ARF tumor suppressor
Encyclopedia
The ARF tumor suppressor is a protein
that is transcribed from an alternate reading frame of the INK4a/ARF locus (CDKN2A
). ARF is induced in response to sustained mitogenic stimulation, such as aberrant growth signaling from MYC
and Ras (protein). It accumulates mainly in the nucleolus
where it forms stable complexes with NPM or Mdm2
. These interactions allow ARF to act as a tumor
suppressor by inhibiting ribosome
biogenesis or initiating p53
-dependent cell cycle
arrest and apoptosis
, respectively. ARF is an atypical protein, in terms of its transcription, its amino acid composition, and its degradation: it is transcribed in an alternate reading frame
of a different protein, it is highly basic, and it is polyubiquinated at the N-terminus.
For a review and figures, please see
suppressor transcript was first identified in humans in 1995, and its protein
product confirmed in mice that same year. Its gene locus is on the short arm of chromosome
9 in humans, and on a corresponding location on chromosome 4 in mice. It is located near the genes
for the tandem repeats INK4a and INK4b, which are 16 kDa (p16INK4a) and 15 kDa (p15INK4b) proteins, respectively. These INK4 proteins directly inhibit the cyclin D-dependent kinases CDK4 and CDK6. There are other INK4 genes on other chromosomes, however these are not linked to cancer
, and so their functions are not likely to be overlapping. An important cyclin-dependent substrate is the retinoblastoma protein
Rb, which is phosphorylated in late gap 1 phase (G1 phase
), allowing G1 exit. The Rb protein limits cell proliferation by blocking the activity of E2F
transcription factors, which activate the transcription of genes needed for DNA
replication. When Rb is phosphorylated by cyclin D and E-dependent kinases during the G1 phase of the cell cycle, Rb can not block E2F-dependent transcription, and the cell can progress to the DNA synthetic phase(S phase
). Therefore, INK4a and INK4b serve as tumor suppressors by restricting proliferation though the inhibition of the CDKs responsible for Rb phosphorylation
.
In addition to the INK4a protein, the unrelated protein, ARF, is transcribed from an alternate reading frame
at the INK4a/ARF locus. INK4a and ARF mRNA each consist of three exons. They share exons 2 and 3, but there are two different exon 1 transcripts, α and β. Exon 1β (E1β) is intercalated between the genes for INK4a and INK4b. Although exon 1α (E1α) and E1β are about the same in terms of content and size, the 5’ AUG (start codon
) of exon 1β has its own promoter and opens an alternative reading frame in exon 2, hence the name ARF (ARF exon 3 is not translated). Because of this, INK4a and ARF have unrelated amino acid
sequences despite overlapping coding regions, and have distinct functions. This dual use of coding sequences is not commonly seen in mammals, making ARF an unusual protein. When the ARF β-transcript was found, it was thought that it probably would not encode a protein. In humans, ARF is translated into the 14kDa, 132 amino acid p14ARF protein, and in mice, it is translated into the 19kDa, 169 amino acid p19Arf. The E1β protein segment of mouse and human ARF are 45% identical, with an overall ARF identity of 50%, compared to a 72% identity between mouse and human INK4a E1α segment, and a 65% overall identity.
Although the INK4a and ARF proteins are structurally and functionally different, they are both involved in cell cycle
progression. Together, their broad inhibitory role may help counter oncogenic signals. As mentioned above, INK4a inhibits proliferation by indirectly allowing Rb to remain associated with E2F
transcription factors. ARF is involved in p53
activation by inhibiting Mdm2
(HDM2 in humans). Mdm2 binds to p53, inhibiting its transcriptional activity. Mdm2 also has E3 ubiquitin
ligase activity toward p53, and promotes its exportation from the cell nucleus
to the cytoplasm
for degradation. By antagonizing Mdm2, ARF permits the transcriptional activity of p53 that would lead to cell cycle arrest or apoptosis
. A loss of ARF or p53, therefore, would give cells a survival advantage.
