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Home , Casein kinase 1, Casein kinase 2

-2 mediates cell survival through 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 and Akt signaling, and its aberrant ently by default. increase in cells has been implicated in 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 , 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 (P), glutamic acid (E), (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 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 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 . 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 (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 (Fig. 1B). This phosphorylation 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 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 CK2 inhibitor (24) dependent fashion with a tripling of catalytic activity at 16 h (Fig. 2D). In wild-type cells resorufin stimulates PARP cleavage following TBB addition. in a concentration dependent fashion. In IP6K2 deleted cells To elucidate the molecular mechanism whereby CK2 phos- PARP cleavage is reduced and does not manifest a concentra- phorylates and destabilizes IP6K2, we conducted mass spectro- tion-response dependence upon resorufin. metric analysis of GST-IP6K2 overexpressed in HEK293 cells. We wondered how IP6K2 mediates cell death associated with The enzyme is phosphorylated at 347 and 356 within the CK2 inhibition. The tumor suppressor protein promyelocytic PEST motif (Fig. 3A). leukemia (PML) is degraded through the ubiquitin pathway To characterize the sites of phosphorylation, we developed an following CK2 phosphorylation at its PEST motif, a consensus antibody that is selective for IP6K2 phosphorylated at S347 and sequence that signals degradation (22). IP6K2 possesses a PEST clearly recognizes CK2-phosphorylated IP6K2 purified from motif at amino acid residues 340–369 with potential CK2 phos- bacteria (Fig. 4A). This phosphorylation is abolished in immuno- phorylation sites (Fig. 3A). Treatment of HEK293 cells with the precipitated Myc-IP6K2 with mutations of both S347 and S356 protein synthesis inhibitor cycloheximide (CHX) leads to a rapid (IP6K2 SS/AA) (Fig. 4B). loss of IP6K2 protein with a half-life of about 2 h (Fig. 3B). We If phosphorylation of serines 347∕356 destabilizes IP6K2, then explored potential influences of CK2 phosphorylation upon the their mutations should slow turnover of the protein. Mutations stability of IP6K2 protein. If CK2 facilitates IP6K2 degradation, of S347 and S356 singly partially alter IP6K2 stability, monitored then one might expect increased IP6K2 levels when CK2 is inhib- in terms of degradation following cycloheximide treatment ited. Treatment with TBB does increase IP6K2 protein concen- (Fig. 4C); whereas the double mutant is stable (Fig. 4D). Wild- tration (Fig. 3C). In another approach to evaluating IP6K2 type IP6K2 displays a half-life of about 2 h, whereas very little stability, we utilized HEK293TRex cells in which IP6K2 is in- degradation of IP6K2-SS347/356AA is evident over an 8 h time duced by treatment with tetracycline (Fig. 3D). Withdrawal of course (Fig. 4E). tetracycline for 4 h depletes IP6K2 protein, while TBB treatment Resistance to degradation of IP6K2 is associated with dimin- prevents this depletion. Endogenous levels of IP6K2 in HEK293 ished ubiquitination. Thus, TBB treatment decreases the ubiqui- cells are markedly augmented by TBB treatment (Fig. 3E). In tin ladder of IP6K2 in wild-type HeLa cells (Fig. 4F). The mutant HCT116 cells TBB treatment also substantially enhances concen- IP6K2-SS347/356AA displays a further reduction of ubiquitina- trations of IP6K2 protein (Fig. 3F). tion in the presence or absence of TBB (Fig. 4F).

2of5 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1019381108 Chakraborty et al. Downloaded by guest on September 24, 2021 AB 0 2 4 6 CHX (h) Myc IP6K2

βTubulin

HEK293 340 RPEVVLDSDAEDLEDLSEESADESAGAYAY 369

C DETet withdrawn (4 h) 0 2 4 16 TBB (h) - - - + TBB (4 h) - + TBB Myc IP6K2 - + + + Tet (24 h) IP6K2 Myc IP6K2 βTubulin βTubulin βTubulin HEK293 HEK293 HEK293 F G H µ IgG IP6K2 ab. + + + Myc-IP6K2 - + TBB (50 M) + + + HA-Ub Myc-IP6K2 50 0 25 50 TBB (µM) Con TBB TBCA GAPDH IP6K2 U2OS HCT116 IP6K2 stable I J - - + MG132 HA - + + Tet (24 h)

