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Department of Anatomy and Cell Biology, Uniformed Services University of Health Sciences, Bethesda, Maryland 20814, USA
1Correspondence: Department of Anatomy and Cell Biology, Uniformed Services University of Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA. E-mail: msrivastava{at}usuhs.mil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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, cyclic AMP-dependent protein kinase, and ecto-protein kinase.
For these and other reasons, nucleolin is fundamental to the survival
and proliferation of cells. Considerable progress has been made in
recent years with the identification of new nucleolin binding proteins
that may mediate these many nucleolin-dependent functions. Nucleolin
also functions as a cell surface receptor, where it acts as a shuttling
protein between cytoplasm and nucleus, and thus can even provide a
mechanism for extracellular regulation of nuclear events. Exploration
of the regulation of this multifaceted protein in a remarkable number
of diverse functions is challenging.—Srivastava, M., Pollard, H. B. Molecular dissection of nucleolin's role in growth and cell
proliferation: new insights.
Key Words: nucleolin • ribosome biogenesis • phosphorylation • proteolysis • binding proteins • ligands • apoptosis • cell cycle
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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. Certain nucleolar components
appear to coordinate the phenomena of dynamic changes that occur in the
nucleolar structure between interphase and the mitotic phase of the
cell cycle (2
, 3)
. Among these candidate nucleolar
components, nucleolin, with a unique multiple domain structure, plays a
fascinating and fundamental role in linking nucleolar activities to
cell proliferation and mitotic dynamics.
Nucleolin is a ubiquitous, nonhistone nucleolar phosphoprotein of
exponentially growing eukaryotic cells and is present in abundance at
the dense fibrillar and granular regions of nucleolus (4
, 5)
. Intact nucleolin is the major species and represents 5% of
nucleolar protein in actively dividing cells. In nondividing cells,
degraded forms of various molecular size are predominantly expressed
due to autodegradation (6
7
8
9)
. The first comprehensive
review of nucleolin was published more than 10 years ago and focused on
the problem of ribosomal RNA transcription, maturation, and assembly
(10)
principally because the expression of this major
nucleolar phosphoprotein was directly correlated with ribosomal DNA
(rDNA) transcription (10
11
12
13
14)
. The focus of nucleolin
research has widened to include chromatin decondensation
(15)
, cytoplasmic nucleolar transport of ribosomal
components and preribosomal particles (12)
, and
nucleogenesis (14)
. In particular, nucleolin has been
shown to be a component of B cell-specific transcription factor
(16
, 17)
, an autoantigen (18
, 19)
, a DNA/RNA
helicase (20)
, DNA-dependent ATPase (21)
, and
a transcriptional repressor (22)
. The protein would
therefore appear to be involved in fundamental aspects of
transcriptional regulation, cell proliferation, and growth. Indeed, an
increasingly complicated literature suggests that these new functions
are just the tip of the iceberg; this review is designed to be a map
through the phenomenology of this amazing protein.
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STRUCTURE-FUNCTION RELATIONSHIPS |
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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, the human alternative splicing factors
ASF and SF2 (24
, 25)
, and the mammalian splicing factors
U2AF and SC35 (26
, 27)
. To date, more than 200 consensus
sequence type RNA binding domains (CS-RBDs) containing proteins have
been found, including proteins with various functions in RNA splicing,
processing, or translation. These RNA binding proteins have a common
structure that is used for nucleic acid binding, with additional
modules attached for various other functions and for determination of
specificity. Since the proteins described above have different
functions and bind to distinctly different RNA molecules, it is not
surprising that sequences are substantially different in regions
outside the consensus sequences. In this respect, nucleolin appears to
be more closely related to hnRNP protein A1 than to the poly(A) binding
protein. This is consistent with the role of hnRNP protein A1 in
packaging newly synthesized RNA compared to the poly(A) binding
protein's association with cytoplasmic RNA.
