Site-specific phosphorylation by intracellular kinases determines the apoptotic activity of IGFBP-3 in prostate cancer

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4393 The IGF axis is known to play an important role in the epidemiology of many tumors, including prostate, lung and breast cancers. IGFBP-3 promotes apoptosis in cancer cells by both IGF-dependent and -independent mechanisms. We have previously shown that IGFBP-3 is rapidly internalized and localized to the nucleus, where its interactions with the nuclear receptor RXRα are important in apoptosis induction. Proteomic and bioinformatic analysis of IGFBP-3 reveals multiple consensus phosphorylation sites for kinases including CK2, PKA, PKC and cdc2. We have previously reported that phosphorylation of IGFBP-3 (S156) by DNA-PK enhances its nuclear accumulation, and is essential for its ability to interact with RXR and induce apoptosis in cultured prostate cancer cells. Indeed, IGFBP-3-S156A is completely unable to induce apoptosis in 22RV1 prostate cancer cells. Using specific chemical inhibitors, we investigated the contribution of other protein kinases to the regulation of IGFBP-3-induced apoptosis. Preventing the activation of CK2 enhanced the apoptotic potential of IGFBP-3. Using web-based proteomics software, we mapped three potential CK2 phosphorylation sites in IGFBP-3: S167, S175 and S177. These sites were mutated to Ala, and the resulting constructs were transfected in to LAPC4 and 22RV1 prostate cancer cells. WtIGFBP-3, IGFBP-3-S175A and IGFBP-3-S177A induced apoptosis to a comparable extent; however, IGFBP-3-S167A was far more potently apoptosis-inducing. Interestingly, IGFBP-3-S167A was able to induce apoptosis even in the absence of active DNA-PK, and IGFBP-3-S156A was able to induce apoptosis when CK2 activity was inhibited chemically or by using siRNA. Together, these data reveal two key regulatory phosphorylation sites in the central region of IGFBP-3. Phosphorylation of S156 by DNA-PK promotes apoptosis, whilst phosphorylation of S167 by CK2 limits the ability of IGFBP-3 to induce apoptosis in prostate cancer. Interestingly, our data suggest that the anti-apoptotic phosphorylation event induced by CK2 is dominant. These studies reveal multi-site phosphorylation of IGFBP-3 that both positively and negatively regulate its apoptotic potential. Understanding such intrinsic regulation of IGFBP-3 action may enhance the development of potential cancer therapies.