Identification of a site critical for kinase regulation on the central processing unit (CPU) helix of the aspartate receptor.

Ligand binding to the homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium generates a transmembrane signal that regulates the activity of a cytoplasmic histidine kinase, thereby controlling cellular chemotaxis. This receptor also senses intracellular pH and ambient temperature and is covalently modified by an adaptation system. A specific helix in the cytoplasmic domain of the receptor, helix alpha6, has been previously implicated in the processing of these multiple input signals. While the solvent-exposed face of helix alpha6 possesses adaptive methylation sites known to play a role in kinase regulation, the functional significance of its buried face is less clear. This buried region lies at the subunit interface where helix alpha6 packs against its symmetric partner, helix alpha6'. To test the role of the helix alpha6-helix alpha6' interface in kinase regulation, the present study introduces a series of 13 side-chain substitutions at the Gly 278 position on the buried face of helix alpha6. The substitutions are observed to dramatically alter receptor function in vivo and in vitro, yielding effects ranging from kinase superactivation (11 examples) to complete kinase inhibition (one example). Moreover, four hydrophobic, branched side chains (Val, Ile, Phe, and Trp) lock the kinase in the superactivated state regardless of whether the receptor is occupied by ligand. The observation that most side-chain substitutions at position 278 yield kinase superactivation, combined with evidence that such facile superactivation is rare at other receptor positions, identifies the buried Gly 278 residue as a regulatory hotspot where helix packing is tightly coupled to kinase regulation. Together, helix alpha6 and its packing interactions function as a simple central processing unit (CPU) that senses multiple input signals, integrates these signals, and transmits the output to the signaling subdomain where the histidine kinase is bound. Analogous CPU elements may be found in other receptors and signaling proteins.

Recombinants in the H-2S/H-2D interval of mouse chromosome 17 define the map position of a gene for cleft palate susceptibility.

The H-2 region of mouse chromosome 17 is known to include one or more genes that affect susceptibility to cortisone-induced cleft palate. We have now studied congenic strains that possess crossovers in the interval between H-2S and H-2D and have observed significant differences in susceptibility among recombinants that had been believed to possess the same H-2 haplotypes. Pregnant mice were injected on days 11 through 14 of gestation with 100 mg of cortisone per kg of body weight. The frequency of cleft palate in B10.A(2R) was significantly greater than in B10.A(1R), despite the fact that both have H-2a/H-2b crossovers in the interval between the S and D loci and have the same alleles at all loci that have been previously characterized. Both B10.BAR5 and B10.BAR12 were significantly more susceptible than B10.A(18R), although these strains also share the same alleles at all loci that have been previously characterized. All three of these strains have H-2b/H-2a recombinant chromosomes, with crossovers in the S/D interval. Genetic linkage between H-2 and the high-susceptibility gene of B10.BAR5 was confirmed by testing H-2 homozygotes derived by intercrossing backcross animals. These data therefore suggest that a gene coding for susceptibility, which we designate Cps-1, maps in the 350-kb interval between H-2S and H-2D, and the congenic strains that we have found to be different have different crossover points within this interval. Alleles at the Cps-1 locus have embryonic effects, but no demonstrable effects on the maternal environment.