The amino-terminal domain controls rDNA transcription
Nucleolin is composed of 707 amino acids, and analysis of
nucleolin cDNA has revealed the presence of three major domains
(6
, 28)
. Due to the high content of negatively charged
amino acids in the amino-terminal domain, nucleolin runs in sodium
dodecyl sulfate-polyacrylamide gel electrophoresis gels with an
apparent molecular mass of 105 kDa, although the actual calculated mass
is 77 kDa from the cDNA sequence (6
, 28)
. The
amino-terminal third of nucleolin contains alpha helical domains
comprising four lengthy acidic stretches, similar to those of certain
high-mobility group proteins. This domain has an analogous
high-mobility group function in inducing nucleolar chromatin
decondensation through ionic interaction of the acidic amino acids with
histone H1 (10
, 15
, 29)
. These acidic regions, which are
variable and less conserved in different species (6
, 28
, 30)
, bind to nontranscribed spacer regions in DNA that separate
the rRNA gene repeats (31
32
33)
. This process organizes
nucleolar chromatin in a way that confers specificity for transcription
of rDNA by RNA polymerase 1 (34)
. These regions also
contain in vivo phosphorylation sites for casein kinase II
(CKII), cdc2, and protein kinase interspersed with basic lysine
residues susceptible to proteolysis (14
, 35
, 36)
. The
importance of these sites lies in the fact that transcription of rDNA
genes starts only when the serine residues are phosphorylated by casein
kinase II and the proteolytic sites in nucleolin are cleaved
(34)
. The phosphorylation of nucleolin seems to enhance
its degradation by proteases concordantly, suggesting that the
stability of nucleolin is dependent on phosphorylation (37
, 38)
.
As is well known, the transcription of rDNA genes by RNA polymerase 1
gives rise to 47S RNA, which is rapidly cleaved to yield the mature
18S, 28S, and 5.8S rRNA species. Several processing sites have been
mapped during this maturation process at the vicinity of 5'-end of the
external transcribed spacer (ETS) region of pre-RNA. The 5'-end of
pre-RNA is assembled into a large ribonucleoprotein complex involving
nucleolin, fibrillarin, and different snoRNPs (small
ribonucleoproteins). The specific interaction of nucleolin with the
rRNA substrate might be the first step in the processing event.
Nucleolin could then recruit other factors, including U3 snoRNP,
required for the cleavage reaction in formation of the processing
complex (39
, 40)
. In this complex, nucleolin interacts
with RNAs as well as other proteins (41
, 42)
. Thus,
nucleolin's involvement in rDNA transcription, pre-RNA processing,
ribosome assembly, and maturation mediated through the interaction of
nucleolin with rDNA (nontranscribed spacer, 5' and 3'-external
nontranscribed spacer, internal transcribed spacer, nascent 45S
pre-RNAs, RNA polymerase 1, 18S, 28S rRNAs) and ribosomal
proteins could be an efficient way for the cell to regulate the
production of large amount of ribosomes needed throughout its life.
The central globular domain controls pre-RNA processing
While the amino-terminal region interacts with chromatin and is
involved in modulating nucleic acid binding activity, the central
globular domain is involved in pre-RNA recognition, condensing, and
packaging (43
44
45)
. The central domain of nucleolin
exhibits alternating hydrophobic and hydrophilic stretches, and also
contains four CS-RBDs (46)
. The latter domains consist of
~80 amino acid residues, each containing two highly conserved
regions, the so-called RNP motifs (47)
. Nucleolin
interacts specifically with an RNA stem-loop structure (NRE) and with
in vitro-selected RNAs containing UCCCGA through its first
two RNA binding domains (CS-RBDs 1 and 2) (32
, 48
, 49)
.
This property may account for nucleolin's association with pre-rRNA in
the nucleolus. The 45 kDa fragment of nucleolin (exon 5–14, containing
the central and carboxyl-terminal domains) binds strongly to G-rich DNA
and ATP. This ATP binding is important for its ATPase activity.
Furthermore, this region of nucleolin directly binds GTP, dATP, and
dGTP but not dCTP, dTTP, or dUTP (50)
. In addition, the
adenosine analog DRB
(5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole), an inhibitor of
hnRNA synthesis, modifies the stability and the amount of nucleolin,
and thus the nucleolar morphology (51)
. This implicates
nucleolin as a key player in nucleolar structure and function.
