Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation.
Deleted in Split hand/Split foot 1 (DSS1) was previously identified as a novel 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible gene with possible involvement in early event of mouse skin carcinogenesis. The mechanisms by which human DSS1 (HsDSS1) exerts its biological effects via regulation of the ubiquitin-proteasome system (UPS) are currently unknown. Here, we demonstrated that HsDSS1 regulates the human proteasome by associating with it in the cytosol and nucleus via the RPN3/S3 subunit of the 19S regulatory particle (RP). Molecular anatomy of HsDSS1 revealed an RPN3/S3-interacting motif (R3IM), located at amino acid residues 15 to 21 of the NH(2) terminus. Importantly, negative charges of the R3IM motif were demonstrated to be required for proteasome interaction and binding to poly-ubiquitinated substrates. Indeed, the R3IM motif of HsDSS1 protein alone was sufficient to replace the ability of intact HsDSS1 protein to pull down proteasome complexes and protein substrates with high-molecular mass ubiquitin conjugates. Interestingly, this interaction is highly conserved throughout evolution from humans to nematodes. Functional study, lowering the levels of the endogenous HsDSS1 using siRNA, indicates that the R3IM/proteasome complex binds and targets p53 for ubiquitin-mediated degradation via gankyrin-MDM2/HDM2 pathway. Most significantly, this work indicates that the R3IM motif of HsDSS1, in conjunction with the complexes of 19S RP and 20S core particle (CP), regulates proteasome interaction through RPN3/S3 molecule, and utilizes a specific subset of poly-ubiquitinated p53 as a substrate.
Wei
Sung-Jen
SJ
Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. wei2@niehs.nih.gov
Williams
Jason G
JG
Dang
Hong
H
Darden
Thomas A
TA
Betz
Bryan L
BL
Humble
Margaret M
MM
Chang
Fang-Mei
FM
Trempus
Carol S
CS
Johnson
Katina
K
Cannon
Ronald E
RE
Tennant
Raymond W
RW
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D019143:Evolution, Molecular; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
C403591:PSMD10 protein, human; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; C484606:SEM1 protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D046988:Proteasome Endopeptidase Complex; C119011:26S proteasome non-ATPase regulatory subunit 13