Regulation of p27Kip1 by tyrosine phosphorylation

The Cdk inhibitor p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Binding of p27 usually inhibits Cdk kinase activity, however it has also been observed that p27 or related proteins are required for activation of cyclin D / Cdk complexes. An activating function of Cdk inhibitor proteins still remains a controversial issue and is mechanistically not understood. We have recently discovered that a tyrosine residue of p27, Y88, can be phosphorylated in vitro and is phosphorylated in vivo. One consequence of Y88 phosphorylation is a reduced ability of p27 to inhibit Cdk kinase activity. This observation leads to our working hypothesis that tyrosine phosphorylation may convert the p27 Cdk inhibitor into an activator of cyclin D/Cdk4,6 kinase complexes. In the proposed project, we aim to investigate novel biochemical properties of Y88-phosphorylated p27 in vitro and in vivo and to determine the role of Y-88 phosphorylation on cell cycle progression and cell cycle reentry. We will set up a biochemical assay system to determine whether Y88 phosphorylated p27 can act as a Cdk activator by assembling active kinase complexes, whereas the unphosphorylated protein acts as an inhibitor. Since inhibitors of the p27 family have been reported to be essential activators for cyclin D-associated kinases in murine fibroblasts, we aim to investigate if the mutant Y88F protein that cannot be phosphorylated can still function as activator. For this purpose, we will generate an allele of p27 that replaces tyrosine-88 with phenylalanine. We will use the allele to generate a homozygous knock- in mouse. To determine the role of Y88 phosphorylation for cell cycle re-entry from quiescence and during the unperturbed cell cycle, we will investigate cell cycle kinetics of cells isolated from p27-Y88F knock-in animals. Embryonic fibroblasts and hematopoietic stem cells of homozygous knock-in animals and wild-type mice will be used to determine the role of Y88 phosphorylation in cell cycle reentry from quiescence and during G1 progression. We will compare cyclin D/Cdk4,6 assembly with that of wt and p27-/- knock out cells. As the related inhibitor p21 may compensate for the assembly function of p27, we will also perform this analysis in p21-/- animals. In a parallel part of the proposed project, we aim to identify tyrosine kinases that directly phosphorylate p27 upon mitogen stimulation and analyze the deregulation of Y88 phosphorylation in human malignancies.

The Cdk inhibitor p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Binding of p27 usually inhibits Cdk kinase activity, however it has also been observed that p27 or related proteins are required for activation of cyclin D / Cdk complexes. An activating function of Cdk inhibitor proteins still remains a controversial issue and is mechanistically not understood. We have recently discovered that a tyrosine residue of p27, Y88, can be phosphorylated in vitro and is phosphorylated in vivo. One consequence of Y88 phosphorylation is a reduced ability of p27 to inhibit Cdk kinase activity. This observation leads to our working hypothesis that tyrosine phosphorylation may convert the p27 Cdk inhibitor into an activator of cyclin D/Cdk4,6 kinase complexes. In the proposed project, we aim to investigate novel biochemical properties of Y88- phosphorylated p27 in vitro and in vivo and to determine the role of Y-88 phosphorylation on cell cycle progression and cell cycle reentry. We will set up a biochemical assay system to determine whether Y88 phosphorylated p27 can act as a Cdk activator by assembling active kinase complexes, whereas the unphosphorylated protein acts as an inhibitor. Since inhibitors of the p27 family have been reported to be essential activators for cyclin D-associated kinases in murine fibroblasts, we aim to investigate if the mutant Y88F protein that cannot be phosphorylated can still function as activator. For this purpose, we will generate an allele of p27 that replaces tyrosine-88 with phenylalanine. We will use the allele to generate a homozygous knock-in mouse. To determine the role of Y88 phosphorylation for cell cycle re-entry from quiescence and during the unperturbed cell cycle, we will investigate cell cycle kinetics of cells isolated from p27-Y88F knock-in animals. Embryonic fibroblasts and hematopoietic stem cells of homozygous knock-in animals and wild-type mice will be used to determine the role of Y88 phosphorylation in cell cycle reentry from quiescence and during G1 progression. We will compare cyclin D/Cdk4,6 assembly with that of wt and p27-/- knock out cells. As the related inhibitor p21 may compensate for the assembly function of p27, we will also perform this analysis in p21-/- animals. In a parallel part of the proposed project, we aim to identify tyrosine kinases that directly phosphorylate p27 upon mitogen stimulation and analyze the deregulation of Y88 phosphorylation in human malignancies.