The carboxyl-terminal domain controls nucleolar localization
The extended carboxyl-terminal domain is rich in glycine residues
and is interspersed with dimethylarginine and phenylalanine. The
function of this domain is to control unstacking of bases and the
unfolding of RNA secondary structure (52
53
54
55)
. These
events permit RNAs access to the RNA binding motifs located in the
central region of nucleolin (44)
. Furthermore, the
intrinsic protease activity of nucleolin for autodegradation has been
mapped to the carboxyl-terminal two-thirds of the nucleolin molecule
(8)
. Nucleolin is accumulated within the nucleolus by
virtue of its binding to other nucleolar components (probably rRNA) via
the two RNA recognition motifs and the glycine-rich domains of the
carboxyl-terminal region (45
, 56)
. Hints as to how the
mammalian nucleolin is involved in the formation and maturation of
preribosomal particles have come from studies of homologous proteins in
yeast. In particular, the glycine- and arginine-rich carboxyl-terminal
domain of nucleolin is shared homologously by the yeast genes nucleolar
phosphoprotein 1/fibrillarin, SSB1, NSR1, Gly/Arg-rich (GAR1), and
GAR2. Disruption of the GAR2 gene in yeast affects normal cell growth
and leads to an accumulation of 35S pre-RNA and a decrease of mature
18S and 40S rRNA (57
, 58)
. Deletion of NSR1 causes a
severe growth defect at cold temperatures and leads to a rapid decrease
in 27S, 20S, and 7S rRNA precursors in the nsr1 strain
(59)
. In addition, this domain may mediate protein/protein
and/or protein/nucleic acid interactions (60)
. A recent
report has shown that nucleolin interacts directly with a subset of
ribosomal proteins through its carboxyl-terminal domain
(61)
.
Nucleolin is a shuttling protein between the plasma membrane,
cytoplasm, and nucleus
Nucleolin has been shown to be a shuttling protein between
the cytoplasm and nucleus and contains only an import signal
(62)
. In addition, nucleolin possesses binding affinity
for some ribosomal proteins and interacts with a subset through its
carboxyl-terminal domain (61)
, suggesting that nucleolin
may play a role in the assembly of ribosomal subunits by bringing
together both ribosomal proteins and RNA. Through its nucleocytoplasmic
shuttling property, nucleolin may act as a carrier either during the
import of ribosomal proteins to the nucleus or during the export of
ribosomal subunits to the cytoplasm (12
, 56
, 63)
.
Nucleolin thus serves as an `adaptator' for specific binding to rRNA.
The bipartite nuclear localization signal, situated between the
amino-terminal domain and RNA binding domain of nucleolin, is necessary
for nucleolin to enter the nucleus (64)
. In the developing
Xenopus embryo, nucleolin has been shown to be localized
cytoplasmically up to the midblastula stage, but thereafter accumulates
in the nucleus. The nucleolar accumulation event starts at the
gastrulation stage (65)
. In addition, massive
phosphorylation by cdc2 or CKII kinases localizes nucleolin to the
cytoplasm, and nuclear translocation of nucleolin accompanies
dephosphorylation (66)
. Laminin alters the distribution of
nucleolin in intestinal epithelial (IEC-6) cells, which may be an early
signal for cell proliferation (67)
.
As mentioned earlier, the yeast NSR1 gene bears a structural similarity
to nucleolin. Like nucleolin, NSR1 in yeast is directly involved in
pre-RNA processing and regulates the nuclear entry of ribosomal
proteins required for proper assembly of pre-rRNA particles
(68)
. Shuttling in the opposite direction begins as soon
as the 47S pre-RNA is assembled into the 80S particle. At this point,
nucleolin might be involved in the transport of these newly assembled
ribosomal subunits into the cytoplasm. Thus, nucleolin's binding to
both chromatin and the 5'-ETS region of the nascent pre-RNA suggests
that it may play a key role in the assembly and/or processing of
preribosomes and transport of ribosomal proteins across the nuclear
envelope (10
11
12)
.
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GENE STRUCTURE |
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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, 69)
. The human nucleolin gene is
localized on chromosome 2q12-qter (69)
, which is not
syntenic with any other RNA binding proteins so far localized.
