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Introduction
Since the late 1970s, when the first gene involved in
tumor development in human was cloned, more than 200
tumor-related genes have been identified. They constitute a
heterogeneous group of regulators of physiological
processes that includes hormones, growth factors, receptors,
cell adhesion molecules, signal transduction mediators, and
transcription factors. Ras is the most widely studied
oncogene in human carcinogenesis, and its discovery
stimulated the search for Ras-related genes. Today, the Ras
superfamily constitutes a numerous group of small guanosine
triphosphatases (GTPases) that comprises over 150
members in humans, but can be found in all
eukaryotes[1,2]. The common feature of Ras-related proteins is a ~20 kDa domain
that, with few exceptions, binds and hydrolyzes GTP. Ras
proteins act as molecular switches, cycling between an
active GTP-bound state and an inactive GDP-bound state.
Activation enables the GTPase to interact with a multitude
of effectors that relay upstream signals to other components,
eliciting diverse cellular responses. Two classes of molecules
modulate the activation/inactivation cycle: guanine
nucleotide exchange factors (GEF) and GTPase-activating
proteins (GAP). In addition, guanine nucleotide-dissociation
inhibitors regulate cycling of some GTPases between
membranes and cytosol. The members of the Ras superfamily
can be divided into several families based on sequence
similarities, such as the extensively studied Ras, Rho, Rab,
Arf, Ran, and Miro families[1,2], and broadly speaking, each
family participates in the regulation of a major cellular process.
Rho GTPases are major regulators of the actin filament
system and consequently of all processes that depend on
the reorganization of the actin cytoskeleton, but they also
participate in signaling pathways that regulate gene
expression, cell cycle progression, apoptosis, and
tumorigenesis[3_5]. Rho GTPases are being extensively studied in
eukaryotes, from plants to mammals. In humans, the family
comprises 21 members that have been grouped into subfamilies: Cdc42-like (Cdc42, TC10, TCL, Chp/Wrch-2,
Wrch-1), Rac-like (Rac1_3, RhoG), Rho-like (RhoA_C), Rnd
(Rnd1_2, Rnd3/RhoE), RhoD (RhoD and Rif), RhoH/TTF and
RhoBTB (RhoBTB1_3)[4]. Although RhoBTB3 is frequently
left outside because of its divergent GTPase domain, there is
compelling architectural, phylogenetic, and possibly
functional evidence for grouping this protein within the RhoBTB
subfamily.
The RhoBTB subfamily constitutes the most recent
addition to the Rho family. It was identified during the study of
Rho-related protein-encoding genes in Dictyostelium
discoideum[6]. Orthologs have been found in numerous
eukaryote clades, but are absent in fungi, plants, and some
metazoa[7]. RhoBTB proteins are remarkable for their
unusual domain architecture: all RhoBTB proteins possess
additional domains beyond the GTPase domain, in particular,
a tandem of broad complex, tramtrack, bric à brac (BTB)
domains (from the Drosophila transcription factors where the
domain was first described) that explains the name given to
the family and fully justifies their inclusion in the group of
so-called atypical Rho GTPases[8].
Interest in the RhoBTB subfamily arose when RHOBTB2,
the gene encoding the homonymous protein, was identified
as the gene homozygously deleted in breast cancer samples
and was proposed as a candidate tumor suppressor gene,
dubbed DBC2 (deleted in breast cancer
2)[9]. The same property has been attributed recently to
RHOBTB1[10]. RhoBTB proteins can be therefore incorporated into the group of Rho
GTPases involved in tumorigenesis, although the
mechanism RhoBTB proteins use differs radically from those of more
typical Rho proteins[11] and may involve the direct targeting
of other proteins for degradation in the 26S proteasome.
In this review we will summarize what we know about
RhoBTB proteins, starting with basic aspects, such as
domain architecture and gene expression, followed by the
evidence that has accumulated during the last few years linking
RhoBTB proteins with cancer. We will then connect this
information with the roles that emerge from functional
studies performed on the mammalian, and more limited, slime mold
and Drosophila orthologs. We will close this review with an
attempt to integrate all the available structural and functional
information into a model that explains the participation of
RhoBTB proteins in tumorigenesis.
Structure of RhoBTB proteins
The most salient feature of RhoBTB proteins is their
domain architecture, which is, in general terms, shared by all
members of the subfamily. In these proteins, the GTPase
domain is followed by a proline-rich region, a tandem of 2
BTB domains, and a conserved C-terminal region (Figure 1).
