Abstract
Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.
Highlights
The hematopoietic system can become dysfunctional via various distinct molecular pathologies
The protein tyrosine kinase oncoproteins (PTKs) associated with a number of different leukaemias and preleukaemias have been identified as critical in the molecular pathology of these diseases. They can result from point mutations, reciprocal chromosomal translocations and internal tandem duplication; in Anaplastic Large Cell Lymphoma (ALCL) the t(2:5) translocation leads to the formation of the NPM/ALK gene which encodes a lymphoma specific NPM/ALK protein with intrinsic and disregulated PTK activity [1]
Each oncogenically-transfected cell line was shown to be independent of Interleukin 3 (IL-3 is normally required for survival and proliferation of Ba/F3 cells) for growth and have the same growth rate as control (MSCV Ba/F3) cells cultured in IL-3 [14]
Summary
The hematopoietic system can become dysfunctional via various distinct molecular pathologies. The protein tyrosine kinase oncoproteins (PTKs) associated with a number of different leukaemias and preleukaemias have been identified as critical in the molecular pathology of these diseases. They can result from point mutations, reciprocal chromosomal translocations and internal tandem duplication; in Anaplastic Large Cell Lymphoma (ALCL) the t(2:5) translocation leads to the formation of the NPM/ALK gene which encodes a lymphoma specific NPM/ALK protein with intrinsic and disregulated PTK activity [1]. A point mutation in the Kit PTK (D816V) is associated with transformation in mast cell leukaemia [2] and in the myeloproliferative disorders the t(9;22) translocation found within Chronic Myeloid Leukaemia (CML) leads to the production of the BCR/ ABL oncogenic PTK [3]. A key question is whether these kinases share a common mode of action? Certainly there is some evidence for common activation of canonical pathways such as the Raf/Erk cellular signalling pathway [7]
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