Several small nucleolar RNAs (snoRNAs), have been shown to be essential
for processing steps that lead to the production of 28S, 18S, and 5.8S
rRNA. These antisense snoRNAs have been shown to play a chaperone role
in the processing of the pre-rRNA (70)
. U20 snoRNA
contains an extended region (21 nucleotides) of perfect complementarity
to the phylogenetically conserved sequence in 18S rRNA
(71)
. The detection of U20 snoRNA in intron 11 of the
mammalian nucleolin gene supports the notion that this particular
genomic organization has some relevance to those functions of nucleolin
that are involved in the production of ribosomes. Perhaps this
observation might provide the basis for regulatory linkages during
ribosome biogenesis or function. Since these snoRNAs are no longer
present in ribosomes, they must have been removed during or after
processing of pre-RNA. Nucleolin is a good candidate to perform this
function, since nucleolin has DNA/RNA helicase activity, and the number
of base pairs in these sequences (up to 21) is fully compatible with
the length of RNA duplexes that nucleolin could unwind in
vitro (20)
. In addition, the presence of a second
intron encoded snoRNA, termed U22, in the nucleolin gene may reflect
the multiplicity of regulatory circuits for ribosome production
mediated by this multifunctional protein throughout the cell cycle and
in different conditions of cell growth (6
, 14
, 36)
.
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REGULATION OF NUCLEOLIN FUNCTION |
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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, 36
, 72)
, proteolysis (34
, 73)
, autodegradation (7
, 8)
, and ADP-ribosylation
(74)
. For example, the amino-terminal domain contains
sites for modification by phosphorylation and proteolysis, whereas the
carboxyl-terminal domain has sites for methylation and proteolysis. The
idea that nucleolin function is coupled to growth control by
phosphorylation is supported by the observation that active rRNA
transcription is positively correlated with highly phosphorylated
nucleolin (75
76
77
78)
. Treatment of human fibroblasts and
keratinocytes with okadaic acid, calyculin A (79)
,
polyamines, or histones induces hyperphosphorylation of N60, a
proteolytic product of nucleolin (80
81
82)
. Therefore, a
diversity in susceptibility to such modifications can underlie
regulation.
Serine phosphorylation of nucleolin occurs during interphase
Cell growth requires CKII-mediated phosphorylation of several
cytosolic and nuclear substrates (69
, 83
84
85)
. In growing
cells, CKII phosphorylates nucleolin, topoisomerase 1 (Topo 1), and RNA
polymerase 1. It may not be coincidental that all three proteins are
localized on chromosomes containing rDNA. These chromosomes are
decondensed and transcribed in daughter cells, and thus appear to be
involved in nucleolar structural organization in the G1 phase
(86
, 87)
. Phosphorylation by CKII also enhances nucleolin
as a substrate for protease to produce active 30 and 72 kDa proteins.
These latter fragments then trigger rDNA transcription by RNA
polymerase 1 (37)
. Both CKII activity and phosphorylation
of nucleolin are enhanced at day 12 of gestation (72)
,
after stimulation with mitogens (70)
, in regenerating rat
liver after partial hepatectomy (88)
, and in tumor cells
(72)
. In addition, CKII phosphorylation of nucleolin and
rRNA synthesis have been reported to be dependent on hormones, such as
dexamethasone (78
, 89)
and androgen (38)
, and
on growth factors such as epidermal growth factor (90)
and
fibroblast growth factor 2 (91)
.
Insulin action may also be mediated by phosphorylation of nucleolin.
Insulin induces serine phosphorylation, possibly via CKII, at
subnanomolar concentrations, whereas insulin effectively promotes
dephosphorylation of nucleolin at the micromolar concentration range.
This dose-response relationship is identical to insulin-induced effects
on the RNA efflux from nuclei, suggesting that the phosphorylation
state of nucleolin might be a regulator of (ribosomal) RNA transport
through the nuclear membrane (92)
. The interaction of CKII
with the FK506 binding protein (FKBP) results in the phosphorylation of
nucleolin, which itself leads to the regulation of cell growth
(93)
. FKBP is the cellular receptor for the
immunosuppressive drugs FK506, cyclosporin, and rapamycin. These
findings strongly suggest that the signals that affect CKII also change
nucleolin's function, which in turn affects rDNA transcription and
ribosome biogenesis.
