We have previously shown that the phosphorylation state of Lyn in B cells is governed by the activity of the BCR associated protein tyrosine phosphatase SHP 1. Interestingly, studies from other groups have demonstrated that the activity of SHP 1 can, in turn, be regulated through phosphorylation at specific Ser/Thr residues. It, therefore, seemed plausible to us that p38 dependent modulation of basal phospho Lyn levels may represent an indirect effect that is mediated through SHP 1. That is, the enhanced basal levels of activated Lyn could represent a consequence of attenuated SHP 1 activity, which is enforced through its phosphorylation by p38. The possibility of a direct inter action between p38 and SHP 1 was supported by our initial results involving confocal microscopy, which revealed that at least a fraction of the molecules repre senting these two proteins were indeed co localized in the proximity of the cell membrane.

Sub sequent immunoprecipitation experiments established that this co localized pool also included that subset of SHP 1 that was constitutively associated with the BCR. Thus, a Western blot analysis of BCR immunoprecipi tated from either unstimulated or anti IgM stimulated CH1 cells also revealed the co precipitation of both SHP 1 and p38. Finally, we could further demonstrate that treatment of the BCR immunoprecipi tate with the p38 inhibitor SB203580 resulted in a sig nificant increase in the associated phosphatase activity. These collective results, therefore, confirm that activity of the BCR associated SHP 1 was indeed under negative control of the co associated p38.

This, in turn, provides a likely explanation for the increased levels of activated Lyn detected in un stimulated CH1 cells. Extracting the core cellular network that mediates BCR dependent cell cycle arrest CH1 cells Our Anacetrapib results so far had helped to characterize at least some of the intermediates that were involved during anti IgM induced signal transduction. In subsequent experiments, we were also able to define the key set of TFs that were responsible for translating the pattern of signaling events generated into the expression of those target genes that were, at least primarily, involved in driving the G1 phase arrest.

Having thus generated the molecular map of the network emanating from the BCR and extending up to the enforcement of the specific cel lular response, we then also identified a feedback inter action between p38 and SHP 1 that functioned, through the regulation of Lyn activity, as a key regulatory motif of this network. These cumulative results, therefore, allowed us to further refine the rather generic network map derived in Figure 3C, and obtain a more precise description of the BCR dependent regulatory network for G1 arrest in CH1 cells.