Characterization of Tumor-associated Chk2 Mutations*

Xianglin WuDagger , Shelley R. WebsterDagger , and Junjie ChenDagger §

From the Dagger  Guggenheim 1342, Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905

Received for publication, October 25, 2000



    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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The integrity of the DNA damage response pathway is essential for prevention of neoplastic transformation. Several proteins involved in this pathway including p53, BRCA1, and ATM are frequently mutated in human cancer. Checkpoint kinase 2 (Chk2) is a DNA damage-activated protein kinase that lies downstream of ATM in this pathway. Recently, heterozygous germline mutations in Chk2 have been identified in a subset of patients with Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype, suggesting that Chk2 is a tumor suppressor gene. In this study, we have reported the biochemical characterization of the four tumor-associated Chk2 mutants. Two of the reported Chk2 mutations identified in Li-Fraumeni syndrome result in loss of Chk2 kinase activity. Whereas one mutation within the Chk2 forkhead homology-associated (FHA) domain, R145W, retains some basal kinase activity, this mutant cannot be phosphorylated at an ATM-dependent phosphorylation site (Thr-68) and cannot be activated following gamma radiation. Wild-type Chk2 exists mainly in a protein complex of Mr ~200,000 whereas the R145W mutant forms a larger, presumably inactive complex in the cell. The other FHA domain mutant, I157T, behaves as wild-type Chk2 in all the assays used here. Because the FHA domain is involved in protein-protein interactions, this mutation may affect associations of Chk2 with other proteins. Additionally, we have shown that Chk2 can also be inactivated by down-regulation of its expression in cancer cells. Thus, Chk2 may be inactivated by multiple mechanisms in the cell.



    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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The maintenance of genomic integrity following DNA damage depends on the coordination of DNA repair and the control of cell cycle progression. Chk21/hcds1, a mammalian homolog of the Saccharomyces cerevisiae rad53 and Schizosaccharomyces pombe cds1 genes, plays a critical role in DNA damage signaling pathways (1-5). Downstream of ATM in response to gamma radiation (1, 4, 6), Chk2 directly phosphorylates and regulates the functions of p53 and BRCA1 (7-9, 11). Moreover, heterozygous germline mutations in Chk2 have been identified in a subset of patients with Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype (12). These studies strongly suggest that Chk2 is a tumor suppressor gene similar to p53.

Several mutations of Chk2 were identified in patients with Li-Fraumeni syndrome and in sporadic colon cancer. Although it has been speculated that these Chk2 mutants are defective in their tumor suppressor functions (12), this possibility has not been addressed directly. Here, we report the biochemical characterization of the four reported Chk2 mutations.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Constructs-- Dr. Jann Sarkaria kindly provided plasmid for the expression of HA-tagged Chk2 in mammalian cells (13). Site-directed mutagenesis (Promega) was performed to introduce mutations into the Chk2 coding sequence. For expressing wild-type or mutant Chk2 as glutathione S-transferase (GST) fusion proteins in insect cells, wild-type or mutant Chk2 coding sequences were cloned into the pDONR201 vector (Life Technologies, Inc.). Gateway cloning technology (Life Technologies, Inc.) was used to subclone these coding sequences into pDEST20 vector, a vector for baculovirus expression of GST fusion proteins. Recombinant baculoviruses encoding GST-fused wild-type and mutants of Chk2 were generated using Bac-to-Bac baculovirus system (Life Technologies, Inc.).

Cell Lines and Culture Conditions-- All cell lines were obtained from American Tissue Culture Collection and cultivated in RPMI 1640 (Biofluids) supplemented with 10% fetal bovine serum. To establish cell lines stably expressing HA-tagged wild-type or mutant Chk2, HCT116 cells were transfected with plasmids encoding the indicated HA-tagged sequences. G418 resistant clones were isolated and analyzed by Western blotting using either anti-HA antibody (Babco) or anti-Chk2 antibody. Clones that express HA-tagged Chk2 at levels similar to that of endogenous Chk2 were used in this study. Where indicated, cells were exposed to gamma radiation from a 137Cs source at a dose of 6.4 gray/min. Following irradiation, cells were returned to the incubator and harvested 1 h later.

Sf9 insect cells were cultivated in Grace's insect media supplemented with 10% fetal bovine serum. For protein expression, Sf9 cells were infected with baculoviruses encoding GST-fused wild-type or mutant Chk2. Cells were collected and lysed 48 h after viral infection. Wild-type or mutant Chk2 was purified using glutathione affinity chromatography.