The function of ARF has primarily been attributed to its Mdm2/p53 mechanism. ARF does, however, also inhibit proliferation in cells lacking p53 or p53 and Mdm2. It has recently been found that one of ARF’s p53-independent functions involves its binding to nucleophosmin/B23 (NPM). NPM is an acidic ribosomal chaperone (protein) involved in preribosomal processing and nuclear exportation independent of p53, and oligomerizes with itself and p19ARF. Nearly half of p19ARF is found in NPM-containing complexes with high molecular mass (2 to 5 MDa). Enforced expression of ARF retards early 47S/45S rRNA precursor processing and inhibits 32S rRNA cleavage. This suggests that p19ARF can bind to NPM, inhibiting rRNA processing. ARF-null cells have increased nucleolar area, increased ribosome
biogenesis, and a corresponding increase in protein
synthesis. The larger size resulting from more ribosomes and protein is not associated with increased proliferation, however, and this ARF-null phenotype occurs even though the normal basal levels of Arf are usually low. Knocking down ARF with siRNA
to exon
1β results in increased rRNA transcripts, rRNA processing, and ribosome nuclear export. The unrestrained ribosome biogenesis seen when NPM is not bound to ARF does not occur if NPM is also absent. Although the induction of ARF in response to oncogenic signals is considered to be of primary importance, the low levels of ARF seen in interphase
cells also has a considerable effect in terms of keeping cell growth in check. Therefore, the function of basal level ARF in the NPM/ARF complex appears to be to monitor steady-state ribosome biogenesis and growth independently of preventing proliferation.
. Without INK4a, Cdk4/6 can inappropriately phosphorylate Rb, leading to increased E2F
-dependent transcription. Without ARF, Mdm2
can inappropriately inhibit p53, leading to increased cell survival.
The tumor
suppressor p53 is mutated in more than 50% of human cancers, and HDM2 is overexpressed in 5-10% of tumors. The INK4a/ARF locus is found to be deleted or silenced in many kinds of tumors. For example, of the 100 primary breast carcinomas, approximately 41% have p14ARF defects. In a separate study, 32% of colorectal adenomas (non-cancerous tumors) were found to have p14ARF inactivation due to hypermethylation
of the promoter. Mouse models lacking p19Arf, p53, and Mdm2 are more prone to tumor development than mice without Mdm2 and p53, alone. This suggests that p19Arf has Mdm2- and p53-independent effects, as well. Investigating this idea lead to the recent discovery of smARF.
interactions. The function of ARF involving p53
-independent death, has now been attributed to the small mitochondrial isoform of ARF, smARF. While full-length ARF inhibits cell growth by cell cycle
arrest or type I apoptotic death, smARF kills cells by type II autophagic death. Like ARF, the expression of smARF increases when there are aberrant proliferation signals. When smARF is overexpressed, it localizes to the mitochondrial matrix
, damaging the mitochondria membrane potential and structure, and leading to autophagic cell death.
The translation of the truncated ARF, smARF, is initiated at an internal methionine
(M45) of the ARF transcript in human and mouse cells. SmARF is also detected in rat, even though an internal methionine is not present in the rat transcript. This suggests that there is an alternate mechanism to form smARF, underscoring the importance of this isoform. The role of smARF is distinct from that of ARF, as it lacks the nuclear localization signal
(NLS) and cannot bind to Mdm2
or NPM. In some cell types, however, full-length ARF can also localize to the mitochondria and induce type II cell death, suggesting that in addition to autophagy
being a starvation or other environmental response, it may also be involved in responding to oncogene
activation.
or Ras (protein), will increase its expression, as will an amplification of mutated p53
or Mdm2
, or p53 loss. ARF can also be induced by enforced E2F
expression. Although E2F expression is increased during the cell cycle
, ARF expression probably is not because the activation of a second, unknown transcription factor might be needed to prevent an ARF response to transient E2F increases. ARF is negatively regulated by Rb-E2F complexes and by amplified p53 activation. Aberrant growth signals also increase smARF expression.