As already mentioned, in humans, the RhoBTB subfamily is
composed of 3 isoforms: RhoBTB1, RhoBTB2, and RhoBTB3.
RhoBTB1 and RhoBTB2 are very similar to each other and to
the Drosophila ortholog (DmRhoBTB), whereas RhoBTB3
and the Dictyostelium discoideum ortholog (RacA) are the
most divergent members. Here we will describe each domain
and will discuss its functionality as well as some variations
found in individual members.
GTPase domain The GTPase domain is perhaps the
region where most divergence is found among members of the
RhoBTB subfamily. Early analyses revealed that this
domain is typically Rac-like in RacA and divergent, but
recognizable as Rho-related in RhoBTB1 and RhoBTB2 as well as
in DmRhoBTB[6]. In RhoBTB3, the GTPase domain appears
extensively erased, to the point that it is virtually
unrecognizable as a GTPase. Only a short stretch at the end of the
domain can be reliably aligned to the GTPase domain of other
subfamily members. Consequently, RhoBTB3 does not bind
GTP in vitro (Berthold J et al,
personal communication). In phylogenetic analyses, the GTPase domain of RacA groups
together with GTPases of the Rac subfamily and all relevant
residues for nucleotide binding and enzymatic activity are
conserved. In RacA the so-called Rho insert, a hypervariable
insertion characteristic for Rho proteins, is shorter (6 amino
acids) than the usual 13 amino acids of most Rac proteins.
As far as it has been examined, the GTPase domain of RacA
behaves like other Rac proteins (see functions of RhoBTB
proteins below).
The GTPase domain of RhoBTB proteins other than
RhoBTB3 and RacA also contains a Rho insert that is longer
than usual (18 residues or more) and rich in charged residues.
Moreover, the GTPase domain of these RhoBTB contains 2
insertions and 1 deletion, as well as a few deviations from
the GTPase consensus of most Rho
GTPases[6]. The insertions are placed immediately before (6 residues) and after (10
residues) the switch I. The deletion (2 residues) affects the
phosphate/magnesium binding region 3 within the switch II;
in particular, one of the deleted residues is the glutamine
equivalent to Q61 in Ras. Also of importance, the glycine
residue equivalent to G12 in Ras appears substituted by
asparagine in RhoBTB1 and RhoBTB2 or threonine in DmRhoBTB. Because these 2 residues are essential for GTP
hydrolysis, these proteins would predictably display impaired
enzyme activity. Indeed, using a blot overlay approach,
Chang and coworkers have shown that the GTPase domain
of RhoBTB2 appears not to bind GTP at all, although this
aspect requires biochemical
confirmation[12].
Proline-rich region The proline-rich region links the
GTPase to the first BTB domain. Sequences rich in proline
are very common recognition motifs involved in
protein_protein interactions. Among the modules that bind
proline-rich regions are the SH3 (Src homology 3) domain, the WW
domain, the Ena/VASP homology 1 domain, profilin, the GYF
domain, ubiquitin enzyme variant (UEV), and the
cytoskeleton-associated protein glycine-rich
domain[13,14]. The proline-rich region of some RhoBTB proteins could act as a SH3
domain-binding site. The SH3 domain is often present in
proteins involved in signal transduction and cytoskeleton
organization. The PxxP motif (where x denotes any amino
acid), initially described as the core binding motif of the SH3
domain, can be found in RhoBTB1, RhoBTB2, and DmRhoBTB, where the proline-rich region is prominent, and
in RacA, but not in RhoBTB3 where this region is very poorly
preserved. Subsequent analyses have defined proline-rich
motifs for a number of different SH3 domains more precisely
as +xΦPxΦP (class I ligands) and ΦPxΦPx+ (class II ligands;
where Φ is a hydrophobic and + is in most cases a basic
residue)[13,15]. Interestingly, RhoBTB1 and RhoBTB2 have a
conserved class II motif. DmRhoBTB has a motif that matches
the more recently recognized class III ligands with the
(R/K)xx(K/R) sequence[14]. Nevertheless, albeit the sequence
analysis strongly suggests that the proline-rich region of
several RhoBTB proteins is a potential SH3 domain-binding
site, this still needs to be verified experimentally.
BTB domain The BTB domain, also known as a poxvirus
and zinc finger domain, is an evolutionary conserved
domain that is widespread among eukaryotes. In humans,
nearly 200 different proteins bear BTB domains, in most cases,
in combination with other domains. Two of the
accompanying domains are particularly frequent, the zinc finger (ZF)
and the Kelch domain. BTB_ZF proteins constitute a large
family of transcription factors, whereas BTB_Kelch proteins
play roles in the dynamics of the actin
cytoskeleton[16,17].