Immunoprecipitation, Immunoblotting, and Kinase Assays-- Preparation of cell lysates, immunoprecipitation, and immunoblotting were performed as described previously (14). Antibodies against Chk2 were raised against GST fusion proteins containing full-length Chk2 (mAB no.7) or the C terminus of Chk2 (residues 193-543, anti-Chk2B). Anti-Chk2 Thr-68 phosphospecific antibodies were provided by Dr. Bin-Bing Zhou. Chk2 kinase assays were performed as described previously (13).

Size Fractionation of Native Chk2 Complexes-- HCT116 and derivative cells were harvested and lysed in NETN buffer (150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.05% Nonidet P-40). Whole cell extracts were loaded onto a Superdex 200 HR 10/30 (Amersham Pharmacia Biotech) column equilibrated with NETN and run in the same buffer with a flow rate of 0.5 ml/min. For each run, a sample of 500 µl was injected, and 500-µl fractions were collected. For column equilibration, low and high molecular weight gel filtration calibration kits (Amersham Pharmacia Biotech) were used, and the column was run under identical conditions.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Frameshift Mutations at the C Terminus of Chk2 Lead to the Loss of Chk2 Kinase Activity-- Because Chk2 is a DNA damage-activated protein kinase that participates in the phosphorylation of several substrates including Cdc25C, p53, and BRCA1, we first examined the kinase activity of Chk2 mutants. Using site-directed mutagenesis, four Chk2 mutants were generated (Fig. 1) that had been previously reported (12). Wild-type and mutant GST·Chk2 proteins were expressed in insect cells and purified using glutathione-Sepharose beads, and kinase activities were assessed using GST·Cdc25C (residues 200-256) as a substrate. As shown in Fig. 2, one FHA domain mutant (I157T) exhibited wild-type activity whereas the other FHA domain mutant (R145W) showed reduced catalytic activity, and the two frameshift mutants lacked kinase activity.



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Fig. 1.   Schematic diagram of Chk2 and its mutants. S/TQ-rich, FHA, and kinase domains are indicated, and corresponding Chk2 residues are labeled. Black boxes indicate unrelated protein sequences caused by frameshift mutations.



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Fig. 2.   In vitro kinase activities of wild-type or mutant Chk2. Wild-type or mutant GST·Chk2 proteins were expressed and purified from insect cells. Upper, Coomassie Blue-stained gel indicating amounts of GST·Chk2 proteins used. Middle, autoradiograph showing the incorporation of 32P into the substrate GST·Cdc25C by input kinases from the upper panel. Lower, Coomassie blue-stained gel (the same gel as shown in the middle panel) indicating equal levels of substrate in each kinase reaction.

The R145W Mutant of Chk2 Is Not Phosphorylated or Activated Following Gamma Radiation-- Chk2 is activated following DNA damage (1). Although Chk2 FHA domain mutants (R145W and I157T) retain some kinase activity (Fig. 2), they may not be activated by DNA damage. To explore this possibility, we have established HCT116 derivative cell lines that stably express comparable levels of HA epitope-tagged wild-type or mutant Chk2 (Fig. 3a). The expression levels of HA-tagged Chk2 in these cells are similar to that of endogenous Chk2 (Fig. 3a). Like wild-type Chk2, the I157T Chk2 mutant was activated following gamma radiation, as demonstrated by its ability to autophosphorylate and to phosphorylate Cdc25C (Fig. 3b). However, the R145W Chk2 mutant was not activated following DNA damage (Fig. 3b).



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Fig. 3.   R145W mutant of Chk2 is not activated following gamma radiation. a, HCT116 derivative cell lines that stably express HA-tagged wild-type or mutant Chk2. Whole cell extracts were prepared from indicated cell lines, and immunoblots were probed with anti-Chk2 mAb or with anti-HA mAb HA11. b, R145W mutant is not activated following gamma radiation. Extracts were prepared from indicated cell lines before and 1 h after gamma radiation. HA-tagged wild-type or mutant Chk2 were immunoprecipitated using anti-HA antibodies, and kinase reactions were performed using GST·Cdc25C as substrate. c, Thr-68 is not phosphorylated in R145W mutant following gamma radiation. Extracts were prepared as described above. Immunoprecipitates with anti-Chk2 or anti-HA antibodies were immunoblotted with anti-phospho-Thr-68 antibodies and anti-Chk2 or anti-HA antibodies.

