TY - JOUR
T1 - Protein kinase Cℓ promotes UBF1-ECT2 binding on ribosomal DNA to drive rRNA synthesis and transformed growth of nonsmall-cell lung cancer cells
AU - Justilien, Verline
AU - Lewis, Kayla C.
AU - Meneses, Kayleah M.
AU - Jamieson, Lee
AU - Murray, Nicole R.
AU - Fields, Alan P.
N1 - Funding Information:
Funding and additional information—This work was supported by NCI, National Institutes of Health, Grants R01 CA081436-22 and R01 CA206267-04 (to A. P. F.), R01 CA140290-05 (to N. R. M.), and R21 CA204938 (to V. J.). A. P. F. is the Monica Flynn Jacoby Professor of Cancer Research, an endowment fund that provides partial support for the investigator’s research program. V. J. was supported in part by a Mayo Clinic Center for Biomedical Discovery Career Development Award and an American Cancer Society Research Scholar Award (RSG-18-201-01). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2020 Justilien et al.
PY - 2020/6/12
Y1 - 2020/6/12
N2 - Epithelial cell-transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho GTPases that is overexpressed in many cancers and involved in signal transduction pathways that promote cancer cell proliferation, invasion, and tumorigenesis. Recently, we demonstrated that a significant pool of ECT2 localizes to the nucleolus of non-small-cell lung cancer (NSCLC) cells, where it binds the transcription factor upstream binding factor 1 (UBF1) on the promoter regions of ribosomal DNA (rDNA) and activates rDNA transcription, transformed cell growth, and tumor formation. Here, we investigated the mechanism by which ECT2 engages UBF1 on rDNA promoters. Results from ECT2 mutagenesis indicated that the tandem BRCT domain of ECT2 mediates binding to UBF1. Biochemical and MS-based analyses revealed that protein kinaseCℓ (PKCℓ) directly phosphorylates UBF1 at Ser-412, thereby generating a phosphopeptide-binding epitope that binds the ECT2 BRCT domain. Lentiviral shRNA knockdown and reconstitution experiments revealed that both a functional ECT2 BRCT domain and the UBF1 Ser-412 phosphorylation site are required for UBF1-mediated ECT2 recruitment to rDNA, elevated rRNA synthesis, and transformed growth. Our findings provide critical molecular insight into ECT2-mediated regulation of rDNA transcription in cancer cells and offer a rationale for therapeutic targeting of UBF1- and ECT2-stimulated rDNA transcription for the management of NSCLC.
AB - Epithelial cell-transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho GTPases that is overexpressed in many cancers and involved in signal transduction pathways that promote cancer cell proliferation, invasion, and tumorigenesis. Recently, we demonstrated that a significant pool of ECT2 localizes to the nucleolus of non-small-cell lung cancer (NSCLC) cells, where it binds the transcription factor upstream binding factor 1 (UBF1) on the promoter regions of ribosomal DNA (rDNA) and activates rDNA transcription, transformed cell growth, and tumor formation. Here, we investigated the mechanism by which ECT2 engages UBF1 on rDNA promoters. Results from ECT2 mutagenesis indicated that the tandem BRCT domain of ECT2 mediates binding to UBF1. Biochemical and MS-based analyses revealed that protein kinaseCℓ (PKCℓ) directly phosphorylates UBF1 at Ser-412, thereby generating a phosphopeptide-binding epitope that binds the ECT2 BRCT domain. Lentiviral shRNA knockdown and reconstitution experiments revealed that both a functional ECT2 BRCT domain and the UBF1 Ser-412 phosphorylation site are required for UBF1-mediated ECT2 recruitment to rDNA, elevated rRNA synthesis, and transformed growth. Our findings provide critical molecular insight into ECT2-mediated regulation of rDNA transcription in cancer cells and offer a rationale for therapeutic targeting of UBF1- and ECT2-stimulated rDNA transcription for the management of NSCLC.
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U2 - 10.1074/JBC.RA120.013175
DO - 10.1074/JBC.RA120.013175
M3 - Article
C2 - 32350115
AN - SCOPUS:85086496798
VL - 295
SP - 8214
EP - 8226
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 24
ER -