The BTB domain has been known for long time as a
protein_protein interaction domain participating in homomeric
and heteromeric associations with other BTB
domains[18]. More recently, a series of papers almost simultaneously
identified this domain as a component of cullin3-dependent
ubiquitin ligase complexes[19_22]. These complexes
constitute a class of the very large family of ubiquitin
ligases[23], which catalyze the addition of ubiquitin, a highly conserved
76-amino acid globular protein, to a number of target proteins.
This post-translational modification labels proteins for
degradation by the 26S proteasome, although other cellular
functions not directly involving protein degradation are also
controlled by this modification[24].
Cullins (of which there are 7 in mammals) function as
scaffolding proteins that bring together the
ubiquitin-conjugating enzyme and substrate-recognition components. The
core ligase of a cullin-dependent complex consists of a cullin
protein that binds through its C-terminus the RING-finger
protein Roc1 (which recruits the ubiquitin-conjugating
enzyme) and through its N-terminus, a linker protein. An
adaptor protein then acts as a bridge between the linker
protein and the substrates. The complex is positively regulated
by covalent attachment of the Nedd8 ubiquitin-like protein
to the cullin subunit. Each cullin family member interacts
with a specific adaptor. The cullin1 and cullin7 complexes
contain the Skp1 linker and an F-box-containing adaptor,
whereas the cullin2 and cullin5 complexes contain the linker
elongin C (along with elongin B) and a
SOCS-box-containing protein. The cullin3 complexes contain a BTB
domain-bearing protein that interestingly functions simultaneously
as a linker and adaptor[25].
Closer inspection of the structure of the BTB domain in
comparison with that of Skp1 and elongin C revealed a
similar folding, and predictably a common interface for
interaction with the corresponding cullin, despite a low degree of
primary sequence conservation. In fact, elongin C and Skp1
are now considered BTB proteins. The common folding of
all these proteins consists of a 95 amino acid globular cluster
of 5 α-helices flanked by 3 short β-strands. The BTB
domains of the BTB_ZF, BTB_Kelch, and RhoBTB proteins
contain an N-terminal extension that folds into 1
α-helix and 1 β-strand, and this extension mediates the formation of
dimers and oligomers[17].
The BTB domains of RhoBTB have some special features.
A tandem of 2 BTB domains as in RhoBTB is not frequently
found within the BTB protein family. Moreover, the first
BTB domain is bipartite, being interrupted by an extension
of unknown function that varies in length and composition
among RhoBTB proteins. In RhoBTB1, RhoBTB2, and DmRhoBTB the insertion is 3 times longer (up to 100 residues)
than in RhoBTB3 and RacA, and is in all cases rich in charged
residues. Because the BTB domains of RhoBTB are of the
extended type, these proteins are predicted to exist as dimers,
and in fact, they are capable of forming homodimers and
heterodimers (Berthold J et al, personal communication). The
role of RhoBTB as components of the cullin3-dependent
complexes will be discussed below.
The C-terminal region Following the second BTB
domain, there is a region conserved in all members of the
RhoBTB subfamily that may constitute a novel domain, but
has not been found so far in any other protein apart from
RhoBTB. The core of the C-terminal domain consists of
approximately 80 amino acids that predictably folds as 4
consecutive α-helices. The last helix ends close before the
prenylation signal of RhoBTB3, but prolongs further in a
predicted β-strand in RhoBTB1, RhoBTB2, and
DmRhoBTB[7].
Although Rho GTPases typically bear a CAAX motif,
only RhoBTB3 conserves this feature. This motif is
recognized by a set of enzymes that introduce a post-translational
modification, isoprenylation, responsible for the targeting
of the modified protein to membranes. Closely upstream of
this motif there is an additional cysteine residue in RhoBTB3,
which suggest that this protein might also be
palmitoylated[7]. The presence of nuclear localization signals in the
C-terminus of some members of the RhoBTB subfamily has been
occasionally reported[8,12], but is controversial because
computer programs commonly used to predict these signals
often yield inconsistent results. Unlike for many BTB proteins
that function as transcription factors, there is no
experimental evidence showing the nuclear localization of RhoBTB.