ARF is a highly basic (pI>12) and hydrophobic protein. Its basic nature is attributed to its arginine content; more than 20% of its amino acids are arginine, and it contains little or no lysine. Due to these characteristics, ARF is likely to be unstructured unless it is bound to other targets. It reportedly complexes with more than 25 proteins, although the significance of each of these interactions is not known. One of these interactions results in sumoylating activity, suggesting that ARF may modify proteins to which it binds. The SUMO protein
is a small ubiquitin
-like modifier, which is added to lysly ε-amino groups. This process involves a three-enzyme
cascade similar to the way ubiquitylation occurs. E1 is an activating enzyme, E2 is a conjugation enzyme, and E3 is a ligase. ARF associates with UBC9, the only SUMO E2 known, suggesting ARF facilitates SUMO conjugation. The importance of this role is unknown, as sumoylation is involved in different functions, such as protein trafficking, ubiquitylation interference, and gene expression changes.
The half-life
of ARF is about 6 hours, while the half-life of smARF is less than 1 hour. Both isoforms are degraded in the proteasome
. ARF is targeted for the proteasome
by N-terminus ubiquitylation. Proteins are usually ubiquinated at lysine
residues. Human p14ARF, however, does not contain any lysines, and mouse p19Arf only contains one lysine. If the mouse lysine is replaced with arginine, there is no effect on its degradation, suggesting it is also ubiquinated at the N-terminus. This adds to the uniqueness of the ARF proteins, because most eukaryotic proteins are acetylated at the N-terminus, preventing ubiquination at this location. Penultimate residues affect the efficiency of acetylation, in that acetylation is promoted by acidic residues and inhibited by basic ones. The N-terminal amino acid sequences of p19Arf (Met-Gly-Arg) and p14ARF (Met-Val-Arg) would be processed by methionine aminopeptidase but would not be acetylated, allowing ubiquination to proceed. The sequence of smARF, however, predicts that the initiating methionine would not be cleaved by methionine aminopeptidase and would probably be acetylated, and so is degraded by the proteasome without ubiquination.
Full-length nucleolar ARF appears to be stabilized by NPM. The NPM-ARF complex does not block the N-terminus of ARF, but likely protects ARF from being accessed by degradation machinery. The mitochondrial matrix
protein p32 stabilizes smARF. This protein binds various cellular and viral proteins, but its exact function is unknown. Knocking down p32 dramatically decreases smARF levels by increasing its turnover. The levels of p19Arf are not affected by p32 knockdown, and so p32 specifically stabilizes smARF, possibly by protecting it from the proteasome
or from mitochondrial proteases.
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
that is transcribed from an alternate reading frame of the INK4a/ARF locus (CDKN2A
CDKN2A
CDKN2A can refer to:* P16 * p14arf...
). ARF is induced in response to sustained mitogenic stimulation, such as aberrant growth signaling from MYC
Myc
Myc is a regulator gene that codes for a transcription factor. In the human genome, Myc is located on chromosome 8 and is believed to regulate expression of 15% of all genes through binding on Enhancer Box sequences and recruiting histone acetyltransferases...
and Ras (protein). It accumulates mainly in the nucleolus
Nucleolus
The nucleolus is a non-membrane bound structure composed of proteins and nucleic acids found within the nucleus. Ribosomal RNA is transcribed and assembled within the nucleolus...
where it forms stable complexes with NPM or Mdm2
Mdm2
Mdm2 is an important negative regulator of the p53 tumor suppressor. It is the name of a gene as well as the protein encoded by that gene. Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain of the p53 tumor suppressor and an inhibitor of...
. These interactions allow ARF to act as a tumor
Tumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
suppressor by inhibiting ribosome
Ribosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
biogenesis or initiating p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
-dependent cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
arrest and apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
, respectively. ARF is an atypical protein, in terms of its transcription, its amino acid composition, and its degradation: it is transcribed in an alternate reading frame
Reading frame
In biology, a reading frame is a way of breaking a sequence of nucleotides in DNA or RNA into three letter codons which can be translated in amino acids. There are 3 possible reading frames in an mRNA strand: each reading frame corresponding to starting at a different alignment...
of a different protein, it is highly basic, and it is polyubiquinated at the N-terminus.
