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Casein Kinase-2 Mediates Cell Survival Through Phosphorylation and Degradation of Inositol Hexakisphosphate Kinase-2

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Casein Kinase-2 Mediates Cell Survival Through Phosphorylation and Degradation of Inositol Hexakisphosphate Kinase-2 Casein kinase-2 mediates cell survival through phosphorylation and degradation of inositol hexakisphosphate kinase-2 Anutosh Chakrabortya,1, J. Kent Werner, Jr.a,1, Michael A. Koldobskiya, Asif K. Mustafaa, Krishna R. Juluria, Joseph Pietropaolia, Adele M. Snowmana, and Solomon H. Snydera,b,c,2 aThe Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205; bDepartment of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and cDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Contributed by Solomon H. Snyder, December 23, 2010 (sent for review November 12, 2010) The inositol pyrophosphate, diphosphoinositol pentakisphosphate, leading to augmentation of the cell death genetic program appar- regulates p53 and protein kinase Akt signaling, and its aberrant ently by default. increase in cells has been implicated in apoptosis and insulin resis- Heretofore posttranslational modifications of IP6K2 have not tance. Inositol hexakisphosphate kinase-2 (IP6K2), one of the major been characterized. We chose to examine phosphorylation of inositol pyrophosphate synthesizing enzymes, mediates p53-linked IP6K2 by casein kinase-2 (CK2) for the following reasons. IP6K2 apoptotic cell death. Casein kinase-2 (CK2) promotes cell survival possesses a proline (P), glutamic acid (E), serine (S), and threo- and is upregulated in tumors. We show that CK2 mediated cell nine (T) (PEST) sequence whose phosphorylation typically facil- survival involves IP6K2 destabilization. CK2 physiologically phos- itates protein degradation (16). The PEST sequence in IP6K2 phorylates IP6K2 at amino acid residues S347 and S356 contained contains a consensus for CK2 phosphorylation. CK2 is a serine/ within a PESTsequence, a consensus site for ubiquitination. HCT116 threonine protein kinase with prominent prosurvival functions cells depleted of IP6K2 are resistant to cell death elicited by CK2 which are mediated in part by phosphorylation of IκBα (17). CK2 inhibitors. CK2 phosphorylation at the degradation motif of IP6K2 is markedly augmented in a wide range of cancers (18) and BIOCHEMISTRY enhances its ubiquitination and subsequent degradation. IP6K2 appears to enhance malignant transformation by creating a more mutants at the CK2 sites that are resistant to CK2 phosphorylation hospitable environment for tumor growth (19). CK2 also stimu- are metabolically stable. lates angiogenesis (20) as well as multidrug resistance (21). Pandolfi and coworkers demonstrated that the tumor suppressor nositol phosphates are messenger molecules mediating a variety promyelocytic leukemia protein (PML) is a physiologic substrate of cell functions, the best known being inositol 1,4,5-trispho- of CK2 (22). CK2 phosphorylation of PML facilitates its degra- I dation through the ubiquitin pathway leading to enhanced cell sphate (IP3) which releases intracellular calcium (1). Inositol survival and increased tumor growth. pyrophosphates contain a pyrophosphate bond with energetic In the present study we demonstrate that CK2 physiologically qualities comparable to ATP (2). The most studied example, phosphorylates IP6K2 at two identified sites. Cell death elicited diphosphoinositol pentakisphosphate (IP7), displays a 5′-dipho- by CK2 inhibitors is prevented by deletion of IP6K2. Inhibition sphate (3, 4). Another isomer of IP7 has been reported with a of CK2 enhances IP6K2 stability, whereas CK2 phosphorylation pyrophosphate at position 1 or 3 (5, 6). The predominant mam- ′ of IP6K2 destabilizes the enzyme. Thus, regulation of IP6K2 malian form contains the 5 -diphosphate and will hereafter be appears to mediate the prosurvival actions of CK2. designated IP7. IP7 is generated in mammals by a family of inositol hexaki- Results sphosphate kinases (IP6Ks) with evidently disparate functions In HEK293 cells labeled with 32P-orthophosphate IP6K2 is (7, 8). IP6K1 generates a pool of IP7 which physiologically inac- prominently radiolabeled (Fig. 1A) with the label abolished by tivates the protein kinase Akt leading to marked upregulation of treatment with λ phosphatase (Fig. 1B). This phosphorylation glycogen synthase kinase beta (GSK3β) and inhibition of the predominantly involves CK2, as treatment with the selective mTOR protein translation pathway (9). IP6K1 mediates insulin CK2 inhibitor TBB (4, 5, 6, 7-tetrabromobenzotriazole) virtually resistance and weight gain, as IP6K1 deleted mice manifest insu- abolishes the labeling of IP6K2 (Fig. 1C). Definitive evidence lin sensitivity and diminished body weight (9). Functions for that CK2 phosphorylates