Expression of RHOBTB genes
Both in humans and mice, all 3 RHOBTB genes are rather
ubiquitously expressed, although with notable differences
in the pattern of tissue levels among the 3 genes. In humans,
where expression has been studied using multiple-tissue
Northern dot blots and quantitative
PCR[7,26,27], RHOBTB1 showed high levels in skeletal muscle and placenta followed
by the stomach, kidney, testis, adrenal gland, and uterus,
whereas RHOBTB3 is highly expressed in the placenta, testis,
pancreas, adrenal and salivary glands, and neural and
cardiac tissues. RHOBTB2 is very weakly expressed, but
relatively high levels were detected in neural and cardiac tissues.
All 3 genes are expressed in fetal tissues.
The expression pattern of the mouse counterparts has
been analyzed in conventional Northern blots and is roughly
comparable to that of the human
genes[7]. Mouse RHOBTB1 is highly expressed in the heart, testis, and kidney, and
moderately in the uterus, liver, lung, stomach, placenta, and
skeletal muscle. Mouse RHOBTB3 is strongly expressed in the
brain, heart, and uterus, and moderately in all other tissues.
As in human tissues, RHOBTB2 is very weakly expressed in
mouse tissues, with relatively higher expression levels in the
brain. In addition, the expression of 1 or more
RHOBTB genes has been reported in numerous human and mouse cell
lines using RT_PCR. In Northern blot analyses, mouse
RHOBTB3 and RHOBTB1 appear as single 5 kb transcripts,
although in most tissues, a less prominent 4 kb
RHOBTB1 transcript is also expressed.
RHOBTB2 is equally expressed both as 4 kb and 5 kb transcripts. The 2 transcripts in these
genes have been explained by the use of alternative
promoters or by alternative splicing in the 5´UTR, but this issue has
not been addressed and remains
speculative[7].
RHOBTB3 has been reported in RNA from whole mouse
embryos in Northern blot analyses, where a transcript was
detected from embryonic d 11.5, declining at d
17.5[7]. RHOBTB2 has been reported in several fetal tissues using
RT_PCR[28]. Using in situ hybridization, the high and
specific expression of RHOBTB2 has been observed in the
central and peripheral nervous system and comparatively weaker
in the gut during mouse embryogenesis, but the mRNA
becomes undetectable at embryonic d
18.5[28]. Although still limited, these data implicate
RHOBTB genes in controlling developmental processes.
With RHOBTB2 having been described as a tumor
suppressor gene involved in breast cancer, it was of interest
studying the expression of this gene during mammogenesis.
Using RT_PCR and Northern blot analysis, St-Pierre
et al[28] found that during mammary gland development in mice,
RHOBTB2 transcripts are expressed at low but constant
levels. However, attempts to study the spatial pattern of the
expression of RHOBTB2 in the mammary gland using
in situ hybridization were inconclusive because of undetectable
mRNA levels. Our own attempts to study the expression of
RHOBTB genes at the cellular level using
in situ hybridization on adult mouse tissues were hampered by the very low
mRNA levels of these genes. While the expression of
RHOBTB2 was undetectable in all of the tissues analyzed,
RHOBTB1 and RHOBTB3 mRNA were found in the
endothelial cells of the heart as well as in spermatocytes and spermatides
in the testis. Additionally, RHOBTB1 and
RHOBTB3 messages were detected in large vessels of the kidney and brain,
respectively (Berthold J et al, personal communication).
RhoBTB in cancer
Since the first report proposing RHOBTB2
as a tumor suppressor gene, evidence is accumulating in support of
members of the RhoBTB subfamily being implicated in
tumorigenesis (Table 1). The RHOBTB2 gene was identified as
the gene homozygously deleted at region 8p21 in breast
cancer samples[9]. This is a region commonly associated with
loss of heterozygosity (LOH) in a wide range of cancers.
Hamaguchi and coworkers performed a representational
deletion analysis on a large sample of breast tumors using DNA
markers for the 8p21 region and found that RHOBTB2
was homozygously deleted in 3.5% of the tumors. A mutation
analysis revealed 2 somatic missense mutations in breast
tumors and 2 more missense mutations each in a breast and
a lung tumor cell line. The expression of RHOBTB2
appeared extinguished in approximately 42% of breast and 50% of lung
cancer cell lines[9]. A more extensive mutation analysis of
breast cancers revealed some polymorphisms as well as 2
novel somatic mutations in the promoter and 5´UTR of
RHOBTB2 in sporadic tumors, but no additional mutations
in the coding region of sporadic or familial
cancers[29].
