p27-Y88 phosphorylation by BCR-Abl, JAK2-V617F and FLT3-ITD in tumor development

Phosphorylation of tyrosine residue 88 (Y88) of the cyclin-dependent kinase (CDK) inhibitor p27 impairs its ability to inactivate CDK/cyclin complexes (Chu et al., 2007; Chu et al., 2008; Grimmler et al., 2007; James et al., 2008; Kardinal et al., 2006). While the Y88-phosphorylated p27 remains bound to the cyclin/CDK complex, its inhibitory 310-helix becomes ejected from the ATP-binding domain of the CDK subunit, permitting ATP access to the CDK and substrate phosphorylation. One substrate of CDK2 is the bound Y88 phosphorylated p27 protein itself. Subsequent phosphorylation of p27 on T187 within the bound cyclin/CDK complex can permit the ubiquitin ligase SCF-Skp2 to ubiquitinate p27 and to initiate its proteasomal degradation (Grimmler et al., 2007). In addition to BCR-Abl, Lyn and cSrc (Chu et al., 2007; Grimmler et al., 2007), the non-receptor kinase JAK2 can phosphorylate Y88 of p27. This may link JAK2-mediated cytokine activated signal transduction to p27 regulation. The mutation JAK2-V617F creates a constitutive active protein which strongly phosphorylates p27 (Jäkel et al., 2011). Expression of BCR-Abl or JAK2-V617F leads to increased p27-Y88 phosphorylation, reduced p27 level and increased cell proliferation in cultured cells. A non-phosphorylatable mutant p27-Y88F was more efficient in inhibiting proliferation of BCR-Abl expressing K562 cells or JAK2-V617F expressing HEL cells as the wt p27 protein (Grimmler et al., 2007; Jäkel et al., 2011). Thus, the Y88-dependent inactivation of p27 may contribute to hyperproliferation induced by oncogenic tyrosine kinases. Recently, we observed that FLT3 or FLT3-ITD bind p27 and expression of these tyrosine kinases can induce p27-Y88 phosphorylation. FLT3 is the first receptor tyrosine kinase identified that induces p27 tyrosine phosphorylation. Using mouse tumor model systems, we now aim to explore the role of p27 tyrosine phosphorylation in tumor development. We have generated knock-in mice where either tyrosine-88 or all three tyrosines of p27 were exchanged to phenylalanine(s). We now wish to investigate if the inability to phosphorylate p27 alters and delays tumor development in mice. For this purpose, we will use mouse tumor models where oncogenesis occurs in response to expression of oncogenic tyrosine kinases JAK2-V617F, BCR-Abl or FLT3-ITD and compare tumor development in wt and knock-in mice. As a second part of the proposed project we aim to investigate molecular mechanisms of the cytokine-receptor - JAK2 - p27 pathway. Activation of cytokine receptors can induce cell proliferation but also promote cell differentiation and cell cycle exit. The physiological state of a cell or modifications of cytokine receoptors or p27 might determine if activation of the receptor results in p27 Y88 phosphorylation. We aim to investigate if and what mechanisms may exist that regulate the p27 / JAK2 interaction and its physiological consequence. Finally, we aim to elucidate molecular details of the p27-FLT3 interaction.

Cell proliferation is essential to maintain human body and organ homeostasis, but excessive proliferation contributes to diseases like cancer. The machinery controlling cell proliferation is composed of protein kinases from the cyclin-dependent kinase (CDK) family. The CDK inhibitor p27Kip1 restricts cell proliferation in response to antiproliferative signalling. Mitogens, on the other hand, can lead to a decline of p27 and induce cell proliferation. Phosphorylation of p27 at tyrosine residue 88 can impair CDK inhibition and initiate subsequent p27 degradation. Within this project, we observed that the receptor tyrosine kinase FLT3 can bind and directly phosphorylate p27 at tyrosine 88. FLT3 is frequently activated in human acute myeloid leukaemia (AML). Ectopic expression of constitutive active FLT3-ITD leads to increased p27 Y88 phosphorylation. Inhibition of FLT3 or FLT3-ITD resulted strongly reduced p27 tyrosine 88 phosphorylation, increased p27 levels and cell cycle arrest. We could also monitor the presence of tyrosine 88 phosphorylated p27 in primary patient samples. Inhibition of FLT3 kinase activity with AC220 significantly reduced p27 tyrosine 88 phosphorylation in cells isolated from FLT3 wild type expressing acute myeloid leukaemia (AML) patients. In FLT3-ITD positive AML patients, p27 tyrosine 88 phosphorylation was reduced in 5 out of 9 subjects, but, surprisingly, increased in 4 of these patients. This indicated that other tyrosine kinases such as Src family kinases might contribute to p27 tyrosine 88 phosphorylation in these FLT3-ITD positive AML cells. In fact, incubation with the Src family kinase inhibitor dasatinib could decrease p27 tyrosine 88 phosphorylation in these patient samples, indicating that p27 phosphorylated on tyrosine 88 may be a therapeutic marker for the treatment of AML patients with tyrosine kinase inhibitors.In order to investigate the role of p27-Y88 phosphorylation in oncogenesis, we generated mouse strains expressing a p27 protein with a single amino acid exchange Y88F. It was our hypothesis that the non-phosphorylatable p27 might attenuate oncogenesis because it cant be inactivated by tyrosine phosphorylation. Surprisingly, Y88F knock-in animals were characterised by reduced p27-Y88F expression in all organs investigated. Opposite to our initial hypothesis, tyrosine kinase-induced oncogenesis was accelerated rather than delayed in these animals, potentially due to the strong overall reduction of p27 expression. We are currently investigating the molecular mechanisms responsible for this rather unexpected phenotype.