RING finger protein 13 RNF13 has been identified Keywords cancer; development; differentiation; E3 ubiquitin ligase; myogenesis; neurogenesis; proliferation; RING finger domain; RNF13; ubi
Trang 1RNF13: an emerging RING finger ubiquitin ligase important
in cell proliferation
Xianglan Jin1,2, He Cheng1, Jie Chen2and Dahai Zhu1
1 National Laboratory of Medical Molecular Biology, Tsinghua University, Beijing, China
2 Department of Pathology, Peking Union Medical College Hospital, Tsinghua University, Beijing, China
Introduction
The classic function of the 76-amino acid globular
polypeptide termed ubiquitin involves participation in
post-translational modification of proteins prior to
proteolytic degradation in the 26S proteasome
com-plex The ubiquitin ligase (E3) enzyme acts in concert
with appropriate ubiquitin-conjugating enzymes (E2)
and ubiquitin-activating enzymes (E1) to mediate
protein ubiquitination It is now well recognized that
polyubiquitination of cellular proteins for proteasomal
degradation represents only one form of protein
modification Apart from protein polyubiquitination,
mono-ubiquitination or multi-ubiquitination mediated
by E3 with appropriate lysine linkages have been reported, and such patterns of protein ubiquitination play important roles in regulating protein–protein interactions in signal transduction, protein trafficking between subcellular compartments and the biological functions of proteins [1]
The ubiquitin ligases of eukaryotes fall into three major families: RING, HECT and U-box proteins Based on genome-wide predictions, > 90% of ubiqu-itin ligases are RING-type proteins including single-subunit and SCF-like multiple-single-subunit E3 ligases [2] RING finger protein 13 (RNF13) has been identified
Keywords
cancer; development; differentiation; E3
ubiquitin ligase; myogenesis; neurogenesis;
proliferation; RING finger domain; RNF13;
ubiquitination
Correspondence
D Zhu, National Laboratory of Medical
Molecular Biology, Institute of Basic Medical
Sciences, Chinese Academy of Medical
Sciences & Peking Union Medical College,
Tsinghua University, 5 Dong Dan San Tiao,
Beijing 100005, China
Fax: +86 10 6510 5083
Tel: +86 10 6529 6949
E-mail: dhzhu@pumc.edu.cn or
dhzhusara@gmail.com
(Received 15 April 2010, revised 13
September 2010, accepted 12 October
2010)
doi:10.1111/j.1742-4658.2010.07925.x
Protein ubiquitination mediated by ubiquitin ligases plays a very important role in a wide spectrum of biological processes including development and disease pathogenesis RING finger protein 13 (RNF13) is a recently identi-fied ubiquitin ligase which contains an N-terminal protease-associated domain and a C-terminal RING finger domain separated by a transmem-brane region RNF13 is an evolutionarily conserved protein Most interest-ingly, RNF13 expression is developmentally regulated during myogenesis and is upregulated in various human tumors These data suggest that RNF13, acting as an ubiquitin ligase, might have profound biological func-tions during development and disease This minireview summarizes recent work on RNF13 functions related to cell proliferation, differentiation and cancer development
Abbreviations
PA, protease-associated domain; RNF13, RING finger protein 13; TM, transmembrane.