In a study addressing RHOBT2 in bladder cancer,
Knowles et al performed a LOH and mutation analysis on tumor
samples and cell lines[30]. They found LOH in the target
region in 42% of informative tumors. A sequence analysis
revealed numerous polymorphisms and 1 missense somatic
mutation. In addition, the expression of RHOBTB2
was found to be reduced by 2 to 20-fold in 9 of 12 cell lines with
predicted LOH in the region of interest. In a study on
primary gastric cancers, LOH was found in 29% of tumors; a
sequence analysis identified several polymorphisms and 1
more missense somatic mutation[31].
In a recent study on head and neck cancer, RHOBTB1
has also been postulated as a tumor suppressor
gene[10]. The 10q21 region where the
RHOBTB1 gene is located has been identified as a hotspot region in head and neck
squamous cell carcinomas (HNSCC) in a genome-wide LOH
analysis[32]. Focusing on
RHOBTB1, Beder et al found a high frequency of LOH with a microsatellite marker located in
intron 7 of the gene[10]. In 12 of 52 tumor samples, LOH could
be demonstrated, and interestingly, 4 samples showed LOH
exclusively for the RHOBTB1 locus. Since almost 50% of
the tumor samples were not informative in the LOH analysis,
it is very likely that the RHOBTB1 locus is affected in a
higher proportion of tumors. A mutation analysis revealed 3
polymorphisms, but no pathogenic mutations. The
expression of RHOBTB1 decreased in 37% of the samples analyzed,
although it increased in 35%, but significantly, all
low-expression samples for which informative allelic loss data were
available displayed LOH.
Although we still have limited information on the status
of RHOBTB genes in tumors, the picture that emerges from
the reports discussed above is one of rare mutations but
common reduced or extinguished expression. This
observation can be made extensive to the third family member,
RHOBTB3. We have determined the expression of
RHOBTB3 in an array of tumor tissues and their matched normal tissues
and have found a moderate but significant decrease of
RHOBTB3 expression in the breast, kidney, uterus, lung,
and ovary tumors (Berthold J et al, personal communication).
It appears that mechanisms other than mutations are more
frequently implicated in the inactivation of these genes. One
such mechanism may be promoter methylation. The hypermethylation of CpG islands results in the
downregulation or complete abrogation of gene expression and is a frequent
epigenetic alteration in primary
tumors[33]. The promoter region of RHOBTB2
has a CpG island, and in RHOBTB1,
the promoter region and exon 1 (an untranslated exon) have a
high GC content and numerous CpG motifs. Interestingly,
the mutations found in the promoter and 5´UTR of
RHOBTB2 in some breast tumors might affect the regulation of gene
expression[29]. The _238G>A polymorphism abolishes a
putative Sp1-1 binding site and creates an additional CpG
dinucleotide. The _121C>T mutation abolishes binding sites
for the transcription factors E2F and snail, and the +48G>A
mutation creates a putative binding site for the bZIP910
transcription factor. Clearly, future work should be directed to
analyze this aspect of RHOBTB gene expression in tumor
tissues and cell lines.
Functions of RhoBTB
The role of RHOBTB genes as tumor suppressors,
initially attributed to RHOBTB2, more recently to
RHOBTB1, and probably extensively also to
RHOBTB3, is receiving increasing support. Nevertheless, the mechanisms by which
RhoBTB proteins exert this and other roles remain largely
speculative. Siripurapu et al have taken a large scale
approach to explore the roles of
RHOBTB2[34]. They constitutively expressed
RHOBTB2 in HeLa cells, followed by silencing of the ectopic gene and then a microarray analysis.
A comparison of the overexpressing and silenced samples
revealed significant alterations in genes belonging to 2
networks: one that regulates cell growth through cell cycle
control and apoptosis and one that is related to
cytoskeleton and membrane trafficking. Although the approach used
in this study is adventurous and rather artificial, evidence is
accumulating in support of the roles for RhoBTB proteins in
the processes revealed by Siripurapu et
al[34]. The identification of RhoBTB2 as a component and substrate of
cullin3-dependent ubiquitin ligase complexes was key for the
mechanistic understanding of RhoBTB
functioning[35]. Although several potential roles of RhoBTB proteins are considered
separately, they are probably interrelated. It is also very
likely that the function as adaptors of cullin3-dependent
ubiquitin ligases constitutes the underlying mechanism for
all other roles, therefore, it will be discussed first and more
extensively.
