BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF THE ONCOSUPPRESSOR GENE PROTEIN TYROSINE PHOSPHATASE GAMMA (PTPRG) AND ITS ROLE IN CHRONIC MYELOGENOUS LEUKEMIA

Aims: 1) to characterize the functional role of PTPRG in the pathogenesis of CML through the reconstruction of the molecular basis of its interference with BCR/ABL signaling and 2) to study the response of CML cells to tyrosine kinase inhibitors (TKIs) and cell permeable PTPRG intracellular domain (ICD) by infrared (IR) microspectroscopy (microFTIR). Background: Chronic Myelogenous Leukemia (CML) is a myeloproliferative disorder of the bone marrow that originates from the t(9;22)(q34;q11) balanced reciprocal translocation. The molecular consequence is the generation of the BCR/ABL oncogene that encodes for the chimeric BCR/ABL protein with a constitutively abnormal tyrosine kinase activity, leading to leukemia. For this reason, our attention has been focused on naturally occurring negative regulators of tyrosine kinase signaling: the protein tyrosine phosphatases (PTPs). PTP receptor type gamma (PTPRG) is a member of the receptor-like PTPs. The results of previous studies carried out by our research group had already indicated that PTPRG is down regulated in CML and that it can interfere with BCR/ABL signaling, thus suggesting a possible role for PTPRG in the pathogenesis of CML. Methods: Protein Purification, Pull-down assay, in vitro phosphatase activity assays, Cell viability assay, confocal microscopy, Flow cytometry, Immunoprecipitation, and Infrared spectroscopy. Results: we have applied protein transduction technology based on TAT Trojan nanovector technology, by cloning and expressing TAT- fused with the recombinant, enzymatically active intracellular domain (rICD) and the enzymatically inactive rD1028A ICD mutant domain of PTPRG protein in BL-21 E.Coli. We purified the recombinant rICDs-TAT proteins by FPLC and tested their biological activity. In cell lines high protein transduction rate was achieved. We then evaluated the inhibitory effects of these two purified rICDs-TAT proteins alone and in combination with the tyrosine kinase inhibitor Imatinib on cell proliferation and cell apoptosis in K562 leukemic cell line. As compared to untreated controls, a 30-40% reduction of cell proliferation was observed in samples treated with the active rICD-TAT at 1-2 μM. Notably, no detectable effect was observed with same concentrations of the enzymatically inactive form rD1028A ICD-TAT. Furthermore, the results of pull-down and co-immune precipitation assays performed with both purified enzymatically active rICD-TAT and inactive rD1028A ICD-TAT forms demonstrated that ICD of PTPRG interacts with BCR/ABL, CRKL, LYN, and RHOA. Finally we tested the ability of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR), a label-free analytical technique, in identifying protein phosphorylation and cell apoptosis signatures as markers of TKIs and/or of rICDs-TAT responses in leukemic cells. Conclusion: To unravel the signaling events associated to the oncosuppressive role hypothesized for PTPRG in CML we used a membrane-permeable nanovector technology. The enzymatically active form of intracellular domain of PTPRG was transduced into the cell with high efficiency. We demonstrated that LYN Kinase might form a tripartite complex with PTPRG, which is able to bind to and dephosphorylate BCR/ABL through binding sites localized in the intracellular domain. These results suggest that PTPRG could be a potential target for diagnostic and therapeutic applications in CML. Finally we successfully applied micro-FTIR as an innovative approach capable to identify molecular signatures of both apoptosis and protein dephosphorylation in individual cells within minimally processed, unstained samples.