Cell division control protein 4
Encyclopedia
Cdc4 is a substrate
recognition component of the SCF (SKP1-CUL1-F-box protein) ubiquitin
ligase complex, which acts as a mediator of ubiquitin transfer to target proteins, leading to their subsequent degradation via the ubiquitin-proteasome pathway. Cdc4 targets primarily cell cycle regulators for proteolysis
. It serves the function of an adaptor that brings target molecules to the core SCF complex.
Cdc4 was originally identified in the model organism Saccharomyces cerevisiae
.
CDC4 gene function is required at G1/S and G2/M transitions during mitosis and at various stages during meiosis.
In the nematode C. elegans, the homologue to Cdc4 is F-box/WD repeat-containing protein sel-10.
, RBX1
, and CDC4.
Its 779 amino acids (in S. cerevisiae) are arranged into one F-box domain (approximately 40 amino acids (“F-box” motif)) and 7 WD repeats.
Cdc4 is a WD-40 repeat F-box protein. Like all members of this family, it contains a conserved dimerization motif called D domain. In yeast Cdc4, the D domain protomers arrange in a superhelical homodimeric manner. SCF (Cdc4) dimerization hardly affects the affinity for target molecules, but significantly increases ubiquitin conjugation. Cdc4 adapts a suprafacial configuration: The substrate-binding sites lie in the same plane AS the catalytic sites, with a separation of 64Å
within and 102Å
between each SCF monomer
.
In Cdc4, the substrate binding domain is built on WD40 domains, which use repeats of 40 amino acids), each forming four anti-parallel beta-strands, to assemble the blades of a so-called beta-propeller. Beta-propellers are a quite frequent form of adaptable surface for interaction between different proteins. This substrate interaction region is located C-terminally.
There are three isoforms of Cdc4 in mammals: α, ß, and γ. These are produced via alternative splicing
of 3 unique 5’ exons to 10 common 3’ exons. This results in proteins that differ only at their N-termini.
Cdc4 protein interacts with Cdc34
, an ubiquitin-conjugating enzyme, and Cdc53 in vivo. (There is a Cdc4p/Cdc53p-binding region on Cdc34p.) All three proteins are stable throughout the cell cycle
.
.
In order for the cell cycle to proceed, several inhibitory proteins, as well as cyclins, have to be eliminated at given time points. Cdc4 assists there by recruiting target molecules via its C-terminal substrate interaction domain (WD40 repeat domain) to the ubiquitination machinery. This causes transfer of ubiquitin molecules to the target, hence marks it for degradation.
Cdc4 recognizes and binds to phosphorylated target proteins.
Cdc4 can be essential
, or nonessential, depending on the organism. For instance, it is essential in S. cerevisiae, while it is non-essential in C. albicans.
It is essential for initiation of DNA replication
and separation of spindle pole bodies, hence for the formation of the poles of the mitotic spindle
. In budding yeast it is also involved in bud development, fusion of zygotic nuclei (karyogamy
) after conjugation, and several aspects of sporulation.
Roughly speaking, in the cell cycle Cdc4 function is required for G1/S and G2/M transition.
Some important interactions in which Cdc4 is involved are:
Ultrasensitivity+in+the+mitogen-activated+protein+kinase+cascade
s. Thus, Sic1
protein degradation is necessary to enter S-phase. SCF (Cdc4) complex’s regulatory function concerning S-phase entry comprises not only degradation
of Sic1, but also degradation of Swi5.
In order for the substrate adapter unit Cdc4 to bind to Sic1, a minimum of any six of the nine cyclin-dependent kinase
sites on Sic1 have to be phosphorylated. In other words: There is a threshold number of phosphorylation
sites in order to achieve receptor-ligand binding. As recently stated, this “suggests that the ultrasensitivity in the Sic1-Cdc4 system may be driven at least in part by cumulative electrostatic interactions”. In general, an ultrasensitive enzyme requires less than 81-fold increase in stimulus to drive it from 10% to 90% activity. "Ultrasensitivity" highlights that the upstroke of the stimulus/response curve is steeper than the one that is obtained for a hyperbolic Michaelis-Menten enzyme. Thus, ultrasensitivity allows a highly sensitive response: A graded input can be transformed into a sharply thresholded output. The development of B-type cyclin–cyclin-dependent kinase activity, as well as the onset of DNA replication, requires degradation of Sic1 in the late G1 phase of the cell cycle. The WD domain of Cdc4 binds to the phosphorylated form of Sic1. Each bond to a Sic1-Phosphate is weak, but together the binding is strong enough to enable Sic1-degradation via the pathway described before. Hence, in this case ultrasensitivity allows precise definition ("fine tuning") of the time point in which destruction of Sic1 occurs, leading to initiation of the next step in the cell cycle (-> DNA replication).