Myc-IP6K2

HEK293T BIOCHEMISTRY Myc-IP6K2

β Tu b u l i n U2OS IP6K2 stable

Fig. 3. IP6K2 stability is enhanced by CK2 inhibition A. CK2 phosphorylation sites at the degradation specific PEST motif of IP6K2. B. Myc-IP6K2 protein displays a half-life of 2 h as measured by cycloheximide treatment of HEK293 cells. C. TBB (50 μM) stabilizes Myc-IP6K2 protein levels in a time-dependent fashion. D. TBB (50 μM) protects tetracycline removal induced IP6K2 protein degradation. E. TBB (50 μM, 6 h) stabilizes endogenous IP6K2 levels in HEK293 cells. F. Immunoprecipitation of endogenous IP6K2 from HCT116 cells reveals increased protein levels after TBB treatment. G. IP6K2 ubiquitination is significantly reduced after TBB (50 μM) or TBCA (10 μM) treatment, H. TBB stabilizes Myc-IP6K2 in U2OS cells. I. The MG132 treatment reveals multiple ubiquitinated Myc-IP6K2. J. TBB increases IP7 levels in U2OS cells stably expressing Myc-IP6K2. (***p < 0.001).

Discussion mechanisms and substrate specificity. Scansite analysis reveals In the present study we obtained evidence that IP6K2 mediates IP6K2 as a substrate for CK2 but not for CK1. Moreover, TBB, the antiapoptotic actions of CK2. Thus, the proapoptotic effects which inhibits CK2 much more potently than CK1, abolishes the of a number of CK2 inhibitors of differing chemical classes all physiologic phosphorylation of IP6K2. We also observe similar are greatly reduced with IP6K2 deletion. CK2 acts by phosphor- effects with the more specific CK2 inhibitor TBCA (23). Thus, ylating IP6K2 to decrease its stability (Fig. 4G). Under normal CK1 does not appear to exert any noticeable effect on IP6K2 circumstances IP6K2 turns over with a half-life of about 2 h. function. Enzyme stability is notably enhanced by inhibition of CK2. CK2 Lindner and coworkers (26) noted that IP6K2 can bind tumor phosphorylates IP6K2 at serines 347 and 356 whose mutation necrosis factor associated factor (TRAF2) in OVCAR-3 leads to marked increases in IP6K2 stability. ovarian carcinoma cells thereby influencing NFκβ via transform- The amino acid sequence in IP6K2 that is phosphorylated by ing β activated kinase. The binding site on IP6K2 CK2 is not present in IP6K1 or IP6K3. IP6K1 can be phosphory- includes its PEST sequence, and mutations of both serines 347 lated by CK2 in vitro (3). Mass spectrometric analysis of IP6K1 and 359, but not of either singly, impair binding with less cell overexpressed in HEK293 cells reveals multiple phosphorylation death. In our study serine 359 was not phosphorylated by CK2, sites, none of which represent CK2 consensus sites. Reactivity of making it difficult to compare our findings with Lindner’s group. IP6K3 to CK2 has not been reported. IP6K2 may be important for organ development. Very recently, The apoptotic actions of IP6K2 are dependent upon IP7 gen- Wente and coworkers (27) obtained evidence that IP6K2 influ- eration. In several studies, kinase-dead IP6K2 fails to enhance ences development of zebrafish and mammals by stimulating apoptosis (12, 14) and the proapoptotic actions of p53 (15). the hedgehog pathway. Thus, IP6K2 deletion disturbs overall Elegant studies of Pandolfi and coworkers (22) established an development and hedgehog signaling, which is increased by over- analogous pathway whereby CK2 phosphorylates and destabilizes expression of IP6K2. the tumor suppressor protein lost or altered in PML. PML Our findings may have clinical implications. CK2 is overex- mutants that resist CK2 phosphorylation display augmented pressed in a large number of tumors (18), and CK2 inhibitors tumor suppression. Moreover, in lung cancer derived cell lines have potential antitumor activity (18, 24, 28). Conceivably, the and primary tumors CK2 catalytic activity and PML protein levels beneficial effects of CK2 inhibitors derive from stabilization of are inversely correlated. IP6K2 to augment its apoptotic effects. Such drugs may also act CK1, like CK2, phosphorylates a wide range of substrates by preventing the destabilizing actions of CK2 upon PML (22). (25). However, the two enzymes differ substantially in catalytic IP6K2 actions can be facilitated by anticancer drugs such as