RhoBTB as adaptors of cullin3-dependent ubiquitin
ligases The identification of the BTB domain as adaptor in
cullin3-dependent ubiquitin ligase complexes prompted
Wilkins and coworkers to investigate whether RhoBTB2 may
also take part in the formation of such
complexes[35]. They identified the N-terminal region of murine cullin3 as an
interacting partner of RhoBTB2 in a yeast 2 hybrid
screening[35]. RhoBTB2 interacts specifically with cullin3, but not other
cullin family members in vivo; the interaction was mapped to
the first BTB domain in a series of pull-down experiments
with deletion constructs. Wilkins et al also provided
evidence that RhoBTB2 is itself a substrate for cullin3-based
ubiquitin ligase complexes, as treatment with proteasomal
inhibitor MG132 or shRNA ablation of cullin3 resulted in
increased levels of RhoBTB2, and RhoBTB2 was
polyubi-quitinylated by cullin3 complexes in
vitro[35]. Our own unpublished data indicate that many of these properties are
shared by all members of the RhoBTB subfamily, including
RhoBTB3.
RHOBTB2 was proposed as a candidate tumor
suppressor gene based on the fact that its re-expression in T-47D (a
breast cancer cell line that lacks RHOBTB2 transcripts)
caused growth inhibition, whereas the expression of the
somatic mutant D299N did not have the same
effect[9]. This mutation is placed in the first BTB domain immediately
before the insertion. In fact, it is interesting that almost all
missense mutations found in the RHOBTB2 locus reside in
the first BTB domain of the protein (Figure 1). The question
arises whether one or more of those mutations result in
impaired interaction with cullin3. This has been investigated
by Wilkins and coworkers who found that the Y284D mutant,
but not the D299N and D368A mutants, failed to
coimmuno-precipitate with cullin3, and consequently, had a longer
half-life than the wild-type
protein[35]. The Y284D mutation resides in the dimerization interface of the first BTB domain
and could prevent proper folding. Analogous mutants have
been shown to abrogate function by impairing folding of the
BTB domain, for example, in the transcription factor
PLZF[36].
We have found a correlation in the expression changes
between RHOBTB3 or RHOBTB1 and CUL3
in tumor tissues (Berthold J et al, personal communication), supporting
the view that RHOBTB genes and CUL3 may be coregulated
and the role of RhoBTB in tumorigenesis is related to its role
as adaptor for cullin3-dependent ubiquitin ligase complexes.
There are approximately 200 genes encoding BTB proteins
in the human genome, suggesting that a significant
proportion of cullin3-dependent complexes might control the
ubiquitinylation and degradation of cancer-related proteins
through multiple mechanisms. In fact, several BTB proteins
have been found to be linked to tumorigenesis, although
their roles in the formation of cullin3-dependent complexes
have generally not been addressed. To cite a few examples,
the tumor suppressor gene HIC1 (hypermethylated in
cancer 1) is located at a region of chromosome 17 that is
frequently hypermethylated or deleted in human tumors. It
works as a transcriptional repressor functionally
cooperating with p53 to suppress the age-dependent development of
cancer[37]. The transcriptional repressor and candidate
oncogene Bcl-6 is an important regulator of lymphoid
development and function. The BCL6 gene is localized in a region
implicated in chromosomal translocations frequently found
in non-Hodgkin's lymphoma of B-cell
type[38]. The Kelch-related Mayven has been proposed to promote tumor growth
through the induction of c-Jun and cyclin
D1[39]. Another example is Kaiso, involved in p120-catenin/Kaiso signaling
pathways that regulate gene expression in development and
carcinogenesis[40]. In fact, the role of cullin3-dependent
complexes in tumorigenesis can be placed in the wider context of
the cullin family where every member has been found to be
implicated in ubiquitinylation of cancer-related substrates
(see Guardavaccaro and Pagano[41] for a comprehensive
review).
