Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase.

Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.

Activation and tyrosine phosphorylation of protein kinase C delta in response to B cell antigen receptor stimulation.

Activation of the B cell through antigen receptor (BCR) crosslinking is known to initiate a prominent tyrosine kinase cascade and lipid second messenger production through the activation of phospholipase Cgamma and phosphatidylinositol 3' kinase. In this study, we demonstrate that protein kinase C delta (PKCdelta) responds to crosslinking of the BCR by becoming activated and tyrosine phosphorylated within 30s of stimulation. PKC6 activation was dependent primarily on phosphatidylinositol 3' kinase, and this in turn was dependent on an upstream tyrosine phosphorylation event. Inhibition of PKCdelta activation by blocking phosphatidylinositol 3' kinase was also accompanied by a decrease in its tyrosine phosphorylation, suggesting that PKCdelta must be activated in order to become tyrosine phosphorylated. Inhibition of phospholipase C activation had an insignificant effect on the activation of PKCdelta, however it attenuated the tyrosine phosphorylation of PKCdelta. This suggests a distinct role for phospholipase C in the regulation of PKCdelta. This report describes a role for PKCdelta in response to the combined signals originated by the BCR.

The association between CD20 and Src-family Tyrosine kinases requires an additional factor.

CD20 is a B cell surface protein which can initiate intracellular signals involving tyrosine kinase activation, and modify B cell growth and differentiation. CD20 is tightly associated with the Src-family kinases Lyn, Fyn and Lck; however, the mechanism of interaction remains to be determined. Association between CD20 and Src-family kinases has been detected in peripheral blood B cells and in 5 out of 8 unrelated B cell lines. The lack of CD20-associated kinase activity in some cell lines offered an opportunity to investigate the mechanism of CD20 associations. All 8 B cell lines were found to express Lyn, and, with one exception, all cell lines also expressed Fyn. Lck, however, was not detected in any of the cell lines in which CD20 failed to coprecipitate kinase activity. To test the possibility that Lck was required for assembly of the CD20 complex, Lck was transfected into one of the 3 CD20/kinase association-deficient lines, namely T51. CD20 did not coprecipitate kinase activity from the transfected T51 cells, despite their expression of high levels of exogenous Lck, as well as endogenous Lyn and Fyn. CD20 cDNA from T51 was sequenced and found to be normal. These data establish that association between CD20 and Src-family kinases requires an additional factor.