For a review and figures, please see
Background
The ARF tumorTumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
suppressor transcript was first identified in humans in 1995, and its protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
product confirmed in mice that same year. Its gene locus is on the short arm of chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
9 in humans, and on a corresponding location on chromosome 4 in mice. It is located near the genes
Gênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
for the tandem repeats INK4a and INK4b, which are 16 kDa (p16INK4a) and 15 kDa (p15INK4b) proteins, respectively. These INK4 proteins directly inhibit the cyclin D-dependent kinases CDK4 and CDK6. There are other INK4 genes on other chromosomes, however these are not linked to cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
, and so their functions are not likely to be overlapping. An important cyclin-dependent substrate is the retinoblastoma protein
Retinoblastoma protein
The retinoblastoma protein is a tumor suppressor protein that is dysfunctional in the majority types of cancer. One highly studied function of pRb is to prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide...
Rb, which is phosphorylated in late gap 1 phase (G1 phase
G1 phase
The G1 phase is a period in the cell cycle during interphase, before the S phase. For many cells, this phase is the major period of cell growth during its lifespan. During this stage new organelles are being synthesized, so the cell requires both structural proteins and enzymes, resulting in great...
), allowing G1 exit. The Rb protein limits cell proliferation by blocking the activity of E2F
E2F
E2F is a group of genes that codifies a family of transcription factors in higher eukaryotes. Three of them are activators: E2F1, 2 and E2F3a. Six others act as suppressors: E2F3b, E2F4-8. All of them are involved in the cell cycle regulation and synthesis of DNA in mammalian cells...
transcription factors, which activate the transcription of genes needed for DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
replication. When Rb is phosphorylated by cyclin D and E-dependent kinases during the G1 phase of the cell cycle, Rb can not block E2F-dependent transcription, and the cell can progress to the DNA synthetic phase(S phase
S phase
S-phase is the part of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. Precise and accurate DNA replication is necessary to prevent genetic abnormalities which often lead to cell death or disease. Due to the importance, the regulatory pathways that govern this...
). Therefore, INK4a and INK4b serve as tumor suppressors by restricting proliferation though the inhibition of the CDKs responsible for Rb phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
.
In addition to the INK4a protein, the unrelated protein, ARF, is transcribed from an alternate reading frame
Reading frame
In biology, a reading frame is a way of breaking a sequence of nucleotides in DNA or RNA into three letter codons which can be translated in amino acids. There are 3 possible reading frames in an mRNA strand: each reading frame corresponding to starting at a different alignment...
at the INK4a/ARF locus. INK4a and ARF mRNA each consist of three exons. They share exons 2 and 3, but there are two different exon 1 transcripts, α and β. Exon 1β (E1β) is intercalated between the genes for INK4a and INK4b. Although exon 1α (E1α) and E1β are about the same in terms of content and size, the 5’ AUG (start codon
Start codon
The start codon is generally defined as the point, sequence, at which a ribosome begins to translate a sequence of RNA into amino acids.When an RNA transcript is "read" from the 5' carbon to the 3' carbon by the ribosome the start codon is the first codon on which the tRNA bound to Met,...
) of exon 1β has its own promoter and opens an alternative reading frame in exon 2, hence the name ARF (ARF exon 3 is not translated). Because of this, INK4a and ARF have unrelated amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
sequences despite overlapping coding regions, and have distinct functions. This dual use of coding sequences is not commonly seen in mammals, making ARF an unusual protein. When the ARF β-transcript was found, it was thought that it probably would not encode a protein. In humans, ARF is translated into the 14kDa, 132 amino acid p14ARF protein, and in mice, it is translated into the 19kDa, 169 amino acid p19Arf. The E1β protein segment of mouse and human ARF are 45% identical, with an overall ARF identity of 50%, compared to a 72% identity between mouse and human INK4a E1α segment, and a 65% overall identity.
