Rewiring of the Protein Kinase C Beta 2 (PKC βII) and Bcr/Abl Signal Transduction Pathways

Abstract 1673

Chronic myeloid leukemia (CML) accounts for 20% of adult leukemias, and is characterized by the presence of the bcr-abl fusion gene. This gene leads to the production of a constitutively active Abl tyrosine kinase, which promiscuously phosphorylates/activates a variety of intracellular signal transduction pathways. The presence of constitutively active Abl is essential for CML blast survival even in advanced disease, and underlies the success that the Bcr/Abl kinase inhibitor imatinib has had. However, resistance to imatinib occurs in a significant number of accelerated phase or blast crisis patients and is a significant clinical obstacle. Instead of trying to inhibit Bcr/Abl signaling, we propose a previously unexplored approach to ‘rewire’ kinase signaling pathways to activate a “suicide” prodrug that would not normally be activated by Bcr/Abl. We have previously shown that direct activation of PKCβII by Phorbol 12-myristate 13-acetate (PMA) drives dendritic cell differentiation in both normal and leukemic progenitors, as well as induces apoptosis and growth arrest. PKCβII is a member of the PKC family of serine/threonine kinases and is kept in an inactive state in the cytosol by interactions between its pseudosubstrate and kinase domains; upon activation, the pseudosubstrate domain releases the kinase domain, and PKCβII translocates to the plasma membrane. Substitution of an alanine at position 25 in the pseudosubstrate domain for a phosphomimetic glutamic acid leads to the constitutive activation of PKCβII. Similarly, we hypothesized that substituting the alanine at position 25 for a phosphorylatable tyrosine (A25Y) along with the corresponding Bcr/Abl kinase target motif (Ala-X-X-Ile-Tyr-X-X-Phe/Pro) into the pseudosubstrate domain of PKCβII, would allow the Bcr/Abl tyrosine kinase to activate the PKCβII signaling pathway. Bcr/Abl mediated activation of PKCβII would then lead to the induction of apoptosis, growth inhibition, and differentiation. Using confocal microscopy, we show that following transfection WT-PKCβII is cytoplasmically located in media alone and addition of PMA leads to translocation to the plasma membrane, indicating activation in both Bcr/Abl⁺ K562 cells, and Bcr/Abl⁻ KG1a cells. However when A25Y-PKCβII constructs are transfected in, A25Y-PKCβII is found at the plasma membrane in K562, but not in KG1a cells in media alone. These observations were then quantified using ImageStream technology, which allows for simultaneous acquisition of both flow cytometric data, and high resolution fluorescent images. Using this technology, we show that A25Y-PKCβII is activated in media alone in K562 cells, and only upon addition of PMA in KG1a cells. Additionally, when Bcr/Abl was stably transfected in KG1a cells, A25Y-PKCβII was able to translocate to the plasma membrane in media alone, indicating activation by Bcr/Abl. Upon activation and translocation to the plasma membrane, PKCβII is rapidly degraded; accordingly, we show that expression of WT PKCβII decreases only by 20% over 72 hours post transfection, whereas expression of A25Y-PKCβII results in an average decrease of 90% over the same 72 hour time course. To test whether activation of A25Y-PKCβII leads to apoptosis and growth arrest, Bcr/Abl⁺ K562, and Bcr/Abl⁻ KG1a cells were transfected with either WT and A25Y-PKCβII and measured for apoptosis with AnnexinV using Flow Cytometry. We found that A25Y-PKCβII induced a maximum of a 4-fold increase of apoptosis when compared to WT PKCβII in K562 cells; however there was no increase observed in KG1a cells. This work demonstrates that rewiring PKCβII to be inducible by Bcr/Abl is feasible, and that activation of PKCβII by Bcr/Abl induces characteristic translocation to the plasma membrane, and induction of apoptosis. Future work will address whether induction of DC differentiation is maintained in Bcr/Abl activated PKCβII cells, as well as the molecular kinetics of this activation.