Proline-mediated proteasomal degradation of the prostate-specific tumor suppressor NKX3.1.
Reduced expression of the homeodomain transcription factor NKX3.1 is associated with prostate cancer initiation and progression. NKX3.1 turnover requires post-translational modifications including phosphorylation and ubiquitination. Here, we demonstrate the existence of a non-canonical mechanism for NKX3.1 turnover that does not require ubiquitination. Using a structure-function approach, we have determined that the conserved, C-terminal 21-amino acid domain of NKX3.1 (C21) is required for this novel ubiquitin-independent degradation mechanism. Addition of C21 decreased half-life of enhanced green fluorescence protein (EGFP) by 5-fold, demonstrating that C21 constitutes a portable degron. Point mutational analyses of C21 revealed that a conserved proline residue (Pro-221) is central to degron activity, and mutation to alanine (P221A) increased NKX3.1 half-life >2-fold. Proteasome inhibition and in vivo ubiquitination analyses indicated that degron activity is ubiquitin-independent. Evaluating degron activity in the context of a ubiquitination-resistant, lysine-null NKX3.1 mutant (NKX3.1(KO)) confirmed that P221A mutation conferred additional stability to NKX3.1. Treatment of prostate cancer cell lines with a C21-based peptide specifically increased the level of NKX3.1, suggesting that treatment with degron mimetics may be a viable approach for NKX3.1 restoration.
Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 21250, USA.
Varsha
V
Rao
Bin
B
Guan
Laura N
LN
Mutton
Charles J
CJ
Bieberich
D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D020125:Mutation, Missense; D010455:Peptides / Q000235:genetics / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D011392:Proline / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D059748:Proteolysis / Q000187:drug effects*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects* / Q000235:genetics
D000970:Antineoplastic Agents; D018398:Homeodomain Proteins; C491178:NKX3-1 protein, human; D010455:Peptides; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D011392:Proline; D046988:Proteasome Endopeptidase Complex