Although the INK4a and ARF proteins are structurally and functionally different, they are both involved in cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
progression. Together, their broad inhibitory role may help counter oncogenic signals. As mentioned above, INK4a inhibits proliferation by indirectly allowing Rb to remain associated with E2F
E2F
E2F is a group of genes that codifies a family of transcription factors in higher eukaryotes. Three of them are activators: E2F1, 2 and E2F3a. Six others act as suppressors: E2F3b, E2F4-8. All of them are involved in the cell cycle regulation and synthesis of DNA in mammalian cells...
transcription factors. ARF is involved in p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
activation by inhibiting Mdm2
Mdm2
Mdm2 is an important negative regulator of the p53 tumor suppressor. It is the name of a gene as well as the protein encoded by that gene. Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain of the p53 tumor suppressor and an inhibitor of...
(HDM2 in humans). Mdm2 binds to p53, inhibiting its transcriptional activity. Mdm2 also has E3 ubiquitin
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
ligase activity toward p53, and promotes its exportation from the cell nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
to the cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
for degradation. By antagonizing Mdm2, ARF permits the transcriptional activity of p53 that would lead to cell cycle arrest or apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
. A loss of ARF or p53, therefore, would give cells a survival advantage.
The function of ARF has primarily been attributed to its Mdm2/p53 mechanism. ARF does, however, also inhibit proliferation in cells lacking p53 or p53 and Mdm2. It has recently been found that one of ARF’s p53-independent functions involves its binding to nucleophosmin/B23 (NPM). NPM is an acidic ribosomal chaperone (protein) involved in preribosomal processing and nuclear exportation independent of p53, and oligomerizes with itself and p19ARF. Nearly half of p19ARF is found in NPM-containing complexes with high molecular mass (2 to 5 MDa). Enforced expression of ARF retards early 47S/45S rRNA precursor processing and inhibits 32S rRNA cleavage. This suggests that p19ARF can bind to NPM, inhibiting rRNA processing. ARF-null cells have increased nucleolar area, increased ribosome
Ribosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
biogenesis, and a corresponding increase in protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
synthesis. The larger size resulting from more ribosomes and protein is not associated with increased proliferation, however, and this ARF-null phenotype occurs even though the normal basal levels of Arf are usually low. Knocking down ARF with siRNA
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
to exon
Exon
An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...
1β results in increased rRNA transcripts, rRNA processing, and ribosome nuclear export. The unrestrained ribosome biogenesis seen when NPM is not bound to ARF does not occur if NPM is also absent. Although the induction of ARF in response to oncogenic signals is considered to be of primary importance, the low levels of ARF seen in interphase
Interphase
Interphase is the phase of the cell cycle in which the cell spends the majority of its time and performs the majority of its purposes including preparation for cell division. In preparation for cell division, it increases its size and makes a copy of its DNA...
cells also has a considerable effect in terms of keeping cell growth in check. Therefore, the function of basal level ARF in the NPM/ARF complex appears to be to monitor steady-state ribosome biogenesis and growth independently of preventing proliferation.
Role in Disease
Very commonly, cancer is associated with a loss of function of INK4a, ARF, Rb, or p53P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
. Without INK4a, Cdk4/6 can inappropriately phosphorylate Rb, leading to increased E2F
E2F
E2F is a group of genes that codifies a family of transcription factors in higher eukaryotes. Three of them are activators: E2F1, 2 and E2F3a. Six others act as suppressors: E2F3b, E2F4-8. All of them are involved in the cell cycle regulation and synthesis of DNA in mammalian cells...
-dependent transcription. Without ARF, Mdm2
Mdm2
Mdm2 is an important negative regulator of the p53 tumor suppressor. It is the name of a gene as well as the protein encoded by that gene. Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain of the p53 tumor suppressor and an inhibitor of...
can inappropriately inhibit p53, leading to increased cell survival.
