Characterisation of inositol polyphosphate 4-phosphatase type II (INPP4B) enzyme activity, regulation, and expression in prostate cancer
2017-01-31T00:16:32Z (GMT) by
The phosphoinositide 3-kinase (PI3K)/Akt signalling pathway promotes cellular growth, proliferation and survival. PI3K/Akt signalling is amplified in many human cancers due to mutations in PIK3CA (PI3K catalytic subunit p110α) or loss of tumour suppressors such as PTEN. PI3K activation leads to the transient appearance of PtdIns(3,4,5)P3 and PtdIns(3,4)P2, signalling phospholipids at the plasma membrane that are required for Akt activation. PtdIns(3,4)P2 appears at the plasma membrane later and for longer periods than PtdIns(3,4,5)P3, although the reasons for this dynamic remain unclear. Inositol polyphosphate 4-phosphatase type II (INPP4B) dephosphorylates PtdIns(3,4)P2 to form PtdIns(3)P and opposes PI3K/Akt signalling, however the kinetics of INPP4B activity towards PtdIns(3,4)P2 are poorly understood. This thesis examines the enzyme kinetics and regulation of INPP4B activity. Purified human recombinant INPP4B specifically dephosphorylated PtdIns(3,4)P2, and followed allosteric kinetics (h = 2.7 ± 0.9). Other allosteric phosphoinositide phosphatases are activated by their product, however PtdIns(3)P did not activate INPP4B. Instead, INPP4B kinetic activity towards PtdIns(3,4)P2 (Vmax = 1.81 μmol/min/mg, K0.5 = 26.7 μM) was non-competitively inhibited by the upstream phosphoinositide PtdIns(3,4,5)P3 (Vmax = 1.27 μmol/min/mg, K0.5 = 24.4 μM). PtdIns(3,4,5)P3 bound the N-terminal C2 domain as well as full length INPP4B. The C2 domain competed for PtdIns(3,4,5)P3 binding and relieved PtdIns(3,4,5)P3-induced inhibition of INPP4B PtdIns(3,4)P2 4-phosphatase activity. Mutation of aspartate 116 to alanine within the C2 domain rendered INPP4B 4-phosphatase activity insensitive to inhibition by PtdIns(3,4,5)P3, revealing the requirement of this region for PtdIns(3,4,5)P3-induced inhibition. Truncated INPP4B lacking the N-terminal C2 domain decreased Akt phosphorylation and in turn increased cell death compared to wild type INPP4B. INPP4B is a negative regulator of PI3K/Akt signalling, and initial reports from transcriptomic and tissue microarray studies show loss of INPP4B in prostate cancer, but its expression in benign prostate and carcinoma is not well characterised. Investigation of INPP4B expression in the prostate by immunohistochemical staining of 97 radical prostatectomy sections revealed INPP4B is present in luminal epithelium within benign ducts and regions of carcinoma. In most cases INPP4B staining also identified clusters of 10-30 cells located subluminally within benign ducts. The cytokeratin profile, androgen receptor status and proliferative index suggested these INPP4B-positive cell clusters represent a progenitor cell type. Within prostate carcinoma, INPP4B expression was reduced ~ 2.8-fold compared to benign epithelium, however INPP4B expression in carcinoma did not significantly correlate with Gleason score. Taken together, the results in this thesis confirm the emerging putative tumour suppressor role of INPP4B in the prostate and show INPP4B phosphatase activity is negatively regulated by PtdIns(3,4,5)P3, which may prolong PtdIns(3,4,5)P3-PtdIns(3,4)P2 signalling and Akt activation. This is the first observation of phosphoinositide-mediated allosteric inhibition among phosphoinositide phosphatases, and may improve the current understanding of the phosphoinositide regulation dynamics following PI3K activation.