Trang 2as a novel RING-based ubiquitin ligase and
overex-pression of the enzyme is apparent in various human
cancers, suggesting that RNF13 may play a significant
role in cancer development Besides RNF13, eight
other related proteins have been reported to belong to
the Goliath family, including RNF128 (GRAIL),
RNF130, RNF133, RNF148, RNF149, RNF150,
RNF167 and RNF204 (Table 1) [2–4] Alignments of
these nine proteins indicate high similarity in the
prote-ase-associated (PA), transmembrane (TM) and RING
domains (Fig 1B) To date, GRAIL remains the
best-characterized member of this protein family, playing a
functional role in controlling the development of T-cell
clonal anergy
RNF13: structure, expression, and
localization
The RNF13 gene is located at chromosome 3q25.1 in
human and is evolutionarily conserved in many
meta-zoans including the chimpanzee, dog, cow, chicken,
zebra fish, fruit fly, mosquito and Arabidopsis thaliana
(NCBI data) The full-length protein encoded by the
RNF13 gene is composed of 381 amino acid residues
(Accession Number NP_009213) RNF13 contains an
N-terminal PA domain and a C-terminal RING finger
domain separated by a TM region (Fig 1A) [5,6] The
PA domain is supposed to mediate substrate binding or
to be involved in protein–protein interactions [7–9]
The RING region is a C3H2C3 type and exerts E3
activity [5,6] Bioinformatics predictions and
experi-mental evidence indicate that RNF13 is a type I TM
glycoprotein in which the PA domain faces the lumen
or extracellular region and the RING domain localizes
to the cytosol [5,6] In addition, RNF13 also has a PEST domain enriched in proline (P), glutamic acid (E), serine (S) and threonine (T), and a nuclear localiza-tion signal [6] Together, these structural features imply that RNF13 is a member of the Goliath family with characteristics of PA-TM-RING domains [2–4,10] Analysis of RNF13 gene expression shows that RNF13 is ubiquitously expressed in various tissues of chicken, mouse and human [5,6,11] (Fig 2A) How-ever, RNF13 expression is spatially regulated during postnatal development in chicken RNF13 is abun-dantly expressed in skeletal muscle tissue at early stages of embryonic myogenesis but its expression gradually decreases during embryonic development and becomes almost undetectable in skeletal muscle tissue after hatching in chickens [11] (Fig 2B) The expression of RNF13 is greater in adult compared with embryonic tissues in the mouse, especially in the liver and brain [6] More interestingly, RNF13 expression is upregulated by tenascin and myostatin [11,12] Myost-atin is a member of the transforming growth factor-b superfamily and functions as an inhibitor of muscle cell proliferation and differentiation [13,14] Tenascin
is an extracellular matrix glycoprotein and its expres-sion is associated with cancer development [15] There-fore, it is conceivable that RNF13 may be involved in controlling cell proliferation, and differentiation by ubiquitination of proteins that play important regula-tory roles in response to extracellular signals such as myostatin and tenascin
Based on bioinformatics predictions, the human RNF13 protein may be encoded by two alternatively
Table 1 General information of the nine Goliath family members ER, endoplasmic reticulum; INM, inner nuclear membrane; n ⁄ a, not avail-able; TSSC5, tumor-suppressing subchromosomal transferable fragment cDNA; UE, ubiquitous expression; aa, amino acid.
Member Alias
Localization
of human genome
Protein size (aa) mRNA Accession No.
Expression profile
E3 activity Substrates
Subcellular localization Ref.
INM ER ⁄ Golgi
[5,6,11,18]
kidney, liver
Yes RhoGDI, CD40L, CD81, CD151, CD83
Recycling endosome
[7,8,42–44]
leukocytes, liver
RNF167 RING105,
DKFZP566H073
kidney, liver
membrane
[47]
Trang 3spliced transcripts with the shorter transcript lacking
an exon in the 5¢ untranslated region (NCBI Accession
NM_007282.4 and NM_183381.2) [16] A pseudogene,
also residing on chromosome 3, has been described
(NCBI Accession NM_007282.4 and NM_183381.2)
[16] In addition, 15 alternatively spliced variants that
might encode 11 distinct RNF13 isoforms have been
annotated using the ace view program (http://
www.ncbi.nlm.nih.gov/IEB/Research/Acembly/) [17]
Structural analysis has revealed that RNF13
con-tains a nuclear localization signaling domain and a
TM region [5,6] Previous reports have provided
experimental evidence showing that RNF13 is a nucleus- and⁄ or membrane-associated protein Data from Tranque’s group indicate that RNF13 is present
in the nuclei of chicken embryonic heart tissue and cultured embryonic cardiocytes [12] Recently, Bocock
et al [6] reported that RNF13 is present in the endosomal–lysosomal system of COS cells, HeLa cells and primary mouse neurons We also showed that RNF13 resides in the endoplasmic reticulum⁄ Golgi system of pancreatic cancer cells [5] Very recently, an intriguing study found that RNF13 is present on the endosome membrane and is dynamically transported
A
B
Fig 1 RNF13 protein domain structure and alignments of nine Goliath family members (A) Schematic structure of RNF13 protein SP, signal peptide; PA, protease-associated domain; TM, transmembrane region; RING, RING finger domain; NLS, nuclear localization signal;
LC, low complexity [5,39] LC region is shown according to SMART [40,41] (B) Alignments of nine Goliath family members.