Threonine phosphorylation of nucleolin occurs during mitosis
Whereas serine phosphorylation is related to nucleolar function in
the control of rDNA transcription, threonine phosphorylation is linked
to mitotic reorganization through condensation of nucleolar chromatin
(94
95
96
97)
. During mitosis, cdc2 kinase phosphorylates
threonine in the TPKK motifs. These TPKK motifs occur nine times in the
amino-terminal domain of nucleolin. For example, mitogenic stimulation
of resting T cells and lipopolysaccharide induction of mitosis in
resting splenic cells have been shown to lead to the induction of
phosphorylation of nucleolin. The degree of phosphorylation is
therefore closely correlated to the degree of cell proliferation
(50
, 75)
. The cdc2 sites play a dual role by enhancing
nuclear translocation exclusively in their dephosphorylated state and
promoting cytoplasmic localization when phosphorylated, thereby
providing a powerful cell-cycle-dependent regulatory element of the
nuclear localization signal. Evidence that nucleolin is extensively
phosphorylated during mitosis by cdc2 kinase further implies that it
controls the mitotic changes in nucleolar structure by condensing
chromosomes. It is possible that sequential cdc2 and CKII
phosphorylation could modulate nucleolin function in controlling
nucleolar structure and activities between interphase and the mitotic
phase during cell growth.
Phosphorylation of nucleolin by PKC- and ecto-kinases:
implications for global function
Nucleolin is a nuclear target protein for cyclic AMP and PKC-.
Cyclic AMP-dependent protein kinase influences the phosphorylation of
nucleolin. This is exemplified during proliferative stimulation
(transition from G1 to S phase) of the rat parotid gland by
isoprenaline (98)
. Recently, it was reported that it is
also a substrate for PKC-, which is required for nerve growth
factor- (NGF) induced differentiation of PC12 cells. It was suggested
that nucleolin might serve to relay NGF signals from cell surface to
nucleus in PC12 cells (99)
. It has been shown that
ecto-protein kinase also phosphorylates nucleolin, which suggests that
nucleolin expressed on the cell surface is regulated by a cell surface
kinase (100)
. Inasmuch as nucleolin functions on the cell
surface as a default LDL receptor in HepG2 cells, it has been
speculated that nucleolin might use this property to verify that
sufficient exogenous lipids are available to support cell growth
(101)
. Being a regulated shuttling nuclear protein,
nucleolin could thus transfer cytoplasmic signals between the cell
surface, the cytoplasm, and the nucleus. Phosphorylation of nucleolin
by CKII and cdc2 kinase has been shown to regulate its helicase
activity (102)
, and phosphorylation by CKII, cdc2 kinase,
PKC-, cyclic AMP-dependent protein kinase, and ecto-protein kinase
may regulate nucleolin's functional abilities in chromatin
organization, rRNA packaging, rDNA transcription, or ribosome assembly.
Modification of nucleolin function by proteolysis: apoptosis and
autodegradation
Nucleolin is also a central player in the process of T
lymphocyte-mediated apoptotic cell death. Nucleolin in the target cell
is physiologically cleaved by granzyme A (7
, 8
, 103)
. The
mechanism involves secretion of perforin and serine proteases by
cytotoxic T cell lymphocytes and natural killer cells. Perforin forms
pores in the target cells, enabling granzyme A to access nucleolin.
Cleaved nucleolin activates autolytic endonucleases, which fragment DNA
to cause apoptosis (104
, 105)
. Recently, nucleolin was
identified as an apoptosis-associated protein involved in
anti-immunoglobulin M antibody-mediated apoptosis in the human Burkitt
lymphoma cell line (106)
. For some unknown reason,
nucleolin has been passed over by the apoptosis reagent supply
companies, but this may change. Nucleolin is stable in actively
dividing cells. In nondividing cells, nucleolin autocatalyzes its own
degradation, which can be inhibited by nuclear extracts prepared from
proliferative cells (7
, 107)
. In proliferating cells,
nucleolin is regulated by the expression of a proteolytic inhibitor
that prevents the self-degradation activity of nucleolin
(7)
. The ability to stabilize nucleolin could also be of
use to cancer cells.