RhoBTB, cell growth, and apoptosis As already
mentioned, Hamaguchi and coworkers reported that the
overexpression of RhoBTB2 in the breast cancer cell line
T-47D effectively suppressed cell growth in
vitro[9]. More recently, Freeman and coworkers have shown that the overexpression
of RhoBTB2 leads to a short-term increase in cell cycle
progression and proliferation, but long-term expression has a
negative effect on proliferation[42]. The growth arrest effect
of RhoBTB2 has been explained by the downregulation of
cyclin D1. Cyclin D1 is upstream of cyclin E, and the
overexpression of any of both prevented the growth arrest
effect of RhoBTB2[43]. The effect on cyclin D1 is probably
post-transcriptional, but only partially dependent on
proteasomal degradation. Moreover, it has not been
investigated whether cyclin D1 is degraded by cullin3-dependent
complexes through direct binding to RhoBTB2. In this
respect it is important to note that one mechanism as to how
cullin3-dependent complexes regulate the cell cycle is
through the targeting of cyclin E for
ubiquitinylation[44]. The downregulation of cyclin D1 is essential for the cell
proliferation suppression effect of RhoBTB2, but this works for
T-47D cells and not for 293 cells. It therefore appears that the
regulation of cyclin D1 is not a universal tumor suppressive
mechanism used by RhoBTB2. The explanation has been
put forward that resistance to RhoBTB2 in some cell lines
may be achieved by rapid destruction of the protein through
26S proteasome-mediated degradation[45]. Further support
for the roles in cell cycle regulation has been provided
recently with the identification of RHOBTB2
as a target of the E2F1 transcription
factor[42]. E2F1 is a member of a class of
E2F implicated in the transcription of genes necessary for
DNA replication and cell cycle progression and can also
promote apoptosis[46]. RhoBTB2 levels increase upon
initiation of prophase and decrease at telophase, and this effect
depends on E2F1[42].
RhoBTB2 levels also increase during drug-induced
apoptosis in an E2F1-dependent manner, and the
downregu-lation of RHOBTB2 delays the onset of
apoptosis[42]. In agreement with an implication in this process,
RhoBTB was found in Drosophila as one of several genes whose
expression was significantly upregulated in a DNA microarray
analysis aimed at identifying genes associated with cell death
induced by the steroid hormone
ecdysone[47]. Interestingly, in this study, additional genes encoding Rho-signaling
components, most notably Rac2, also appeared upregulated.
However, the role of RhoBTB as a candidate cell death
regulator was not investigated further.
RhoBTB and vesicle transport Chang
et al have addressed the potential role of RhoBTB2 in vesicle transport in
a fluorescent recovery after photobleaching analysis with
the help of a vesicular stomatitis virus glycoprotein (VSVG)
fused to GFP[12]. VSVG is extensively used to study
anterograde transport from the endoplasmic reticulum to the Golgi
apparatus. Knockdown of endogenous RhoBTB2 hindered
the ER to Golgi apparatus transport and resulted in the
altered distribution of the fusion protein. In this study, the
authors found that GFP_RhoBTB2 was distributed in a
vesicular pattern when expressed at low levels. Some of the
vesicles appeared adjacent to microtubules and an intact
microtubule network seemed to be required for the mobility
of RhoBTB.
The localization of RhoBTB1 and RhoBTB2 in vesicular
structures had been postulated before. Aspenström and
coworkers reported the accumulation of the
ectopically-expressed proteins at perinuclear structures that did not
colocalize with lysosomal or Golgi apparatus
markers[48]. These structures apparently represent aggregates, and can
be also induced upon the ectopic expression of RhoBTB3.
However, when RhoBTB3 is expressed at moderate levels, it
displays a vesicular pattern. Many of the vesicles colocalize
with early endosome markers, and localization in close
vicinity of microtubules is also apparent. As mentioned earlier,
RhoBTB3 ends with a prenylation motif, and the C-terminal
extension of RhoBTB3 is necessary and sufficient for the
attaching of the protein to vesicles (Berthold J
et al, personal communication). However, prenylation might not be
the only mechanism required for the targeting of RhoBTB to
vesicles as RhoBTB1 and RhoBTB2 lack a prenylation motif.
Further, in support of a role in vesicle trafficking,
RhoBTB has been identified as one of the genes that suppress the
neuromuscular junction overgrowth phenotype induced in
Drosophila larvae by the expression of a dominant negative
form of the N-ethylmaleimide sensitive factor
(NSF)[49]. NSF is an ATPase that participates in vesicle trafficking through
binding to the SNARE complex and is also important for the
regulation of receptor trafficking[50]. Interestingly,
NSF is one gene whose expression appeared altered in the study of
Siripurapu et al discussed
earlier[34], which is suggestive of a conserved mechanism that requires further investigation.
If a role for these proteins in vesicle trafficking gains support,
then RhoBTB will engross the growing list of Rho GTPases
involved in this process. The mechanism remains obscure,
but will be most likely an unusual one.
RhoBTB and the actin filament system Although very
atypical, RhoBTB proteins are members of the Rho family,
therefore, the first aspect that was investigated was their
effect on the organization of the actin filament system.
