Regulation of PDZ-Binding Kinase (PBK) in Hematopoietic Tumors.

Abstract

We have focused on a protein kinase called PBK or TOPK, believed to be an intermediate in the Ras - MAP Kinase signaling cascade which is implicated in malignant cell growth,. PBK was originally identified in our laboratory as a differentially expressed gene in Burkitt's lymphoma cells as compared to hyperplastic tonsillar B cells. PBK protein expression was detected in 9 out of 12 primary AML samples, 3 out of 3 ALL samples and one sample each of plasmacytoma and blastic-type Mantle cell lymphoma that were analyzed. We demonstrated inhibition of p38 MAPK phosphorylation utilizing an inducible construct involving dominant negative PBK mutant in 293 cells. Homology with the MAP Kinase Kinase, MKK3, protein- protein interactions with c-Raf and its observed effect on p38 MAP Kinase suggest that PBK possibly functions as a MAP Kinase Kinase which utilizes p38 MAP Kinase as a downstream phosphorylation target.Presently, the regulation and function of PBK have been examined with a goal to elucidate its role in the growth of hematopoietic neoplasms. We have found that under conditions of stymied proliferation of HL60, promyelocytic leukemia cells either by genotoxic drugs e.g doxorubicin or by induction of differentiation with TPA along macrophage pathway, PBK is strongly downregulated while housekeeping proteins like actins remain constant. Accumulation of dephosphorylated ( Tyr15) form of Cdc2 Kinase under treatment with doxorubicin indicated cell cycle arrest at the G2/M boundary. Electrophoretic mobility shift assay (EMSA) has demonstrated the binding of ATF/CREB family of transcription factors upstream of PBK transcription start site. A variety of cell lines derived from multiple tissue origin when analyzed, demonstrated codistribution of PBK and transcription factor CREB indicating that CREB factor may be involved in PBK gene expression. To further analyze the regulatory mechanisms involving PBK gene expression, a 2.5 kb putative promoter for the PBK gene has been amplified utilizing human genomic DNA and cloned next to click beetle red-emitting luciferase coding unit. Preliminary data indicate that 0.6kb promoter containing the CRE element retains significant activity compared to 2.5kb promoter. Assay of luciferase activity utilizing different subfragments of the promoter is underway. Efforts have been made to examine PBK expression following ectopic expression of dominant negative CREB mutants. To that end, stable clones have been isolated from 293, human embryonic kidney cells under neomycin selection. Further experiments to monitor PBK expression by western immunoblot analysis will clarify the role of CREB factors in controlling PBK expression. In order to obtain knock down expression of PBK using inducible shRNA, a 51bp long double stranded oligomer derived from the PBK coding region has been cloned next to the Tet-responsive promoter with an object to study possible function of PBK in tumor cells which express the PBK protein.