Enhanced Phosphorylation and Altered Localization Lead to Impairment of p27kip Activity in CML Progenitor Cells Despite Enhanced Protein Translation and Expression.

Abstract The cyclin-dependent kinase inhibitor p27kip is a key regulator of hematopoietic progenitor proliferation and pool size. The activity of p27 can be regulated by modulation of its expression and localization in different cellular compartments. The levels of p27 protein expression are reported to be reduced in BCR-ABL expressing cell lines. In contrast p27 levels have been reported to be elevated in BCR/ABL expressing CML progenitor cells. However, CML progenitors paradoxically demonstrate enhanced proliferation despite having elevated levels of p27. Therefore the regulation of p27 protein expression and activity in primary CML progenitor cells is not well understood and requires further investigation. We have therefore performed a careful assessment of p27 expression, phosphorylation and localization in CD34+ cells from CML patients as well as in cord blood CD34+ cells transduced with retroviral vectors to ectopically express the BCR-ABL gene. We observed that p27 protein levels were markedly increased in both primary CML CD34+ cells and BCR/ABL transduced cells. CML CD34+ cells and BCR/ABL transduced CD34+ cells also demonstrated increased phosphorylation of p27 on T157 (an Akt-dependent phosphorylation site) and T187 (a cdk2-dependent site) on western blotting as well as increased tyrosine phosphorylation of p27 in immunoprecipitation studies. Immunofluorescence microscopy analysis of cells labeled with anti-p27 antibodies demonstrated primarily nuclear localization of p27 in normal CD34+ cells, but markedly reduced nuclear and increased cytoplasmic localization of p27 in CML CD34+ cells and BCR/ABL transduced CD34+ cells. Cdk2 activity was maintained, cdk4 activity was increased and phosphorylated RB levels were increased in BCR-ABL expressing hematopoietic cells, indicating CDKI activity was reduced in these cells despite increased total p27 levels. Consistent with this BCR-ABL expressing CD34+ cells demonstrated enhanced cell cycling compared to controls. Mutation of the Y177 residue in BCR-ABL (BCR-ABL-Y177F), which abrogates Grb2 binding and reduces Ras and Akt activation by BCR-ABL, reversed BCR-ABL induced abnormalities in p27 expression, phosphorylation and localization. Treatment of BCR-ABL transduced CD34+ cells with the PI-3K inhibitor LY294002 resulted in reduced phosphorylation of p27 on T157 but not T187, and restored nuclear localization of p27, further indicating an important role for AKT signaling in abnormal cytoplasmic localization of p27. Quantitative reverse transcription PCR analysis indicated that p27 mRNA levels were similar in BCR/ABL expressing and control CD34+ cells, suggesting that p27 expression is regulated mainly at the posttranscriptional level. Metabolic labeling of cells with S35-methionine showed that p27 translation was increased in BCR/ABL expressing cells, explaining the observed increase in total p27 protein levels. We conclude that p27 CDKI activity is reduced in BCR-ABL expressing progenitors as a result of altered p27 phosphorylation at important regulatory sites and a shift in cellular localization of p27 from the nucleus to the cytoplasm. The enhanced level of total p27 in primary CML progenitors reflects enhanced protein translation, which may be a compensatory response to reduced p27 CDKI activity. These results provide important insights into p27 deregulation in CML progenitors and its potential role in the expansion of the hematopoietic progenitor pool size in CML.