The DNA-dependent protein kinase (DNA-PK) is a mammalian serine/threonine kinase that is implicated in the repair of DNA double-strand breaks, DNA replication, transcription, and V(D)J recombination. To determine the role of the DNA-binding subunit of DNA-PK in vivo, we targeted Ku80 in mice. In mutant mice, T and B lymphocyte development is arrested at early progenitor stages and there is a profound deficiency in V(D)J rearrangement. Although Ku80-/- mice are viable and reproduce, they are 40-60% of the size of littermate controls. Consistent with this growth defect, fibroblasts derived from Ku80-/- embryos showed an early loss of proliferating cells, a prolonged doubling time, and intact cell-cycle checkpoints that prevented cells with damaged DNA from entering the cell-cycle. The unexpected growth phenotype suggests a new and important link between Ku80 and growth control.

The Fc receptor on B lymphocytes, Fc gamma RIIB (beta 1 isoform), helps to modulate B-cell activation triggered by the surface immunoglobulin complex. Crosslinking of membrane immunoglobulin by antigen or anti-Ig F(ab')2 antibody induces a transient increase in cytosolic free Ca2+, a rise in inositol-3-phosphate, activation of protein kinase C, and enhanced protein tyrosine phosphorylation. Crosslinking Fc gamma RIIB with the surface immunoglobulin complex confers a dominant signal that prevents or aborts lymphocyte activation triggered through the ARH-1 motifs of the signal transduction subunits Ig-alpha and Ig-beta. Here we show that Fc gamma RIIB modulates membrane immunoglobulin-induced Ca2+ mobilization by inhibiting Ca2+ influx, without changing the pattern of tyrosine phosphorylation. A 13-amino-acid motif in the cytoplasmic domain of Fc gamma RIIB is both necessary and sufficient for this effect. Tyrosine at residue 309 in this motif is phosphorylated upon co-crosslinking with surface immunoglobulin; mutation of this residue aborts the inhibitory effect of Fc gamma RIIB. This inhibition is directly coupled to signalling mediated through Ig-alpha and Ig-beta as evidenced by chimaeric IgM/alpha and IgM/beta molecules. The 13-residue motif in Fc gamma RIIB controls lymphocyte activation by inhibiting a Ca2+ signalling pathway triggered through ARH-1 motifs as a result of recruitment of novel SH2-containing proteins that interact with this Fc gamma RIIB cytoplasmic motif.

SummaryImmunoglobulin (Ig) antigen receptors are composed of a noncovalently-associated complex of Ig and two other proteins, Igo~ and Ig3. The cytoplasmic domain of both of these Ig associated proteins contains a consensus sequence that is shared with the signaling proteins of the T cell and Fc receptor. To test the idea that Igc~-IgB heterodimers are the signaling components of the Ig receptor, we have studied Ig mutations that interfere with signal transduction. We find that specific mutations in the transmembrane domain of Ig that inactivate Ca 2+ and phosphorylation responses also uncouple IgM from Igoe-Ig/3. These results define amino acid residues that are essential for the assembly of the Ig receptor. Further, receptor activity can be fully reconstituted in Ca 2+ flux and phosphorylation assays by fusing the cytoplasmic domain of Igct with the mutant Igs. In contrast, fusion of the cytoplasmic domain of Ig~ to the inactive Ig reconstitutes only Ca z+ responses. Thus, Igor and IgB are both necessary and sufficient to mediate signal transduction by the Ig receptor in B cells. In addition, our results suggest that IgoL and IgB can activate different signaling pathways.