The increase of Chk2 kinase activity is coincident with its phosphorylation following gamma radiation (1). Both activation and phosphorylation of Chk2 depend on intact ATM kinase, strongly suggesting that ATM may phosphorylate Chk2 and activate its kinase activity following DNA damage (1). In vivo, Thr-68 of Chk2 is phosphorylated in an ATM-dependent manner following gamma radiation (6). Thus, we examined whether the Chk2 mutants were phosphorylated at Thr-68 following gamma radiation. Cells expressing either wild-type or mutant HA-tagged Chk2 were irradiated. Wild-type and mutant Chk2 were immunoprecipitated using either anti-Chk2 or anti-HA antibody. Phosphorylation of Chk2 at Thr-68 was detected by Western blotting using anti-Thr-68 phosphospecific antibody. In agreement with its activation following gamma radiation, ectopically expressed HA·Chk2 was phosphorylated at Thr-68 (Fig. 3c). Moreover, consistent with the above findings that HA·Chk2 containing the I157T mutation but not the R145W mutation can be activated by DNA damage (Fig. 3b), the I157T mutant but not the R145W mutant was phosphorylated at Thr-68 (Fig. 3c). In addition, expression of the R145W mutant did not affect the phosphorylation of endogenous Chk2 in these cells (Fig. 3c), suggesting that the mutant protein may not exhibit dominant-negative activity.

FHA Domain Mutants of Chk2 Localize Normally in Nuclei-- Chk2 normally localizes to the nuclei. We examined the subcellular localization of wild-type and mutant HA-tagged Chk2 stably expressed in HCT116 derivative cell lines. Immunostaining using anti-HA antibodies revealed that wild-type and the R145W and I157T mutants of Chk2 all localized normally to nuclei (Fig. 4). Furthermore, the localization of wild-type or mutant Chk2 did not change following gamma radiation (data not shown).



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Fig. 4.   Nuclear localization of exogenously expressed wild-type or mutant Chk2. HCT116 and its derivative cells were permeabilized, fixed, and stained with DAPI (left panels) and anti-HA mAb HA11 (right panels) to reveal the localization of HA·Chk2 proteins.

Chk2 Exists As a Protein Complex of Apparent Mr ~200,000 in the Cell-- Because Chk2 R145W and I157T mutants are missense mutations within the FHA domain (involved in protein-protein interaction, Refs. 10 and 15), we speculated that these Chk2 mutations might affect the association of Chk2 with other proteins. To investigate this possibility, we first used size-exclusion chromatography to determine the native size of Chk2 in HeLa and HCT116 cells. As shown in Fig. 5, endogenous Chk2 eluted from a Superdex 200 column mainly as a protein complex with an apparent Mr ~200,000, although a smaller portion of Chk2 eluted as a protein complex of Mr ~600,000. Because only a very small amount of Chk2 eluted where monomeric Chk2 is predicted to elute, we conclude that the majority of Chk2 exists in complex(es) with other proteins. Alternatively, Chk2 may exist as multimers.



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Fig. 5.   Native sizes of wild-type or mutant Chk2 in the cell. Extracts prepared from HeLa, HCT116, and HCT116 derivative cells were analyzed by size-exclusion chromatography. Immunoblots were probed with either anti-Chk2 or anti-HA antibody.

We then examined the elution profiles of the stably expressed Chk2 mutants in these HCT116 derivative cell lines. As a control, HA-tagged wild-type Chk2 eluted with an apparent molecular weight identical to endogenous Chk2 (Fig. 5). The Chk2 I157T mutant eluted in fractions similar to that of wild-type Chk2. In contrast, the Chk2 R145W mutant eluted as a much larger protein complex (Fig. 5), suggesting that this mutant may affect the association of Chk2 with other proteins.

Chk2 Expression Is Down-regulated in HCT15 Cells-- The R145W mutation of Chk2 was identified in a colon cancer cell line HCT15 (12). Based on sequence analysis, HCT15 carries one mutant allele (R145W) and one wild-type allele of Chk2 (12). It is speculated that the Chk2 mutation in HCT15 may contribute to tumorigenesis either as a result of reduced gene dosage or through a dominant-negative effect (12).