Aspenström and coworkers observed a moderate influence,
if at all, on the morphology and actin organization of porcine
aortic endothelial cells upon the ectopic expression of
RhoBTB1 and RhoBTB2[48], an observation that we have
made extensive to RhoBTB3 and several other cell lines
(Berthold J et al, personal communication). Not surprisingly,
neither RhoBTB1 nor RhoBTB2 were found to interact with
the GTPase-binding domain of WASP, PAK1, or Rhotekin, 3
well-known effectors of many typical Rho
GTPases[48]. Confirming that, at least in metazoa, RhoBTB proteins do not
play a major role in the organization of the actin filament
system, DmRhoBTB was found among the proteins whose
depletion had no effect on lamellae morphology in
Drosophila S2 cells[51]. These cells can be induced to spread when
plated on a concanavalin A-coated surface and constitute
then an appropriate system to study the formation of lamellae.
Unlike metazoan RhoBTB, the
Dictyostelium ortholog RacA may be directly implicated in the regulation of the actin
cytoskeleton, although the evidence is indirect and no
functional studies have been published yet. The
racA gene is very weakly expressed throughout the life cycle of
Dictyostelium[6], but the protein is present at all stages. The GTPase domain
of RacA, which as already mentioned, is very closely related
to members of the Rac subfamily, is able to interact with the
Rac-binding domain of WASP and kinases of the PAK
family in yeast 2 hybrid assays[52_54], although these
interactions remain to be demonstrated in
vivo. Unlike metazoan RhoBTB, RacA is susceptible to regulation by RhoGEF and
RhoGAP, and in vitro interaction with a RhoGEF, GxcDD,
has been reported recently[55]. We speculate that RacA
represents a "primitive" cytoskeleton-regulating stage of the
RhoBTB subfamily that was replaced in the evolved
metazoan RhoBTB proteins by roles in cell proliferation and
vesicle trafficking.
Conclusion
There is increasing evidence linking Rho-regulated
signal transduction pathways to tumorigenesis and
metastasis[11,56,57]. Rho GTPases play a role in the acquisition of an
invasive phenotype of tumor cells, either directly via their
effects on the cytoskeleton, or indirectly via changes in gene
transcription. It is noteworthy that with the exception of
RacH and perhaps Rac1, no mutations in typical Rho GTPases
have been found to be associated specifically with tumors.
It is rather alterations in the expression or activation levels
of these proteins which characterizes many tumors. For
example, the expression of Rac1b (an alternative splice variant
of Rac1) increases in colorectal
tumors[58], the overexpression of RhoC correlates with the invasiveness of non-small cell
lung cancer[59], and Cdc42 is overexpressed in
HNSCC[60].
Unlike typical Rho GTPases, those of the RhoBTB
subfamily appear to play a part in the carcinogenic process
through a mechanism that involves the downregulation or
loss of function. Taking into consideration the ability of
RhoBTB proteins to constitute cullin3-dependent complexes,
a model emerges in which these proteins recruit substrates
for degradation in the 26S proteasome (Figure 2). While the
first BTB domain is involved in recruitment of cullin3 and
associated components, other regions of the protein, such
as the GTPase domain and the C-terminal conserved region
or even the second BTB domain, would function as
substrate recognition domains. The insertion of the first BTB
domain probably folds away from the globular BTB core and
might also be implicated in substrate recognition, whereas
the proline-rich region of RhoBTB1 and RhoBTB2 could play
regulatory roles through interaction with SH3
domain-bearing proteins.
RhoBTB proteins would be required to maintain constant
levels of putative substrates, thus exerting regulatory roles
during the cell cycle, vesicle transport, and in lower
eukaryotes, cytoskeleton homeostasis. It is easy to understand that
situations that result in the impaired expression of
RHOBTB genes, or more rarely, mutations that result in impaired
functioning (binding to cullin3, dimerization, interaction with
substrates, targeting) of the protein might lead to the
accumulation of RhoBTB substrates and alterations of the
cellular homeostasis. Such a regulatory mechanism could be the
basis of the tumor suppressor role of RhoBTB proteins and
is analogous to the well-studied role of the von
Hippel_Lindau (VHL) tumor suppressor. VHL is an adaptor for
cullin2-dependent ubiquitin ligase complexes that target the
hypoxia-inducible factor for degradation. Nearly 70% of
naturally-occurring cancer-predisposing mutations of VHL
disrupt the formation of these
complexes[61]. Obviously, if we wish to clear the mechanisms as to how the malfunction
of RhoBTB proteins results in tumor formation, we
imperatively need to know how these proteins are regulated at all
levels (transcriptional, translational, and post-translational)
and what their substrates are.
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