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RUNX family members are covalently modified and regulated by PIAS1-mediated sumoylation.

Kim JH, Jang JW, Lee YS, Lee JW, Chi XZ, Li YH, Kim MK, Kim DM, Choi BS, Kim J, Kim HM, van Wijnen A, Park I, Bae SC - Oncogenesis (2014)

Bottom Line: Importantly, PIAS1 failed to sumoylate some RUNX1 mutants associated with breast cancer.In nude mice, tumorigenicity was promoted by RUNX3 bearing a mutation in the sumoylation site, but suppressed by wild-type RUNX3.Our results suggest that RUNXs are sumoylated by PIAS1, and that this modification could play a critical role in the regulation of the tumor-suppressive activity of these proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, South Korea.

ABSTRACT
Transcription factors of the RUNX family (RUNXs), which play pivotal roles in normal development and neoplasia, are regulated by various post-translational modifications. To understand the molecular mechanisms underlying the regulation of RUNXs, we performed a large-scale functional genetic screen of a fly mutant library. The screen identified dPias (the fly ortholog of mammalian PIASs), an E3 ligase for the SUMO (small ubiquitin-like modifier) modification, as a novel genetic modifier of lz (the fly ortholog of mammalian RUNX3). Molecular biological analysis revealed that lz/RUNXs are sumoylated by dPias/PIAS1 at an evolutionarily conserved lysine residue (K372 of lz, K144 of RUNX1, K181 of RUNX2 and K148 of RUNX3). PIAS1-mediated sumoylation inhibited RUNX3 transactivation activity, and this modification was promoted by the AKT1 kinase. Importantly, PIAS1 failed to sumoylate some RUNX1 mutants associated with breast cancer. In nude mice, tumorigenicity was promoted by RUNX3 bearing a mutation in the sumoylation site, but suppressed by wild-type RUNX3. Our results suggest that RUNXs are sumoylated by PIAS1, and that this modification could play a critical role in the regulation of the tumor-suppressive activity of these proteins.

No MeSH data available.


Related in: MedlinePlus

RUNX3 is sumoylated on lysine 148 that is evolutionarily conserved. (a) Schematic representation of human RUNX3. Lysine residues around the Runt domain are indicated. To identify the region required for sumoylation, Myc-RUNX3 deletion mutants were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX3 sumoylation was analyzed by IB. (b) Each lysine (K) residue of RUNX3 was individually mutated to arginine (R), and each Myc-RUNX3-KR mutant was coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX3 sumoylation was analyzed by IB. (c) Amino acid sequence around the RUNX3 sumoylation site (K148) was compared among RUNX3 family members. c, chicken; d, Drosophila melanogaster; h, human; m, mouse; r, rat; x, Xenopus laevis; z, zebra fish. The conserved lysine residues used for RUNX3 sumoylation are indicated by shadow. GenBank accession numbers for each protein: human RUNX1, NP_001001890.1; human RUNX2, NP_001265407; human RUNX3, NP_004341.1; mouse, NP_062706.2; rat, NP_569109.1; chicken, XP_001232978, Xenopus: NP_001182313.1; zebrafish, NP_571679.2; Drosophila, AAC47196. (d) Myc-RUNX1-WT and Myc-RUNX1-K144R were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX1 sumoylation was analyzed by IB. (e) Myc-RUNX2-WT and Myc-RUNX2-K187R were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX2 sumoylation was analyzed by IB. (f) Myc-lz-WT and Myc-lz-372 were coexpressed with HA-dPias and FLAG-dSumo in HEK293 cells. The lz sumoylation was analyzed by IB.
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fig2: RUNX3 is sumoylated on lysine 148 that is evolutionarily conserved. (a) Schematic representation of human RUNX3. Lysine residues around the Runt domain are indicated. To identify the region required for sumoylation, Myc-RUNX3 deletion mutants were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX3 sumoylation was analyzed by IB. (b) Each lysine (K) residue of RUNX3 was individually mutated to arginine (R), and each Myc-RUNX3-KR mutant was coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX3 sumoylation was analyzed by IB. (c) Amino acid sequence around the RUNX3 sumoylation site (K148) was compared among RUNX3 family members. c, chicken; d, Drosophila melanogaster; h, human; m, mouse; r, rat; x, Xenopus laevis; z, zebra fish. The conserved lysine residues used for RUNX3 sumoylation are indicated by shadow. GenBank accession numbers for each protein: human RUNX1, NP_001001890.1; human RUNX2, NP_001265407; human RUNX3, NP_004341.1; mouse, NP_062706.2; rat, NP_569109.1; chicken, XP_001232978, Xenopus: NP_001182313.1; zebrafish, NP_571679.2; Drosophila, AAC47196. (d) Myc-RUNX1-WT and Myc-RUNX1-K144R were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX1 sumoylation was analyzed by IB. (e) Myc-RUNX2-WT and Myc-RUNX2-K187R were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX2 sumoylation was analyzed by IB. (f) Myc-lz-WT and Myc-lz-372 were coexpressed with HA-dPias and FLAG-dSumo in HEK293 cells. The lz sumoylation was analyzed by IB.

Mentions: To identify the sumoylation site of RUNX3, we tested the PIAS1-mediated sumoylation of truncated RUNX3 derivatives. The results revealed that the sumoylation site resides within amino acids18 1–187 of RUNX3 that contains seven lysine residues (Figure 2a).


RUNX family members are covalently modified and regulated by PIAS1-mediated sumoylation.