Expression of this R145W mutant in HCT116 cells did not affect the phosphorylation (see Fig. 3c) or the activation (data not shown) of endogenous Chk2, arguing that this mutant may not behave as a dominant-negative mutant. We also observed that, when the same amount of DNA encoding either wild-type or R145W mutant of Chk2 were used in transient transfection experiments, the expression level of R145W mutant was only 10-20% that of wild-type Chk2 (data not shown). These results suggest that the R145W mutation of Chk2 may affect the stability of this mutant Chk2 protein. Thus, it is possible that this mutant contributes to tumorigenesis because of haploid insufficiency.

To examine whether mutation in HCT15 cells results in reduced levels of Chk2 protein, we compared Chk2 protein levels in HCT15 cells with that in K562, HCT116, or HeLa cells. If the presumed wild-type allele of Chk2 in HCT15 cells were expressed normally, we would expect to observe at most a 2-fold reduction in Chk2 protein levels. However, as shown in Fig. 6a, Chk2 protein was barely detectable in the extract of HCT15 cells, whereas Chk2 protein was readily detected in extracts of K562, HeLa, and HCT116 cells. We estimate that the steady-state level of Chk2 in HCT15 cells is only 5-10% of that in other cell lines. Additionally, Chk2 kinase activity was undetectable in HCT15 cells (Fig. 6b and data not shown). These data strongly suggest that the Chk2 expression from the second Chk2 allele is greatly reduced, if not absent, in HCT15 cells. The mechanism for the down-regulation of Chk2 expression is unknown. Because HCT15 carries inactivating mutations in both hMSH6 alleles, it is also possible that genomic instability in these cells may lead to the mutation in the second Chk2 allele. Such mutation, either at the promoter or in the coding sequence of Chk2, could result in reduced levels of Chk2 protein.



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Fig. 6.   Down-regulation of Chk2 in HCT15 cells. a, Chk2 expression is down-regulated in HCT15 cells. Extracts prepared from HeLa, HCT15, HCT116, and K562 cells were subjected to Western blotting using two independent anti-Chk2 antibodies (anti-Chk2 no. 7 and anti-Chk2B). b, HCT15 has no detectable Chk2 kinase activity before or after gamma radiation. Extracts were prepared from indicated cell lines before and 1 h after gamma radiation. Kinase reactions were performed using anti-Chk2B immunoprecipitates.



    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have characterized the four reported Chk2 mutations. Two mutations identified in Li-Fraumeni patients that lead to frameshifts at the C-terminal kinase domain result in loss of kinase activity. In contrast, the R145W Chk2 mutant still retains some kinase activity in vitro but is incapable of being activated following gamma radiation in vivo, most likely because it is not phosphorylated at Thr-68 by ATM kinase. This mutant also behaves differently from wild-type Chk2 in size-fractionation experiments, suggesting that this mutation may also affect associations of Chk2 with other cellular proteins.

The I157T Chk2 mutant behaves similar to wild-type Chk2 in all the assays used in this study. The I157T mutation may be a rare polymorphism that does not affect Chk2 functions. Alternatively, this mutation may affect associations of Chk2 with certain cellular proteins in a way that does not result in apparent changes in the sizes of Chk2-containing protein complexes as revealed by size-exclusion chromatography. Identification of Chk2-associated proteins will provide us with some insights in this regard.

It is interesting that the two mutations with the Chk2 FHA domain behave differently in our assays. The FHA domain of Chk2 may have multiple functions. FHA domain is involved in protein-phosphoprotein interaction (10, 15). These interactions may be essential for transmitting DNA damage signals to Chk2. Any alternation of association of Chk2 with upstream signaling proteins could lead to the failure of Chk2 activation following DNA damage, as in the case of R145W mutant. In addition, the FHA domain may also mediate transmitting signals from Chk2 to downstream effectors such as p53, BRCA1, and Cdc25C. It is reasonable to speculate that the I157T mutant may be defective in this aspect of Chk2 function. One could examine whether any Chk2-dependent events, such as stabilization of p53 following gamma radiation (8), are defective in cells that carry only the I157T mutant of Chk2. Such experiments will provide insights into the mechanism of this Chk2 mutant.


    ACKNOWLEDGEMENTS

We thank Drs. Scott Kaufmann, Larry Karnitz, and Jann Sarkaria for stimulating conversations and Dr. Bin-Bing Zhou for antibodies against phosphorylated Thr-68 of Chk2.