Trang 4from multivescular endosomes to recycling endosomes
and inner nuclear membrane in response to
4b-phor-bol 12-myristate 13-acetate stimulation [18] It has
become evident that protein trafficking into the inner
nuclear membrane is an important mechanism
regulat-ing gene expression, transducregulat-ing signals from the
plasma membrane to the nucleus in response to
vari-ous stimuli For example, amphiregulin and HB-EGF,
members of the epidermal growth factor family, are
both plasma membrane-anchored proteins, and
partic-ipate in transcriptional and epigenetic regulation of
target genes by traveling between the plasma
mem-brane and the inner nuclear memmem-brane [19,20]
There-fore, further investigation of the dynamic regulation
of RNF13 sublocalization within cells will shed light
on the functional roles of RNF13 as a
membrane-anchored E3 ubiquitin ligase regulating gene
expres-sion by ubiquitination of nuclear proteins during
development and disease [21]
RNF13: functional roles
Work from the laboratory of Erickson and ours has
shown that RNF13 is a novel RING-containing E3
ligase [5,6] and that RNF13 expression is associated
with myogenesis, neuronal development and tumori-genesis [5,6,11,22] Emerging evidence suggests that the ubiquitin ligase RNF13 plays critical roles in the regu-lation of development and human disease
Function of RNF13 in regulating skeletal muscle growth and neuronal development
We have recently shown that RNF13 is highly expressed in proliferating myoblasts and its expression gradually decreases during skeletal myogenesis Inter-estingly, our work has also demonstrated that RNF13 expression is upregulated by the muscle growth inhibi-tor myostatin in chicken primary myoblasts and that ectopic expression of RNF13 in vitro inhibits myoblast proliferation with an E3 activity-dependent manner [11] Given that myostatin, as a cytokine, inhibits skel-etal muscle proliferation and upregulates RNF13 expression in myoblast cells, it is very likely that RNF13 may play an important role in pathways involved in myostatin signal transduction
In addition, RNF13 is expressed in embryonic and adult brain tissues [5,6,11,12], and overexpression of RNF13 induces spontaneous neurite outgrowth in PC12 cells [22] Moreover, RNF13 presents an elevated level in B35 neuroblastoma cells showing extension of neurites after treatment with dibutyryl-cAMP [6] Together, these results highlight the functional signifi-cance of RNF13 in regulating skeletal muscle growth and neuronal development Therefore, identification of RNF13 substrates is becoming a critical step in obtain-ing a better molecular insight into RNF13 functions during development
RNF13 and cancer development Ubiquitin ligases play critical roles in cancer develop-ment and the well-studied enzymes are MDM2 and SCFSkp2[23] Elevated expression of MDM2 is appar-ent in various tumors and is particularly associated with late-stage and highly drug-resistant tumors [24]
A possible molecular role for MDM2, as a ubiquitin ligase controlling tumor development, is degradation
of the tumor suppressor protein p53 by ubiquitination [25,26] Skp2, another ubiquitin ligase, is also overex-pressed in human cancers and functions as an onco-protein by regulating the stability of several tumor suppressor proteins including p21, p57, p130 and FOXO1 [27–31]
Recent studies have shown that RNF13 gene expres-sion is associated with cancer development Our labo-ratory first reported a link between RNF13 expression
A
B
Fig 2 Spatial and temporal expression patterns of RNF13.
(A) Western blot analysis of RNF13 protein in multiple human
tissues using anti-RNF13 IgG [5] (B) Expression pattern analysis of
RNF13 during skeletal muscle development The pectoralis muscles
of White Leghorn chickens were obtained from different
develop-mental stages (10-, 12-, 14-, 16- and 18-day embryos, as well as
from chicks 1 day and 1, 2, 3, 5 and 7 weeks after hatching) The
transcript and protein levels of RNF13 were determined by northern
and western analysis, respectively The ethidium bromide staining
of 18S and 28S ribosomal RNAs and immunoblotting of tubulin
were used as equal loading controls [11].
