The cyclin-dependent kinase inhibitor p27Kip1 plays a critical role in the progression of T-cells through the G1 phase of the cell cycle. The murine p27Kip1 gene consist of three exons and spans more than 5.6 kb. The 5' flanking region lacks a TATA box but contains 2 Sp1, CRE, Myb and NF?B potential binding sites. A positive regulatory element is located between nt -326 and 615. transfection experiments suggests that IL-2 regulates the expression of p27Kip1 and increased levels of the inhibitor accumulate in T-cells activated in the absence of IL-2. Cyclin-dependent kinase 2 is the primary target of the inhibitory effects of p27Kip1 which contains a binding site for the enzyme within amino acid residues 52-85. The inhibitory capacity of the kinase activity of the holoenzyme is also localized within the same region which does not bind the cdk2 activating partner cyclin E. Point mutation at amino acid residue 64 essentially abolishes the kinase inhibitory function of p27Kip1 as well as its capacit to inhibit colony formation in transfected HeLa cells. During these studies a 40kDa protein was found to consistently bind to p27Kip1 and was identified as an isoform of cdk2 resulting from alternative splicing. A significant proportion of quiescent T-cells derived from the spleens of old mice when polyclonally activated failed to traverse the G1 phase of the cells cycle. In activated T-cells from old animals the catalytic activi of cyclin-dependent kinase 2 was decreased during mid G1 and extended to the G1/S transition. Kinase activity was determined by estimating the phosphorylation of retinoblastoma protein 1 and 2 which in the underphosphorylated state are known to prevent G1 progression by sequestering essential transcription factors. As compared to T-cells derived from young mice, there was no detectable decrease in the cellular levels of cdk2 or cyclin E during the G1 phase of the cell cycle of polyclonally activated T-cells from old mice. Lysates from early to mid G1 T-cells of old mice contained high levels of the cyclin dependent kinase inhibitor p27Kip1 and in late G1 increased levels of the inhibitor were associated with cdk2 suggesting that the decreased levels of catalytic activity may be due in part to this specific inhibitor.
