PubMed · 12205466
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Tasuku Honjo. 2002. Does AID need another aid?. https://doi.org/10.1038/ni0902-800
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Voltage-activated proton currents are reported for the first time in human peripheral blood T and B lymphocytes and in the human leukaemic T cell line Jurkat E6-1. The properties of H(+) currents studied using tight-seal voltage-clamp recording techniques were similar in all cells. Changing the pH gradient by one unit caused a 47 mV shift in the reversal potential, demonstrating high selectivity of the channels for protons. H(+) current activation upon membrane depolarisation had a sigmoidal time course that could be fitted by a single exponential function after a brief delay. Increasing pH(o) shifted the activation threshold to more negative potentials, and increased both the H(+) current amplitude and the rate of activation. In lymphocytes studied at pH(i) 6.0, the activation threshold was more negative and the H(+) current density was three times larger than at pH(i) 7.0. Increasing the intracellular Ca(2+) concentration to 1 microM did not change H(+) current amplitude or kinetics detectably. Extracellularly applied Zn(2+) and Cd(2+) inhibited proton currents, slowing activation and shifting the voltage-activation curve to more positive potentials. The H(+) current amplitude was 100 times larger in CD19+ B lymphocytes and in Jurkat E6-1 cells than in CD3+ T lymphocytes. Following stimulation with the phorbol ester PMA, the H(+) current density in peripheral blood T lymphocytes and Jurkat T cells increased. In contrast, the H(+) current density of phorbol ester (PMA)-stimulated B lymphocytes was reduced and activation became slower. The pattern of expression of H(+) channels in lymphocytes appears well suited to their proposed role of charge compensation during the respiratory burst.
Studies of RII alpha-deficient B lymphoid cells and stable transfectants expressing the type II alpha regulatory subunit (RII alpha) of cAMP-dependent protein kinase (PKA), which is targeted to the Golgi-centrosomal area, reveal that the presence of a Golgi-associated pool of PKA type II alpha mediates a change in intracellular transport of the plant toxin ricin. The transport of ricin from endosomes to the Golgi apparatus, measured as sulfation of a modified ricin (ricin sulf-1), increased in RII alpha-expressing cells when PKA was activated. However, not only endosome-to-Golgi transport, but also retrograde ricin transport to the endoplasmic reticulum (ER), measured as sulfation and N-glycosylation of another modified ricin (ricin sulf-2), seemed to be increased in cells expressing RII alpha in the presence of a cAMP analog, 8-(4-chlorophenylthio)-cAMP. Thus, PKA type II alpha seems to be involved in both endosome-to-Golgi and Golgi-to-ER transport. Because ricin, after being retrogradely transported to the ER, is translocated to the cytosol, where it inhibits protein synthesis, we also investigated the influence of RII alpha expression on ricin toxicity. In agreement with the other data obtained, 8-(4-chlorophenylthio)-cAMP and RII alpha were found to sensitize cells to ricin, indicating an increased transport of ricin to the cytosol. In conclusion, our results demonstrate that transport of ricin from endosomes to the Golgi apparatus and further to the ER is regulated by PKA type II alpha isozyme.
The class II transactivator (CIITA) is a master regulator of MHC class II expression. CIITA also modulates the expression of MHC class I genes, suggesting that it may have a more global role in gene expression. To determine whether CIITA regulates genes other than the MHC class II and I family, DNA microarray analysis was used to compare the expression profiles of the CIITA expressing B cell line Raji and its CIITA-negative counterpart RJ2.2.5. The comparison identified a wide variety of genes whose expression was modulated by CIITA. Real time RT-PCR from Raji, RJ2.2.5, an RJ2.2.5 cell line complemented with CIITA, was performed to confirm the results and to further identify CIITA-regulated genes. CIITA-regulated genes were found to have diverse functions, which could impact Ag processing, signaling, and proliferation. Of note was the identification of a set of genes localized to chromosome 1p34-35. The global modulation of genes in a local region suggests that this region may share some regulatory control with the MHC.