    FOOTNOTES

* This work was supported by the Mayo Foundation, Mayo Cancer Center, and Division of Oncology Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Guggenheim 1342, Division of Oncology Research, Mayo Clinic, 200 First St., S. W., Rochester, MN 55905. Tel.: 507-538-1545; Fax: 507-284-3906; E-mail: Chen.junjie@mayo.edu.

Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M009727200


    ABBREVIATIONS

The abbreviations used are: Chk2, checkpoint kinase 2; ATM, ataxia telangiectasia-mutated protein; LFS, Li-Fraumeni syndrome; FHA, forkhead homology-associated; HA, hemagglutinin; GST, glutathione S-transferase.


    REFERENCES
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1. Matsuoka, S., Huang, M., and Elledge, S. J. (1998) Science 282, 1893-1897[Abstract/Free Full Text]
2. Tominaga, K., Morisaki, H., Kaneko, Y., Fujimoto, A., Tanaka, T., Ohtsubo, M., Hirai, M., Okayama, H., Ikeda, K., and Nakanishi, M. (1999) J. Biol. Chem. 274, 31463-31467[Abstract/Free Full Text]
3. Chaturvedi, P., Eng, W. K., Zhu, Y., Mattern, M. R., Mishra, R., Hurle, M. R., Zhang, X., Annan, R. S., Lu, Q., Faucette, L. F., Scott, G. F., Li, X., Carr, S. A., Johnson, R. K., Winkler, J. D., and Zhou, B. B. (1999) Oncogene 18, 4047-4054[CrossRef][Medline] [Order article via Infotrieve]
4. Brown, A. L., Lee, C. H., Schwarz, J. K., Mitiku, N., Piwnica-Worms, H., and Chung, J. H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3745-3750[Abstract/Free Full Text]
5. Blasina, A., de Weyer, I. V., Laus, M. C., Luyten, W. H., Parker, A. E., and McGowan, C. H. (1999) Curr. Biol. 9, 1-10[CrossRef][Medline] [Order article via Infotrieve]
6. Zhou, B. B., Chaturvedi, P., Spring, K., Scott, S. P., Johanson, R. A., Mishra, R., Mattern, M. R., Winkler, J. D., and Khanna, K. K. (2000) J. Biol. Chem. 275, 10342-10348[Abstract/Free Full Text]
7. Chehab, N. H., Malikzay, A., Appel, M., and Halazonetis, T. D. (2000) Genes Dev. 14, 278-288[Abstract/Free Full Text]
8. Hirao, A., Kong, Y. Y., Matsuoka, S., Wakeham, A., Ruland, J., Yoshida, H., Liu, D., Elledge, S. J., and Mak, T. W. (2000) Science 287, 1824-1827[Abstract/Free Full Text]
9. Lee, J. S., Collins, K. M., Brown, A. L., Lee, C. H., and Chung, J. H. (2000) Nature 404, 201-204[CrossRef][Medline] [Order article via Infotrieve]
10. Durocher, D., Henckel, J., Fersht, A. R., and Jackson, S. P. (1999) Mol. Cell 4, 387-394[Medline] [Order article via Infotrieve]
11. Shieh, S. Y., Ahn, J., Tamai, K., Taya, Y., and Prives, C. (2000) Genes Dev. 14, 289-300[Abstract/Free Full Text]
12. Bell, D. W., Varley, J. M., Szydlo, T. E., Kang, D. H., Wahrer, D. C., Shannon, K. E., Lubratovich, M., Verselis, S. J., Isselbacher, K. J., Fraumeni, J. F., Birch, J. M., Li, F. P., Garber, J. E., and Haber, D. A. (1999) Science 286, 2528-2531[Abstract/Free Full Text]
13. Busby, E. C., Leistritz, D. F., Abraham, R. T., Karnitz, L. M., and Sarkaria, J. N. (2000) Cancer Res. 60, 2108-2112[Abstract/Free Full Text]
14. Chen, J., Silver, D. P., Walpita, D., Cantor, S. B., Gazdar, A. F., Tomlinson, G., Couch, F. J., Weber, B. L., Ashley, T., Livingston, D. M., and Scully, R. (1998) Mol. Cell 2, 317-328[Medline] [Order article via Infotrieve]
15. Sun, Z., Hsiao, J., Fay, D. S., and Stern, D. F. (1998) Science 281, 272-274[Abstract/Free Full Text]


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