Chakraborty et al. PNAS Early Edition ∣ 3of5 Downloaded by guest on September 24, 2021 A B all enhance sensitivity of cells to lethal stimuli, cell survival is - + CK2 WT SS/AA IP6K2 uniquely augmented by deletion of IP6K2 but not the other two p-IP6K2 (S347) ab. p-IP6K2 (S347) ab. isoforms (12). IP6K1 does exert antitrophic effects by physiolo- His-IP6K2 Myc-IP6K2 gically inhibiting Akt (9). Our principal experiments on cell death employed HCT116 cells in which essentially all IP7 formation is in vitro HEK293 mediated by IP6K2 so that the other isoforms do not contribute CDto the apoptotic phenotype. IP6K2 levels are higher in brain than other mammalian organs WT S347A S356A IP6K2 WT SS/AA IP6K2 - + - + CHX (4 h) (7). Accordingly, activation of IP6K2 may play a role in neuronal - + - + - + CHX (4 h) death in stroke and neurodegenerative disease so that centrally Myc-IP6K2 Myc-IP6K2 active IP6K2 inhibitors might be beneficial. βTubulin βTubulin Materials and Methods HEK293 HEK293 Reagents. [32P]orthophosphates were from Perkin Elmer; CK2 and λ phospha- tase were from NEB, TBB and TBCA were from EMD Biosciences; all the other EFbiochemical reagents were from Sigma unless otherwise mentioned. Antibo- WT SS/AA IP6K2 0 1 2 4 6 8 CHX (h) dies: α-Myc from Roche; α-PARP from ; and IP6K2 polyclonal - + - + TBB (25 µM) IP6K2-WT antibody was developed in our lab. IP6K2-SS/AA GAPDH Experimental Procedures. Cell lines and culture conditions. HEK293, HEK293T, HEK293 Trex and HeLa cells were cultured in DMEM supplemented with 10% FBS. Tran- Myc-IP6K2 sient transfections were carried out using Polyfect (Qiagen). U2OS and HeLa HCT116 cell culture were done following standard procedures (15).

G Orthophosphate labeling of HEK293 cells. HEK293 cells were labeled for 4 h with [32P]orthophosphate 48 h post transfection of control or Myc-IP6K2 plasmids. Cells were lysed in the presence of a phosphatase inhibitor cocktail, and immunoprecipitated with α-Myc antibody for 2 h. Beads were washed 3x with wash buffer, boiled, and run on SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and phosphorylation was detected by autoradiography. Total protein was detected by immunoblotting with α-Myc antibody. To remove radiolabeled phosphates from immunoprecipitated Myc-IP6K2 Fig. 4. IP6K2 destabilization is associated with CK2 phosphorylation at ser- λphosphatase treatment of immunoprecipitated proteins was carried out ines 347 and 356 A. CK2 phosphorylation of bacterially purified His-tagged in the presence of 1X buffer and the enzyme at 37 °C for 1 h. Beads were α IP6K2 is detected by -pIP6K2 (S347) antibody. Total protein was detected by processed as described above. coommassie blue staining. B. Myc-IP6K2 S347/356AA double mutant is not recognized by the α-pIP6K2 (S347) antibody. WT and mutant IP6K2 were over- expressed in HEK293 cells and were immunoprecipitated using α-Myc anti- CK2 phosphorylation of IP6K2 in vitro. Immunoprecipitated Myc-IP6K2 after λ body. C. IP6K2 S347A and S356A single mutants display partial but not phosphatase treatment was used as a substrate for CK2 in the presence μ μ 32 γ total stability after cycloheximide treatment. D. IP6K2 S347/356AA double of 10 Units of the enzyme, 250 MMg-ATP,and1 Ci [ P] ATP at 37 °C mutant does not degrade after 4 h cycloheximide treatment. E. Substantial for 30 min. Proteins were run on SDS-PAGE and processed for phosphoryla- increase in half-life of IP6K2 S347/356AA double mutant. The half-life of the tion and total protein detection as described earlier. mutant is 8 h as compared to 2 h for WT IP6K2. F.TBB decreases ubiquitination To detect CK2 phosphorylation at S347 site by the phospho-specific of WT-IP6K2, whereas the mutant is resistant to ubiquitination and hence IP6K2 antibody, recombinant bacterially purified 6XHis-IP6K2 was used as unaffected by TBB treatment. G. Model showing CK2 phosphorylation a CK2 substrate. CK2 kinase reaction, SDS-PAGE, and Western transfer were mediated ubiquitination and degradation of IP6K2. carried out as described above. Phosphorylation of IP6K2 was detected using the antibody at 1∶1;000 dilutions. cisplatin and novobiocin that block its binding to HSP90, as this binding inhibits catalytic activity of IP6K2 (14). One might obtain CK2 inhibitor treatment for cell death induction. Varying concentrations of CK2 Δ synergistic antitumor activity by combining drugs that block inhibitors TBB, TBCA, or resorufin were incubated with WT and IP6K2 HCT116 cells for indicated time periods. Cell viability was assessed by MTT HSP90-IP6K2 binding together with CK2 inhibitors that stabilize assay (14, 15). Each experiment was repeated at least three times. IP6K2. In support of this notion, cisplatin induced cell death is To detect cleaved PARP protein as an indicator of apoptotic cell death (15), potentiated by the CK2 inhibitor TBB (24). cells were lysed after indicated treatments. Equal amount of proteins were CK2 promotes cell survival in part by activating the Akt and loaded on SDS-PAGE and were blotted with α-PARP antibody. α-GAPDH or α-β (29, 30). Inositol pyrophosphates inhibit were used as loading controls. Akt which activates GSK3β (9), an important component of the Wnt pathway. In this signaling cascade, IP7 competes with Cycloheximide treatment. Cycloheximide (20 μg∕mL) was added for indicated phosphatidylinositol (3,4,5)-trisphosphate for binding to the time periods to cells overexpressing IP6K2. Cells were lysed and equal Pleckstrin homology domain of Akt thereby reducing phosphor- amounts of proteins were loaded on SDS-PAGE. ylation and activation of Akt (9). As Akt physiologically inhibits GSK3β, IP7 signaling stimulates GSK3β. GSK3β phosphorylates Immunoprecipitation of endogenous IP6K2 from HCT116 cells. Endogenous and facilitates degradation of β-catenin, an essential component IP6K2 was immunoprecipitated from HCT116 cells after TBB treatment for indicated time periods. Immunoprecipitation was done using α-IP6K2 of the Wnt signaling pathway. Interestingly, CK2 phosphorylates antibody using standard procedure (15). both Akt and β-catenin to activate Wnt signaling (30, 31). Thus, CK2 may regulate Akt/GSK3β/β-catenin signaling directly by β Detection of intracellular inositol phosphates. U2OS cells overexpressing tetra- phosphorylating Akt and -catenin and indirectly by enhancing cycline inducible Myc-IP6K2 were used for the experiment. Cells were plated IP7 levels via IP6K2 stabilization. at 60% density and incubated with 100 μCi [3H]myoinositol for 3 d in Among the three IP6Ks, IP6K2 appears to influence cell death presence of 1 μg∕mL tetracycline. Cells were processed for inositol phosphate selectively. Although overexpression of IP6K1, IP6K2, and IP6K3 detection by HPLC (9).