The tumor
Tumor
A tumor or tumour is commonly used as a synonym for a neoplasm that appears enlarged in size. Tumor is not synonymous with cancer...
suppressor p53 is mutated in more than 50% of human cancers, and HDM2 is overexpressed in 5-10% of tumors. The INK4a/ARF locus is found to be deleted or silenced in many kinds of tumors. For example, of the 100 primary breast carcinomas, approximately 41% have p14ARF defects. In a separate study, 32% of colorectal adenomas (non-cancerous tumors) were found to have p14ARF inactivation due to hypermethylation
Methylation
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...
of the promoter. Mouse models lacking p19Arf, p53, and Mdm2 are more prone to tumor development than mice without Mdm2 and p53, alone. This suggests that p19Arf has Mdm2- and p53-independent effects, as well. Investigating this idea lead to the recent discovery of smARF.
smARF
Until recently, the two known effects of ARF were growth inhibition by NPM interactions and apoptosis induction by Mdm2Mdm2
Mdm2 is an important negative regulator of the p53 tumor suppressor. It is the name of a gene as well as the protein encoded by that gene. Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain of the p53 tumor suppressor and an inhibitor of...
interactions. The function of ARF involving p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
-independent death, has now been attributed to the small mitochondrial isoform of ARF, smARF. While full-length ARF inhibits cell growth by cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
arrest or type I apoptotic death, smARF kills cells by type II autophagic death. Like ARF, the expression of smARF increases when there are aberrant proliferation signals. When smARF is overexpressed, it localizes to the mitochondrial matrix
Mitochondrial matrix
In the mitochondrion, the matrix contains soluble enzymes that catalyze the oxidation of pyruvate and other small organic molecules.The mitochondrial matrix also contains the mitochondria's DNA and ribosomes. The word "matrix" stems from the fact that this space is viscous, compared to the...
, damaging the mitochondria membrane potential and structure, and leading to autophagic cell death.
The translation of the truncated ARF, smARF, is initiated at an internal methionine
Methionine
Methionine is an α-amino acid with the chemical formula HO2CCHCH2CH2SCH3. This essential amino acid is classified as nonpolar. This amino-acid is coded by the codon AUG, also known as the initiation codon, since it indicates mRNA's coding region where translation into protein...
(M45) of the ARF transcript in human and mouse cells. SmARF is also detected in rat, even though an internal methionine is not present in the rat transcript. This suggests that there is an alternate mechanism to form smARF, underscoring the importance of this isoform. The role of smARF is distinct from that of ARF, as it lacks the nuclear localization signal
Nuclear localization signal
A nuclear localization signal or sequence is an amino acid sequence which 'tags' a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different...
(NLS) and cannot bind to Mdm2
Mdm2
Mdm2 is an important negative regulator of the p53 tumor suppressor. It is the name of a gene as well as the protein encoded by that gene. Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain of the p53 tumor suppressor and an inhibitor of...
or NPM. In some cell types, however, full-length ARF can also localize to the mitochondria and induce type II cell death, suggesting that in addition to autophagy
Autophagy
In cell biology, autophagy, or autophagocytosis, is a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. It is a tightly regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance...
being a starvation or other environmental response, it may also be involved in responding to oncogene
Oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, they are often mutated or expressed at high levels.An oncogene is a gene found in the chromosomes of tumor cells whose activation is associated with the initial and continuing conversion of normal cells into cancer...
activation.
Biochemistry
ARF expression is regulated by oncogenic signaling. Aberrant mitogenic stimulation, such as by MYCMyc
Myc is a regulator gene that codes for a transcription factor. In the human genome, Myc is located on chromosome 8 and is believed to regulate expression of 15% of all genes through binding on Enhancer Box sequences and recruiting histone acetyltransferases...
or Ras (protein), will increase its expression, as will an amplification of mutated p53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
or Mdm2
Mdm2
Mdm2 is an important negative regulator of the p53 tumor suppressor. It is the name of a gene as well as the protein encoded by that gene. Mdm2 protein functions both as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation domain of the p53 tumor suppressor and an inhibitor of...