Kim JH, Jang JW, Lee YS, Lee JW, Chi XZ, Li YH, Kim MK, Kim DM, Choi BS, Kim J, Kim HM, van Wijnen A, Park I, Bae SC - Oncogenesis (2014)

RUNX3 is sumoylated on lysine 148 that is evolutionarily conserved. (a) Schematic representation of human RUNX3. Lysine residues around the Runt domain are indicated. To identify the region required for sumoylation, Myc-RUNX3 deletion mutants were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX3 sumoylation was analyzed by IB. (b) Each lysine (K) residue of RUNX3 was individually mutated to arginine (R), and each Myc-RUNX3-KR mutant was coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX3 sumoylation was analyzed by IB. (c) Amino acid sequence around the RUNX3 sumoylation site (K148) was compared among RUNX3 family members. c, chicken; d, Drosophila melanogaster; h, human; m, mouse; r, rat; x, Xenopus laevis; z, zebra fish. The conserved lysine residues used for RUNX3 sumoylation are indicated by shadow. GenBank accession numbers for each protein: human RUNX1, NP_001001890.1; human RUNX2, NP_001265407; human RUNX3, NP_004341.1; mouse, NP_062706.2; rat, NP_569109.1; chicken, XP_001232978, Xenopus: NP_001182313.1; zebrafish, NP_571679.2; Drosophila, AAC47196. (d) Myc-RUNX1-WT and Myc-RUNX1-K144R were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX1 sumoylation was analyzed by IB. (e) Myc-RUNX2-WT and Myc-RUNX2-K187R were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX2 sumoylation was analyzed by IB. (f) Myc-lz-WT and Myc-lz-372 were coexpressed with HA-dPias and FLAG-dSumo in HEK293 cells. The lz sumoylation was analyzed by IB.
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fig2: RUNX3 is sumoylated on lysine 148 that is evolutionarily conserved. (a) Schematic representation of human RUNX3. Lysine residues around the Runt domain are indicated. To identify the region required for sumoylation, Myc-RUNX3 deletion mutants were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX3 sumoylation was analyzed by IB. (b) Each lysine (K) residue of RUNX3 was individually mutated to arginine (R), and each Myc-RUNX3-KR mutant was coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX3 sumoylation was analyzed by IB. (c) Amino acid sequence around the RUNX3 sumoylation site (K148) was compared among RUNX3 family members. c, chicken; d, Drosophila melanogaster; h, human; m, mouse; r, rat; x, Xenopus laevis; z, zebra fish. The conserved lysine residues used for RUNX3 sumoylation are indicated by shadow. GenBank accession numbers for each protein: human RUNX1, NP_001001890.1; human RUNX2, NP_001265407; human RUNX3, NP_004341.1; mouse, NP_062706.2; rat, NP_569109.1; chicken, XP_001232978, Xenopus: NP_001182313.1; zebrafish, NP_571679.2; Drosophila, AAC47196. (d) Myc-RUNX1-WT and Myc-RUNX1-K144R were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX1 sumoylation was analyzed by IB. (e) Myc-RUNX2-WT and Myc-RUNX2-K187R were coexpressed with HA-PIAS1 and FLAG-SUMO1 in HEK293 cells. RUNX2 sumoylation was analyzed by IB. (f) Myc-lz-WT and Myc-lz-372 were coexpressed with HA-dPias and FLAG-dSumo in HEK293 cells. The lz sumoylation was analyzed by IB.
Mentions: To identify the sumoylation site of RUNX3, we tested the PIAS1-mediated sumoylation of truncated RUNX3 derivatives. The results revealed that the sumoylation site resides within amino acids18 1–187 of RUNX3 that contains seven lysine residues (Figure 2a).

Bottom Line: Importantly, PIAS1 failed to sumoylate some RUNX1 mutants associated with breast cancer.In nude mice, tumorigenicity was promoted by RUNX3 bearing a mutation in the sumoylation site, but suppressed by wild-type RUNX3.Our results suggest that RUNXs are sumoylated by PIAS1, and that this modification could play a critical role in the regulation of the tumor-suppressive activity of these proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju, South Korea.

ABSTRACT
Transcription factors of the RUNX family (RUNXs), which play pivotal roles in normal development and neoplasia, are regulated by various post-translational modifications. To understand the molecular mechanisms underlying the regulation of RUNXs, we performed a large-scale functional genetic screen of a fly mutant library. The screen identified dPias (the fly ortholog of mammalian PIASs), an E3 ligase for the SUMO (small ubiquitin-like modifier) modification, as a novel genetic modifier of lz (the fly ortholog of mammalian RUNX3). Molecular biological analysis revealed that lz/RUNXs are sumoylated by dPias/PIAS1 at an evolutionarily conserved lysine residue (K372 of lz, K144 of RUNX1, K181 of RUNX2 and K148 of RUNX3). PIAS1-mediated sumoylation inhibited RUNX3 transactivation activity, and this modification was promoted by the AKT1 kinase. Importantly, PIAS1 failed to sumoylate some RUNX1 mutants associated with breast cancer. In nude mice, tumorigenicity was promoted by RUNX3 bearing a mutation in the sumoylation site, but suppressed by wild-type RUNX3. Our results suggest that RUNXs are sumoylated by PIAS1, and that this modification could play a critical role in the regulation of the tumor-suppressive activity of these proteins.

No MeSH data available.


Related in: MedlinePlus