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NUCLEOLIN, HSP70, AND NUCLEAR TRANSPORT |
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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appear to have similar ATP binding domains, contain
serum responsive elements in their promoters, and encode small
nucleolar RNAs in their introns. Their gene expressions are both
coactivated during prereplicative stages of hepatocytes
(109)
. In addition, both nucleolin and hsp70 contain
regions that resemble the `nucleolar targeting' sequence of HIV tat
protein (110)
. Recently, nucleolin has been suggested to
be functional as potential receptor in the HIV binding process by
virtue of its capacity to interact with the V3 loop of gp120
(111)
. Hsp 70 acts during mitochondrial transport to help
maintain nascent precursors in the extended conformation required for
translocation (112)
. A large number of ribosomal proteins,
which are karyophilic proteins, have the propensity to aggregate. They
may require hsp70 and nucleolin to prevent hydrophobic interactions and
translocate them into the nucleolus. Therefore, it is conceivable that
hsp70 and nucleolin could play a role in nuclear transport and
contribute to the efficiency of ribosomal biosynthesis. |
NUCLEOLIN AND CELL GROWTH |
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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. Indeed, nucleolin is
used in studies of different cancer cell lines as a useful marker for
cell proliferation (114
115
116
117)
. Nucleolin is present at low
levels in nondividing cells and is preferentially associated with
chromatin (117)
. The amount of nucleolin is low in
serum-deprived cells; nucleolin expression is induced by v-src in mid
and late G1, and thus is likely to be necessary for cell cycle
progression into G1 (118)
. In ABAE cells undergoing the
Go-G1 transition, basic FGF enters the nucleolus and stimulates
nucleolin, which in turn activates the transcription of ribosomal genes
(13)
. Even in plants, Nucms1 (a plant homologue of
nucleolin) expression is induced in the G1 phase on mitogenic
stimulation of Go-arrested leaf cells. No expression has been reported
in cells that have exited the cell cycle and are undergoing
differentiation or polar growth (119)
. In addition,
retinoic acid and dibutyryl cyclic AMP increase neurite outgrowth
during differentiation of human neuroblastoma cells and have an
inhibitory effect on cell proliferation. Simultaneously, the expression
of N-myc, nucleolin, and hsp70 are down-modulated, indicating a
possible association of expression of these three genes
(120)
. In cancer, the relationship of nucleolin (a major
nucleolar Ag-NOR protein) and cell proliferation represents a reliable
parameter predicting the tumor growth rate
Nucleolin expression is regulated post-transcriptionally and
transcriptionally in the developing embryo. For example, mRNA levels in
Xenopus, are high in adult tissues, where protein expression
is exceedingly low (14)
. Ribosome synthesis is activated
during oogenesis and embryogenesis. These data indicate that nucleolin
participates in nucleogenesis (14)
. Nucleolin appears
before rDNA transcription and ribosome synthesis start. Maximal
accumulation of nucleolin coincides at gastrulation with nucleolar
reformation.
During mouse embryogenesis, CKII and nucleolin increase
concomitantly at day 12 of gestation (72
, 121)
. In chicken
embryos, transcriptional down-regulation of nucleolin has been observed
between days 3 and 11, with a decrease in both mRNA and protein levels
of nucleolin. Nucleolin mRNA progressively decreases during
spermatogenesis. Nucleolin message is present only up to the round
spermatid stage and is absent from mature sperm
(122)
.These findings suggest that nucleolin is needed for
cell proliferation and nucleogenesis and is down-modulated during
differentiation (123)
along with N-myc and hsp70.
