig-beta). In murine B-cell hybridoma LK 35.2 cells, antibody-mediated cross-linking of mIg, CD8/Ig-alpha, or CD8/Ig-beta induced similar increases in intracellular calcium levels and protein tyrosine phosphorylation. Substitution of alanine for the conserved leucine, but not the conserved isoleucine, residue within the putative activation motif of CD8/Ig-beta destroyed signaling ability. In murine T-cell hybridoma DO-11.10 cells, cross-linking of the T-cell antigen receptor, CD8/Ig-alpha, or CD8/Ig-beta stimulated equivalent protein tyrosine phosphorylation and interleukin-2 production. Thus, the cytoplasmic domains of Ig-alpha and Ig-beta are equally capable of initiating early signaling events downstream of B- and T-cell antigen receptors as well as evoking a complete biological effector response in lymphocytes.

B-cell activation by wild type and mutant Ig-beta cytoplasmic domains.

In B lymphocytes, the cytoplasmic domains of the membrane immunoglobulin-associated heterodimeric Ig-alpha and Ig-beta proteins link membrane immunoglobulin to intracellular signalling molecules. We constructed chimeric genes encoding the extracellular and transmembrane domain of human CD8 alpha and the cytoplasmic domain of Ig-alpha or Ig-beta and examined the ability of the chimeric proteins to induce signalling in the murine B-cell lymphoma A20. Crosslinking of CD8/Ig-alpha or CD8/Ig-beta induced both calcium mobilization and protein tyrosine phosphorylation, although induction by CD8/Ig-alpha was somewhat stronger. We also carried out mutagenesis of residues within the "Reth" motif of the CD8/Ig-beta cytoplasmic domain and determined the effects of these mutations on signalling in the murine B-cell hybridoma LK 35.2. Mutants in which alanine was substituted for glutamine 202, threonine 205, and isoleucine 209 retained the ability to induce protein tyrosine phosphorylation and calcium mobilization. In contrast, substitution of alanine for leucine 198 abrogated these responses, suggesting a critical role for this residue in interaction with cytoplasmic signalling proteins.

Genetic characterization of the vaccinia virus DNA polymerase: cytosine arabinoside resistance requires a variable lesion conferring phosphonoacetate resistance in conjunction with an invariant mutation localized to the 3'-5' exonuclease domain.

In this report, we describe the isolation, molecular genetic mapping, and phenotypic characterization of vaccinia virus mutants resistant to cytosine arabinoside (araC) and phosphonoacetic acid (PAA). At 37 degrees C, 8 microM araC was found to prevent macroscopic plaque formation by wild-type virus and to cause a 10(4)-fold reduction in viral yield. Mutants resistant to 8 microM araC were selected by serial passage of a chemically mutagenized viral stock in the presence of drug. Because recovery of mutants required that initial passages be performed under less stringent selective conditions, and because plaque-purified isolates were found to be cross-resistant to 200 micrograms of PAA per ml, it seemed likely that resistance to araC required more than one genetic lesion. This hypothesis was confirmed by genetic and physical mapping of the responsible mutations. PAAr was accorded by the acquisition of one of three G-A transitions in the DNA polymerase gene which individually alter cysteine 356 to tyrosine, glycine 372 to aspartic acid, or glycine 380 to serine. AraCr was found to require one of these substitutions plus an additional T-C transition within codon 171 of the DNA polymerase gene, a change which replaces the wild-type phenylalanine with serine. Congenic viral stocks carrying one of the three PAAr lesions, either alone or in conjunction with the upstream araCr lesion, in an otherwise wild-type background were generated. The PAAr mutations conferred nearly complete resistance to PAA, a slight degree of resistance to araC, hypersensitivity to aphidicolin, and decreased spontaneous mutation frequency. Addition of the mutation at codon 171 significantly augmented araC resistance and aphidicolin hypersensitivity but caused no further change in mutation frequency. Several lines of evidence suggest that the PAAr mutations primarily affect the deoxynucleoside triphosphate-binding site, whereas the codon 171 mutation, lying within a conserved motif associated with 3'-5' exonuclease function, is postulated to affect the proofreading exonuclease of the DNA polymerase.

Genetic characterization of the vaccinia virus DNA polymerase: identification of point mutations conferring altered drug sensitivities and reduced fidelity.

We determined that 85 microM aphidicolin was sufficient to block macroscopic plaque formation by vaccinia virus and to cause a 10(4)-fold reduction in viral yield from a wild-type infection. A chemically mutagenized viral stock was passaged sequentially in the presence of drug, and plaque-purified viral stocks resistant to aphidicolin were isolated and characterized. By use of a marker rescue protocol, the lesion in each mutant was found to map within the same 500-bp fragment within the DNA polymerase gene. All of the mutants were found to contain a single nucleotide change in the same codon. In nine of these mutants, the alanine residue at position 498 was changed to a threonine, whereas a 10th mutant sustained a valine substitution at this position. Congenic viral strains which carried the Aphr lesion in an unmutagenized wild-type background were isolated. The Thr and Val mutations were found to confer equivalent levels of drug resistance. In the presence of drug, viral yields were 25% of control levels, and the levels of viral DNA synthesized were 30 to 50% of those seen in control infections. The two mutations also conferred an equivalent hypersensitivity to the cytosine analog 1-beta-D-arabinofuranosylcytosine (araC); strains carrying the Thr mutation were moderately hypersensitive to the pyrophosphate analog phosphonoacetic acid and the adenosine analog araA, whereas the Val mutation conferred acute hypersensitivity to these inhibitors. The Val mutation also conferred a mutator phenotype, leading to a 20- to 40-fold increase in the frequency of spontaneous mutations within the viral stock.