This result confirmed
the earlier finding that in the anergic cells p21Cip1 did not appear to be acting through cdk inhibition. To determine whether p21Cip1 inhibited proliferation in the secondary cultures through interaction with and inhibition of PCNA, p21Cip1 coprecipitation with PCNA was also examined. Most of the PCNA did not associate with p21Cip1 in either control Th1 cells or anergic Th1 cells, regardless of restimulation (Fig. 5b). In addition, the amount of PCNA that was associated with p21Cip1 was not higher in the anergic Th1 cells than the control cells. This result suggested that in the anergic Th1 cells p21Cip1 was BIBW2992 in vitro not acting through preferential PCNA binding and inhibition. As a third possible mechanism, p21Cip1 interactions with members of the MAPK pathway were studied. Under the same experimental conditions in which p21Cip1–cdk2 and p21Cip1–PCNA interactions Selleck Palbociclib were studied, p21Cip1–JNK coprecipitation was examined. The majority of JNK protein was not associated
with p21Cip1 in any of the groups. However, a small amount of JNK coprecipitated with p21Cip1 in 2-hr restimulated anergic Th1 cells (Fig. 5b). As a control, another MAPK that is reported to interact with p21Cip1in vitro,15 namely p38, was examined for its interaction with p21Cip1 in the anergic Th1 cells. Little p38 could be detected in the p21Cip1 immunoprecipitates except a small band that was present equally in all groups (Fig. 5b). Most of the 4-Aminobutyrate aminotransferase p38 in all the lysates was not associated with p21Cip1. This result suggested that the low level p21Cip1–JNK interaction observed in the anergic restimulated Th1 cells was specific for JNK and did not encompass another MAPK p38. Unlike JNK and p38, which are present in relatively unchanged levels throughout T-cell
activation, phosphorylated versions of MAPK such as p-JNK and p-c-jun are only found in T cells for the initial few hours following stimulation. The interaction of p21Cip1 with JNK in the anergic Th1 cells was detected early in restimulation and was not present in the absence of restimulation, so the possibility that p21Cip1 preferentially associated with p-JNK was explored. Among the three experimental groups, only the 2-hr restimulated anergic Th1 cells contained p-JNK as expected (Fig. 5b). Interestingly, more than half of the p-JNK in the anergic restimulated Th1 cells was found to be associated with p21Cip1. The interaction between p21Cip1 and p-c-jun was also examined. Similar to p-JNK, only the 2-hr restimulated anergic Th1 cells contained p-c-jun. Almost all of the p-c-jun in the anergic group appeared to be associated with p21Cip1. Hence, unlike cdk and PCNA, certain members of the MAPK pathway, especially in their phosphorylated forms, appeared to bind p21Cip1 in anergic Th1 cells.