Trang 5and pancreatic cancer progression by showing that
pancreatic ductal adenocarcinoma has high-level
expression of RNF13 (41.7% of 72 human samples)
and that such expression is related to histological
grad-ing [5] Our data also revealed that RNF13 is present
in precancerous lesions including chronic pancreatitis
and pancreatic intraepithelial neoplasia, suggesting that
RNF13 is involved in inflammation-associated
carcino-genic change Interestingly, Ralf et al screened
candi-date genes, the expression of which is associated with
hepatocellular carcinoma in a mouse model, and
iden-tified RNF13 as one such gene [32] Moreover, our
unpublished data from experiments with human tissue
microarrays also suggest an association between
RNF13 expression and human colon cancer
develop-ment, because higher levels of RNF13 protein are
detected in human colon cancer samples than in
con-trol samples Most intriguingly, microarray analysis of
RNF13 gene expression in multiple tumor samples,
shown in Fig 3 (F.M Marincola, personal
communi-cation), indicates that RNF13 overexpression is
com-mon in various human tumors Such expression is
much higher in renal cell carcinoma and esophageal
carcinoma cells compared with normal tissues
respec-tively Similarly, RNF13 levels are elevated in several
other malignant tumors, including basal cell
carci-noma, melanoma and ovarian carcinoma using normal
peripheral blood mononuclear cell and immune cell
subsets as controls (Fig 3) Based on the accumulating
observations, it seems that the ubiquitin ligase RNF13
may be of profound significance in regulating tumori-genesis in vivo
More evidence implicating RNF13 in cancer devel-opment has come from the observation of a close relationship between RNF13 and tenascin C, an extra-cellular matix molecule highly expressed in the stroma
of most solid tumors and linked to various features of cancer including uncontrolled proliferation and metas-tasis [5,33] RNF13 expression is not only induced by tenascin [12], but such expression also significantly overlaps with tenascin C in pancreatic ductal adeno-carcinoma samples [5] Our recent studies have indi-cated a functional role for RNF13 in regulating cell proliferation and invasive growth in vitro [5,11], and several other RING-type ubiquitin ligases have also been reported to participate in cancer invasion and metastasis These enzymes include BCA2, Hakai and HEI10 [34–38] Therefore, a considerable body of data provides significant information that lays the groundwork for further experimental investigation of RNF13 function involved in the regulation of cancer development
Perspectives Recently, RNF13 has been identified as a novel E3 ubiquitin ligase and its expression patterns suggest that RNF13 may exert profound biological functions during development and the course of diseases includ-ing myogenesis and tumorigenesis However, the state
of current knowledge on RNF13 raises even more questions For example, how is expression of the RNF13gene regulated during development? What con-trols the enzymatic activity of RNF13 ubiquitin ligase? What is the biological significance of RNF13 traffick-ing between subcellular compartments? What are the actual functions of RNF13 in vivo and what molecular mechanisms underlie its action? To answer these ques-tions, the next crucial step is the generation of RNF13 transgenic⁄ knockout mice and identification of its substrates in vivo In this manner, further molecular and biochemical analysis of RNF13 functions in trans-genic⁄ knockout animals will greatly facilitate our understanding of RNF13 actions during development and human disease states such as cancer
Acknowledgements
We thank Dr Francesco M Marincola (Department of Transfusion Medicine, Clinical Center, National Insti-tutes of Health) for sharing the unpublished data shown in Fig 2 We also thank Dr Yuchang Zhou (Institute of Basic Medical Sciences, Chinese Academy
Fig 3 Microarray analysis of RNF13 expression in several
malig-nant human tumors.
Trang 6of Medical Sciences) for valuable comments This
work was supported by grants from the National Basic
Research Program of China (Nos 2005CB522405,
2005CB522505, 2007CB946903, 2009CB 941602 and
2009CB825403), the National Natural Science
Founda-tion of China (Nos 30721063 and 30471970), the
Chinese National Programs for High Technology
Research and Development (Nos 2006AA10A121 and
2007AA02Z109), and the National Key Technology
R&D program (No 2006BAI02A14)
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