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The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

Dubey P, Hendrickson RC, Meredith SC, Siegel CT, Shabanowitz J, Skipper JC, Engelhard VH, Hunt DF, Schreiber H - J. Exp. Med. (1997)

Bottom Line: The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb.Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation.This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Chicago, Illinois 60637, USA.

ABSTRACT
The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

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Predicted primary amino acid sequence of wild-type murine  p68 RNA helicase. The eight regions of homology to other DEAD-box  proteins are outlined by the thin box. The IQ domain, found at the  COOH-terminal end of the protein is outlined by the thick box. Within  this IQ box, the eight–amino acid stretch from which the mutant peptide  is derived is underlined. In the 8101-RE tumor, the second serine was  changed to a phenylalanine in this eight–amino acid stretch.
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Figure 9: Predicted primary amino acid sequence of wild-type murine p68 RNA helicase. The eight regions of homology to other DEAD-box proteins are outlined by the thin box. The IQ domain, found at the COOH-terminal end of the protein is outlined by the thick box. Within this IQ box, the eight–amino acid stretch from which the mutant peptide is derived is underlined. In the 8101-RE tumor, the second serine was changed to a phenylalanine in this eight–amino acid stretch.

Mentions: The primary amino acid sequence of the murine p68 protein is shown in Fig. 9. The first eight boxed motifs show the domains of homology of p68 with other DEAD box proteins, which play a central role in cell growth in a wide variety of organisms. p68 has been shown to undergo dramatic changes in nuclear localization during telophase, when it translocates from the nucleoplasma to the nucleoli (49). In addition, a stretch of amino acids, called the IQ domain (50) is located within the 139 carboxy-terminal amino acids that extend beyond the region of homology with other DEAD box proteins, and which distinguishes p68 from these proteins (41). This domain, which is also found in molecules such as neurogranin and neuromodulin, is subject in vitro to calmodulin (CaM) binding and phosphorylation by protein kinase C (PKC) (50). Experimental evidence suggests that CaM and/or PKC may regulate at least some of the activity of p68 during the cell cycle, through this domain (50). The mutation changes one of the two serines in the IQ domain to a phenylalanine (thick box in Fig. 9), but we do not yet know whether the mutation of S to F affects the physiologic function or localization of the protein. In addition to being an RNA helicase, p68 is also a powerful inhibitor of DNA helicases (51). This activity is quite similar to that of the p53 tumor suppressor gene which also prevents DNA helicase activity (51). It has been suggested that the general role of p53 is to safeguard the integrity of the genome by monitoring and stopping replication when DNA is damaged (52), and it is possible that p68 may serve a similar function as a tumor suppressor gene.


The immunodominant antigen of an ultraviolet-induced regressor tumor is generated by a somatic point mutation in the DEAD box helicase p68.

Dubey P, Hendrickson RC, Meredith SC, Siegel CT, Shabanowitz J, Skipper JC, Engelhard VH, Hunt DF, Schreiber H - J. Exp. Med. (1997)

Predicted primary amino acid sequence of wild-type murine  p68 RNA helicase. The eight regions of homology to other DEAD-box  proteins are outlined by the thin box. The IQ domain, found at the  COOH-terminal end of the protein is outlined by the thick box. Within  this IQ box, the eight–amino acid stretch from which the mutant peptide  is derived is underlined. In the 8101-RE tumor, the second serine was  changed to a phenylalanine in this eight–amino acid stretch.
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Related In: Results  -  Collection

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Figure 9: Predicted primary amino acid sequence of wild-type murine p68 RNA helicase. The eight regions of homology to other DEAD-box proteins are outlined by the thin box. The IQ domain, found at the COOH-terminal end of the protein is outlined by the thick box. Within this IQ box, the eight–amino acid stretch from which the mutant peptide is derived is underlined. In the 8101-RE tumor, the second serine was changed to a phenylalanine in this eight–amino acid stretch.
Mentions: The primary amino acid sequence of the murine p68 protein is shown in Fig. 9. The first eight boxed motifs show the domains of homology of p68 with other DEAD box proteins, which play a central role in cell growth in a wide variety of organisms. p68 has been shown to undergo dramatic changes in nuclear localization during telophase, when it translocates from the nucleoplasma to the nucleoli (49). In addition, a stretch of amino acids, called the IQ domain (50) is located within the 139 carboxy-terminal amino acids that extend beyond the region of homology with other DEAD box proteins, and which distinguishes p68 from these proteins (41). This domain, which is also found in molecules such as neurogranin and neuromodulin, is subject in vitro to calmodulin (CaM) binding and phosphorylation by protein kinase C (PKC) (50). Experimental evidence suggests that CaM and/or PKC may regulate at least some of the activity of p68 during the cell cycle, through this domain (50). The mutation changes one of the two serines in the IQ domain to a phenylalanine (thick box in Fig. 9), but we do not yet know whether the mutation of S to F affects the physiologic function or localization of the protein. In addition to being an RNA helicase, p68 is also a powerful inhibitor of DNA helicases (51). This activity is quite similar to that of the p53 tumor suppressor gene which also prevents DNA helicase activity (51). It has been suggested that the general role of p53 is to safeguard the integrity of the genome by monitoring and stopping replication when DNA is damaged (52), and it is possible that p68 may serve a similar function as a tumor suppressor gene.

Bottom Line: The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb.Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation.This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Chicago, Illinois 60637, USA.

ABSTRACT
The genetic origins of CD8+ T cell-recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A- 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor-specific mutation in the coding region of a nucleic acid helicase.

Show MeSH
Related in: MedlinePlus