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NUCLEOLIN BINDING PROTEINS AND LIGANDS |
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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. The interaction of CKII with nucleolin is promoted
by spermine and inhibited by heparin. The nature of the interaction
shows a similarity to CKII association with p53, DNA topoisomerase II,
and HSP90 (37)
. Interaction of the 540–628 bp region of
nucleolin with the 194–239 bp region of B23, an additional nucleolar
protein, may represent a nucleolar-targeting mechanism for nucleolin to
enter nucleolus in which B23 acts as a nucleolar localization signal
binding protein (125)
. Topo 1 is involved in the
regulation of DNA supercoiling, gene transcription, and rDNA
recombination. The interaction of the 166–210 aa region of Topo 1 with
nucleolin may be needed for Topo 1 to enter the nucleus. Thereafter,
nucleolin could target Topo 1 to sites of transcription
(126)
. It has recently been reported that the Gar2 gene
(nucleolin-like protein) from Schizosaccharomyces pombe
results in a mutant that is defective in cytokinesis and nuclear
division (127)
. Nucleolin fragments (70, 48, 47 kDa) and
heterogeneous nuclear ribonucleoprotein C (39, 38 kDa) specifically
interact with the 3'-untranslated region of amyloid precursor protein
(APP) mRNA and stabilize the APP's mRNA. This is a mechanism for
post-transcriptional regulation (128)
. Midkine and heparin
binding growth-associated molecules bind to nucleolin, which then
translocates these associated molecules to the nucleus in PYS-2, 3T3,
and L cells (129)
. Furthermore, nucleolin is proposed to
be a nuclear matrix binding protein (130)
. In addition,
nucleolin binds to a 222-nucleotide motif within the nonstructural unit
of the viral (minus) strand of minute virus of the mouse. This suggests
that nucleolin may be involved in the regulation of the parvoviral life
cycle (131)
Nucleolin is a cell surface receptor
Although nucleolin is localized predominantly in the nucleolus, it
has also been shown to be localized in a phosphorylated form on the
cell surface of different cells (132)
. Cell surface
expression of nucleolin has been further substantiated by other
investigators. Nucleolin is the protein that specifically binds apoB-
and apoE-containing lipoprotein to the surface of the HepG2 cells
(101)
. Nucleolin serves as a substrate for an ecto-protein
kinase on the cell surface of HeLa cells (100)
. The
neurite-promoting IKVAV site of laminin-1 (basement membrane protein)
binds to nucleolin on the cell surface and has been found to promote
the differentiation of primary neurons and a variety of neural cell
lines (133)
. The significant levels of nucleolin in mature
brain and in differentiating neural cells suggest that nucleolin may
not only function in signaling by extracellular matrix molecules, but
may also be important in differentiation and maintenance of neural
tissue (134)
.
Viral infection of host cells depends primarily on binding of the virus
to a specific cell surface protein. Recently, human nucleolin has been
shown to interact with the amino-terminal domain of hepatitis delta
antigens and modulate hepatitis delta virus replication
(135)
. Although polio and Sendai viruses do not bind to
nucleolin, Coxsackie B viruses does bind to nucleolin specifically on
the cell surface (112)
. The fructosyllysine-specific
binding protein from cell membrane of the monocyte-like cell line U937
is identical to nucleolin (136)
. These findings indicate
that nucleolin itself or putative splicing products might function as
cell surface receptors or binding proteins and thus could act as
mediators to provide a potential mechanism for extracellular regulation
of nuclear events
Nucleolin binds to lipopolysaccharides, immunoglobulin switch
repeats, matrix attachment region, and telomeres
In mitogenically stimulated murine splenocytes with bacterial
lipopolysaccharides, nucleolin has been found to be up-regulated in its
DNA and ATP binding properties as a result of increased stability and
synthesis of nucleolin. Nucleolin also exhibits binding specificity for
the immunoglobulin isotype switch DNA repeats (16
, 50)
.
Because nucleolin specifically binds to switch region DNA, it might act
as a switch region targeting factor in the B cell-specific DNA
recombination complex called SWAP (16
, 17)
. It has also
been shown that transcription at switch regions is important for
recombination (137
, 138)
. The fact that nucleolin is
capable of binding to switch repeats indicates it could potentially
play a role in active or passive repression of transcription at switch
regions. Such actions may attenuate or inhibit switch recombination
processes. As a positive regulator of recombination, nucleolin, with
its four nucleic acid binding domains, can bind and hold the cleaved
strands together during the process of scission and ligation. As a
negative regulator, it can play a role as an inhibitor by preventing
the recognition of switch repeats. Alternatively, it can protect switch
regions from premature or extensive degradation during or prior to
recombination. Nucleolin has been shown to be a transcriptional
repressor for the alpha-1 acid glycoprotein gene (22)
. In
addition, nucleolin also binds to single-stranded T-rich regions of the
matrix attachment region, to the pre-mRNA 3'-splice site sequence
r(UUAG/G), and to the human telomeric DNA sequence d(TTAGGG)n
(139)
. These findings suggest that nucleolin may function
not only in the control of rRNA transcription and ribosome assembly in
the nucleolus, but may also be involved in processes taking place
outside the nucleolus.
