Figure 4.

Pim inhibitors 4a and 16a cause translocation of p27^Kip1 to the nucleus and reduce Cdk2 activity. A, 4a and 16a reduce the Pim-1-mediated phosphorylation of p27^Kip1. The ability of Pim-1 to phosphorylate p27^Kip1 in the presence of DMSO (control), 4a (5 μmol/L), or 16a (5 μmol/L) was assayed in the presence of [γ³²P]ATP as described in Materials and Methods. Autoradiography of a SDS-PAGE gel is shown. B, 4a or 16a treatment causes the translocation of p27^Kip1 to the nucleus. K562, U937, and MV4;11 cells were treated with DMSO (control), 4a (5 μmol/L), or 16a (5 μmol/L) for 72 h in RPMI containing 10% FCS. Cytoplasmic (C) and nuclear (N) fractions were prepared as described in Materials and Methods, and the level of p27^Kip1 in the cytoplasmic and nuclear fraction was determined by SDS-PAGE followed by Western blotting. β-Tubulin and lamin B1 serve as cytoplasmic and nuclear markers, respectively. C, Cdk2 activity in K562 cells treated with DMSO, 4a (5 μmol/L), or 16a (5 μmol/L) for 72 h in RPMI containing 10% FCS. Cdk2 was immunoprecipitated from cell lysates and its activity was measured using histone H1 as a substrate in the presence [γ³²P]ATP as described in Materials and Methods. Scanning densitometry of the ³²P-labeled histone H1 band was used to quantitate the degree of inhibition of activity induced by 4a and 16a addition to cells. Representative of 3 experiments showing similar results.