4of5 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1019381108 Chakraborty et al. Downloaded by guest on September 24, 2021 Ubiquitination of IP6K2. Myc-IP6K2 and HA-ubiquitin were cooverexpressed in Identification of phosphorylation sites in IP6K2 by mass spectrometry. HEK293T cells. Forty-eight h posttransfection, cells were treated with DMSO, Phosphorylation sites at IP6K2 were identified by LC/MS/MS at the Taplin TBB (50 μM) or TBCA (10 μM) for 3 h. Cells were harvested, lysed, and equal Mass Spectrometry facility at Harvard Medical School. GST-IP6K2 was trans- amount of proteins were loaded on SDS-PAGE. Myc-IP6K2 was immunopre- fected in HEK293 cells. The protein was pulled down and run on SDS-PAGE. α cipitated and ubiquitination detected by -HA antibody. The coomassie stained band was cut and analyzed. To detect ubiquitinated IP6K2, tetracycline inducible U2OS cells expressing Myc-IP6K2 were treated with 20 μM MG132 for 3 h after 24 h tetracycline induction. IP6K2 was immunoprecipitated using a α-Myc antibody and was Statistical analysis. All results are presented as the mean and standard error run on SDS-PAGE. Ubiquitinated IP6K2 was detected with a α-Myc antibody. of at least three independent experiments. Statistical significance was calcu- lated by Student’s t-test using the “Sigmaplot software” (***p < 0.001, **p < 0.01,*p < 0.05). Generation of IP6K2 phospho-specific antibody. Phospho-specific antibodies against S347 and S356 sites were generated in rabbits by 21st Century Bio- chemicals using the phospho-peptides VLD[pS]DAEDLED and AEDLEDL[pS] ACKNOWLEDGMENTS. We thank Katherine Sixt for providing reagents and EESA respectively. The second peptide did not generate an acceptable Molee Chakraborty for technical help. This work was supported by Public antibody titer. Antibodies against S347 site showed robust responses and Health Service Grant DA-000266 and Research Scientist Award DA00074 were affinity purified. (to S.H.S.).

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