IP6K2 is apparent utilizing λ phospha- IP6K3 have not been elucidated. An enzyme called VIP1 gener- tase treated immunoprecipitated Myc-IP6K2 in which added ates the 1∕3 isomer and is functionally most prominent in yeast CK2 directly phosphorylates IP6K2 (Fig. 1D). Specificity for this where it regulates phosphate disposition (10). action is evident in its inhibition by treatment with TBB (Fig. 1E). Inositol hexakisphosphate kinase-2 (IP6K2), first identified by Previously, we developed preparations of the HCT116 colon Lindner and coworkers (11) as a proapoptotic gene, sensitizes cancer cell line with targeted deletion of IP6K2 and showed that cells to apoptotic stimuli (12). Mice with targeted deletion of the cells are resistant to the apoptotic actions of agents acting IP6K2 display relative resistance to ionizing radiation and an through p53 (15). The IP6K2 deleted cells are markedly resistant increased incidence of aerodigestive tract carcinoma (13). Cell to the apoptotic effects of the CK2 inhibitor TBB in terms of cell survival associated with heat shock protein 90 (HSP90) involves survival monitored by MTT (Fig. 2A). Cleavage of PARP,another IP6K2 (14). Thus, HSP90 physiologically binds IP6K2 to inhibit its catalytic activity. Drugs and mutations that block this binding Author contributions: A.C., J.K.W.J., and S.H.S. designed research; A.C., J.K.W.J., M.A.K., activate IP6K2 leading to cell death (14). A.K.M., and J.P. performed research; M.A.K., K.R.J., and A.M.S. contributed new IP6K2 killing is selective for p53 mediated cell death (15). reagents/analytic tools; A.C., J.K.W.J., and S.H.S. analyzed data; and A.C., J.K.W.J., and Thus, HCT116 colon cancer cells with deletion of the IP6K2 gene S.H.S. wrote the paper. manifest decreased sensitivity to agents that kill via p53 but not to The authors declare no conflict of interest. agents whose apoptotic actions involve different mechanisms. 1A.C. and J.K.W. J. contributed equally to this work. IP6K2 prevents activation of p53-associated cell arrest thereby 2To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1019381108 PNAS Early Edition ∣ 1of5 Downloaded by guest on September 24, 2021 A B - + λ Phosphatase A B WT ∆IP6K2 - + Myc-IP6K2 + + Myc-IP6K2 0 48 72 0 48 72 TBB (50 µM) Autoradiograph Cleaved PARP Autoradiograph GAPDH HCT116 WB: Myc ab. WB: Myc ab. Orthophosphate: Orthophosphate: HEK293 HEK293 C D C D WT ∆IP6K2 - + CK2 - - + TBB 0 40 60 80 0 40 60 80 Resorufin (µM) - + + Myc-IP6K2 + + λ Phosphatase + + Myc-IP6K2 Cleaved PARP Autoradiograph GAPDH Autoradiograph HCT116 WB: Myc ab. Orthophosphate: HEK293 WB: Myc ab. E in vitro Fig. 2. IP6K2 mediates cell death associated with CK2 inhibition A. IP6K2Δ - + TBB HCT116 cells display resistance to inhibitory effect of TBB on cell survival + + CK2 compared to WT HCT cells. (***p < 0.001). B. TBB mediated PARP cleavage + + Myc-IP6K2 is absent in IP6K2Δ HCT116 cells. C. TBCA’s inhibitory effect on cell survival is greatly reduced in IP6K2Δ HCT116 cells. (***p < 0.001). D. Resorufin in- Autoradiograph duced apoptosis, as indicated by PARP cleavage, is reduced in IP6K2Δ HCT116 cells. WB: Myc ab. To examine whether IP6K2 degradation involves the ubiquitin in vitro pathway, we monitored its ubiquitination (Fig. 3G). Treatment with both TBB and the more potent derivative TBCA diminishes Fig. 1. CK2 phosphorylates IP6K2 A. Myc-IP6K2 phosphorylation in HEK293 IP6K2 ubiquitination consistent with the notion that CK2 desta- cells detected by orthophosphate labeling. B. Specificity of IP6K2 phos- bilizes IP6K2 by increasing its ubiquitin conjugation. phorylation is confirmed by λ phosphatase treatment of the radiolabeled IP6K2 protein after orthophosphate labeling. C. TBB treatment (50 μM, 3 h) To further substantiate the stabilization of IP6K2 protein by abolishes Myc-IP6K2 phosphorylation in HEK293 cells. D. CK2 phosphory- CK2 inhibition, we employed U2OS cells in which IP6K2 is stably lation of immunoprecipitated and λ phosphatase treated Myc-IP6K2 in vitro. expressed. In these cells treatment with TBB greatly increases E. TBB (5 μM) inhibits CK2 phosphorylation of Myc-IP6K2 in vitro. levels of IP6K2 (Fig. 3H). To demonstrate ubiquitination directly, we treated cells with the proteasome inhibitor MG132 which index of cell death, is greatly diminished in IP6K2 mutants causes a pronounced laddering of overexpressed IP6K2 (Fig. 3I). (Fig. 2B). We also employed TBCA, a much more potent and To assess whether IP6K2 protein stabilized by TBB represents selective derivative of TBB (23) (Fig. 2C). TBCA substantially the functional enzyme, we monitored its catalytic activity (Fig. 3J). reduces HCT116 cell survival at concentrations as little as 5 μM. TBB increases IP7 generation presumably by IP6K2 in a time- We evaluated resorufin, a structurally distinct
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