(A) FLT3-ITD phosphorylates p27 on Y88

(A) FLT3-ITD phosphorylates p27 on Y88. in 5 out of 9 subjects, but, surprisingly, was increased in 4 patients. This indicated that other tyrosine kinases such as Src family kinases might contribute to p27 tyrosine 88 phosphorylation in FLT3-ITD positive AML cells. In fact, incubation with the Src family kinase inhibitor dasatinib could decrease p27 WAY 170523 tyrosine 88 phosphorylation in these patient samples, WAY 170523 indicating that p27 phosphorylated on tyrosine 88 may be a therapeutic marker for the treatment of AML patients with tyrosine kinase inhibitors. Introduction Cell proliferation and cell cycle progression are tightly regulated by the sequential activation and inactivation of specific cyclin-dependent kinases (CDKs).2 Binding of the CDK inhibitor p27Kip1 (p27) can regulate CDK activity and thereby control cell cycle progression from G0/G1 phase to S phase. p27 regulates not only CDK activity, but also transcription and cell motility.2,3 p27 levels are elevated in non-proliferating cells and decline when cells progress towards S phase.4 Whereas p27 mRNA levels are frequently not altered during the cell cycle, protein levels of p27 can fluctuate dramatically.2,4 The rapid elimination of p27 at the G1/S transition is triggered through ubiquitin-dependent proteasomal degradation by the SCFSkp2 WAY 170523 E3 ligase complex.5 Cyclin-dependent kinase inactivation by p27 involves the insertion of a 310-helix of the inhibitor into the catalytic cleft of Rabbit polyclonal to CIDEB the kinase, thereby blocking access of ATP.6 Interestingly, phosphorylation of p27 on residue tyrosine 88 (pY88) leads to the ejection of the inhibitory 310-helix from the catalytic cleft, permitting access of ATP7 and partial activation of p27-bound CDK complexes.7C11 The partially active cyclin-CDK2 can now phosphorylate substrates, including the bound p27 on T187.7 T187-phosphorylation is a prerequisite for p27 ubiquitination by SCFSkp2, initiating its proteasomal degradation.5 This mechanism directly couples mitogen-induced activation of tyrosine kinases to cell cycle control, but can also be used during oncogenic transformation of cancer cells.12 The non-receptor tyrosine kinases JAK2, Abl, BCR-Abl, Lyn, Yes, Src, and Brk can phosphorylate p27 on Y88 and likely employ this mechanism to inactivate p27 and to promote cell proliferation.7,8,11,13 The Fms-like tyrosine kinase 3 (FLT3) is a member of the WAY 170523 class III subfamily of receptor tyrosine kinases and is activated by FLT3 ligand (FL).14 FLT3 is expressed in early hematopoietic progenitor cells in the bone marrow.14 High FLT3 levels have been detected in acute myeloid leukemia (AML),15,16 where activating FLT3 mutations can be found in approximately 30% of the patients.14,17 In fact, the most common mutation in AML is the internal tandem duplication (ITD) in the juxtamembrane domain name of FLT3 with a 20C27% occurrence. FLT3-ITD serves as a prognostic marker since it positively correlates with higher blast counts, increased relapse rate, and worse overall survival.17C19 Several activating point mutations in the tyrosine kinase domain (TKD) have also been identified.14 Acute myeloid leukemia cells show increased proliferation and survival, as well as impaired hematopoietic differentiation.14 FLT3-ITD or FLT3 activation confers proliferative and survival advantages to cells14,20 by activating Src family tyrosine kinases (SFKs), the PI3K/Akt-, mitogen-activated protein kinase (MAPK) pathways, and, in the case of FLT3-ITD, also Stat5.20 Identifying the downstream targets of FLT3 and FLT3-ITD is essential to understanding the mechanisms through which they promote leukemia development. In the present study, we identified p27 as a novel direct substrate of FLT3 and FLT3-ITD. FLT3 inhibitor treatment efficiently reduced pY88-p27 in FLT3-ITD expressing cell lines and increased p27 protein levels. Analysis of cells from AML patients demonstrates for the first time that p27 is usually phosphorylated on Y88 in primary patient material. This uncovers a novel pathway with which FLT3 can promote hyperproliferation of AML cells. Methods Cell lines and primary cells Cells were incubated at 37C with 5% CO2 in DMEM (293T, U2OS) or RPMI (MV4;11, U937, Ba/F3, 32D) medium including 10% FCS. Primary blast cells were obtained from bone marrow aspirates or peripheral blood of AML patients. WAY 170523 Written informed consent was obtained from all individuals in accordance with the Declaration of Helsinki. The use of human material was approved by the ethics committees of the Medical University of Innsbruck (AN2014-0362 344/4.22.