, or p53 loss. ARF can also be induced by enforced E2F
E2F
E2F is a group of genes that codifies a family of transcription factors in higher eukaryotes. Three of them are activators: E2F1, 2 and E2F3a. Six others act as suppressors: E2F3b, E2F4-8. All of them are involved in the cell cycle regulation and synthesis of DNA in mammalian cells...
expression. Although E2F expression is increased during the cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
, ARF expression probably is not because the activation of a second, unknown transcription factor might be needed to prevent an ARF response to transient E2F increases. ARF is negatively regulated by Rb-E2F complexes and by amplified p53 activation. Aberrant growth signals also increase smARF expression.
ARF is a highly basic (pI>12) and hydrophobic protein. Its basic nature is attributed to its arginine content; more than 20% of its amino acids are arginine, and it contains little or no lysine. Due to these characteristics, ARF is likely to be unstructured unless it is bound to other targets. It reportedly complexes with more than 25 proteins, although the significance of each of these interactions is not known. One of these interactions results in sumoylating activity, suggesting that ARF may modify proteins to which it binds. The SUMO protein
SUMO protein
Small Ubiquitin-like Modifier or SUMO proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function...
is a small ubiquitin
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
-like modifier, which is added to lysly ε-amino groups. This process involves a three-enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
cascade similar to the way ubiquitylation occurs. E1 is an activating enzyme, E2 is a conjugation enzyme, and E3 is a ligase. ARF associates with UBC9, the only SUMO E2 known, suggesting ARF facilitates SUMO conjugation. The importance of this role is unknown, as sumoylation is involved in different functions, such as protein trafficking, ubiquitylation interference, and gene expression changes.
The half-life
Half-life
Half-life, abbreviated t½, is the period of time it takes for the amount of a substance undergoing decay to decrease by half. The name was originally used to describe a characteristic of unstable atoms , but it may apply to any quantity which follows a set-rate decay.The original term, dating to...
of ARF is about 6 hours, while the half-life of smARF is less than 1 hour. Both isoforms are degraded in the proteasome
Proteasome
Proteasomes are very large protein complexes inside all eukaryotes and archaea, and in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks...
. ARF is targeted for the proteasome
Proteasome
Proteasomes are very large protein complexes inside all eukaryotes and archaea, and in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks...
by N-terminus ubiquitylation. Proteins are usually ubiquinated at lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....
residues. Human p14ARF, however, does not contain any lysines, and mouse p19Arf only contains one lysine. If the mouse lysine is replaced with arginine, there is no effect on its degradation, suggesting it is also ubiquinated at the N-terminus. This adds to the uniqueness of the ARF proteins, because most eukaryotic proteins are acetylated at the N-terminus, preventing ubiquination at this location. Penultimate residues affect the efficiency of acetylation, in that acetylation is promoted by acidic residues and inhibited by basic ones. The N-terminal amino acid sequences of p19Arf (Met-Gly-Arg) and p14ARF (Met-Val-Arg) would be processed by methionine aminopeptidase but would not be acetylated, allowing ubiquination to proceed. The sequence of smARF, however, predicts that the initiating methionine would not be cleaved by methionine aminopeptidase and would probably be acetylated, and so is degraded by the proteasome without ubiquination.
Full-length nucleolar ARF appears to be stabilized by NPM. The NPM-ARF complex does not block the N-terminus of ARF, but likely protects ARF from being accessed by degradation machinery. The mitochondrial matrix
Mitochondrial matrix
In the mitochondrion, the matrix contains soluble enzymes that catalyze the oxidation of pyruvate and other small organic molecules.The mitochondrial matrix also contains the mitochondria's DNA and ribosomes. The word "matrix" stems from the fact that this space is viscous, compared to the...
protein p32 stabilizes smARF. This protein binds various cellular and viral proteins, but its exact function is unknown. Knocking down p32 dramatically decreases smARF levels by increasing its turnover. The levels of p19Arf are not affected by p32 knockdown, and so p32 specifically stabilizes smARF, possibly by protecting it from the proteasome
Proteasome
Proteasomes are very large protein complexes inside all eukaryotes and archaea, and in some bacteria. In eukaryotes, they are located in the nucleus and the cytoplasm. The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks...
or from mitochondrial proteases.