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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, 141)
. Autoimmune serum from systemic lupus erythematosus
patients recognize the 387–461bp domain of nucleolin
(19)
. In scleroderma-like chronic graft-vs.-host disease,
three cases have been reported to have antibodies to nucleolar antigen
nucleolin. Four cases are reported to possess antibodies to
topoisomerase 1. Two cases have antibodies to PM-Sc1. One case has
antibodies to La/SSB (142)
. Recently, human nucleolin has
been shown to interact with the amino-terminal domain of hepatitis
delta antigens and modulate the hepatitis delta virus (a satellite
virus of hepatitis virus associated with fulminant hepatitis and
chronic liver necrosis) replication (135)
The development of combination of autoantibodies in a given disease has
been explained by the assembly of multiple autoantigens into dynamic
particles that which drive the immune response (143)
. Many
proteins that form dynamic particles and become targets of high titer
immunoglobulin G autoantibodies share amino acid sequence similarities
with nucleolin, e.g., U2 snRNP-B, nuclear RNP (U1 snRNP-70K and A),
SS-A/Ro (60K-Y RNA), SS-B/La (48 kDa phosphoprotein complexed with
nascent RNA polymerase III transcripts), and fibrillarin (U3 RNP-34K)
(143
, 144)
. In addition, nucleolin itself forms dynamic
particles with several autoantigens, including histone H1,
topoisomerase 1, RNA polymerase 1, and DNA (10)
. These
correlations predict that 1) the RNA recognition motif in
the nucleolin may be an autoantibody epitope, 2)
anti-nucleolin autoantibodies may form `sets' with certain other
anti-nuclear autoantibodies, and 3) nucleolin can share
epitopes with other viral proteins. In addition, multiple endocrine
neoplasia type 2A (MEN 2A) encodes a 415 aa protein similar in sequence
to nucleolin (145)
. The correlation observed between
anti-nucleolin antibodies and disease activity points to its important
role in disease.
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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that
serves to relay NGF signals from cell surface to nucleus in PC12 cells
and is a substrate for ecto-protein kinase on the cell surface.
All experimental evidence gathered to-date supports an essential role
for nucleolin in cell proliferation. Control of nucleolin expression is
very complex, phosphorylation being only one of several regulatory
elements in the nucleolin tool box. Extensive phosphorylation of
nucleolin by a casein kinase (CKII) in interphase and by cdc2 kinase
during mitosis suggests that phosphorylation may be a mechanism for
regulating nucleolin function during the cell cycle. More recently we
have come to realize that molecular regulators of nucleolin function
can contribute further to the complexity of nucleolin regulation by
interaction with nucleolin protein. Recent data concerning the
structure and cell biology of nucleolin suggest that as an autoantigen,
nucleolin is ideally positioned to play a central role in the
development of other autoantibodies. In addition, it is a switch
region-targeting factor in the recombination complex of a B
cell-specific transcription factor, LR1. To illustrate these many
functions, we have prepared the diagram shown in Fig. 1
.
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Clearly, more investigation into these correlations is required to identify the mechanisms by which nucleolin performs these disparate tasks. The multifunctional nature of nucleolin could be due to the specialized domains within nucleolin performing different functions. Although it is energetically favorable for a cell to use a single protein for unrelated different functions, understanding the basis of multifunctionality of nucleolin will have to await the phenotype of the knockout mice and its interaction with other macromolecules. From this knowledge, profoundly important insights to cell biology and medicine will undoubtedly be derived.
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TOPABSTRACTINTRODUCTIONSTRUCTURE-FUNCTION RELATIONSHIPSGENE STRUCTUREREGULATION OF NUCLEOLIN FUNCTIONNUCLEOLIN, HSP70, AND NUCLEAR...NUCLEOLIN AND CELL GROWTHNUCLEOLIN BINDING PROTEINS AND...AUTOIMMUNITY AND VIRAL...CONCLUSIONREFERENCES |
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