, is responsible for proteolysis
at that stage.
However, experimental data suggests that Cdc4 function in G2/M transition may be linked to the degradation of Pds1 (anaphase
inhibitor). And what is more, CDC4 and CDC20
, an activator of APC, interact genetically.
Cdc4 recruits several other substrates than Sic1 to the SCF core complex, including the Cln-Cdc28 inhibitor / cytoskeletal scaffold protein
Far1, the transcription factor
Gcn4, and the replication
protein Cdc6.
In addition to those functions mentioned above, Cdc4 is involved in some other degradation-dependent events in S. cerevisiae like for instance unfolded protein response.
, and the Notch
intracellular domain are substrates of Cdc4. Due to its involvement in degradation of various cell cycle regulators, as well as several compounds of signaling pathways (e.g. Notch), Cdc4 is a highly sensible component of every organism in which it functions.
The cdc4 gene is a haplo-insufficient tumor suppressor gene. Knock-out of this gene in mice leads to an embryonic lethal phenotype
.
CDC4 mutations occur in a number of cancer types. They are described best in colorectal tumors, and also have been found to be a mutational target in pancreatic cancer.
Recently, it was found out that E3 has an additional function to its primary role in the degradation of certain cell cycle regulators: It is also involved in formation of the neural crest. Hence, Cdc4 is a protein “with separable but complementary functions in control of cell proliferation and differentiation” . This evokes the assumption -beyond regulating cell cycle progression- Cdc4 as a tumor suppressor protein may extend its ability to directly regulate tissue differentiation. However, its concrete role in diseases is still to be elucidated.
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
recognition component of the SCF (SKP1-CUL1-F-box protein) 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 complex, which acts as a mediator of ubiquitin transfer to target proteins, leading to their subsequent degradation via the ubiquitin-proteasome pathway. Cdc4 targets primarily cell cycle regulators for proteolysis
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
. It serves the function of an adaptor that brings target molecules to the core SCF complex.
Cdc4 was originally identified in the model organism Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
.
CDC4 gene function is required at G1/S and G2/M transitions during mitosis and at various stages during meiosis.
Homologues
The human homologue of the cdc4 gene is called FBXW7. The corresponding gene product is the F-box/WD repeat-containing protein 7.In the nematode C. elegans, the homologue to Cdc4 is F-box/WD repeat-containing protein sel-10.
Some general features
Cdc4 has a molecular weight of 86'089Da, an isoelectric point of 7.14, and consists of 779 amino acids. It resides exclusively in the nucleus because of a single monopartite nuclear localisation sequence (NLS) comprising amino acids 82-85 in the N-terminal domain.Structure
Cdc4 is one component of the E3 complex SCF (CDC4), which comprises CDC53, SKP1Skp1
Skp1 is a small protein of approximately 160 amino acids. As a core component of the SCF-type E3 ubiquitin ligases that mediate protein degradation by the 26S proteosome, Skp1 plays key roles in cell-cycle progression, transcriptional regulation, signal transduction, and many other cellular...
, RBX1
RBX1
RING-box protein 1 is a protein that in humans is encoded by the RBX1 gene.-Interactions:RBX1 has been shown to interact with CUL4A, CAND1, CUL1, CUL2, P70-S6 Kinase 1, DCUN1D1, CUL5 and CUL7.-Further reading:...
, and CDC4.
Its 779 amino acids (in S. cerevisiae) are arranged into one F-box domain (approximately 40 amino acids (“F-box” motif)) and 7 WD repeats.
Cdc4 is a WD-40 repeat F-box protein. Like all members of this family, it contains a conserved dimerization motif called D domain. In yeast Cdc4, the D domain protomers arrange in a superhelical homodimeric manner. SCF (Cdc4) dimerization hardly affects the affinity for target molecules, but significantly increases ubiquitin conjugation. Cdc4 adapts a suprafacial configuration: The substrate-binding sites lie in the same plane AS the catalytic sites, with a separation of 64Å
Å
Å represents various sounds in several languages. Å is part of the alphabets used for the Alemannic and the Bavarian-Austrian dialects of German...
within and 102Å
Å
Å represents various sounds in several languages. Å is part of the alphabets used for the Alemannic and the Bavarian-Austrian dialects of German...
between each SCF monomer
Monomer
A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer; the term "monomeric protein" may also be used to describe one of the proteins making up a multiprotein complex...
.
In Cdc4, the substrate binding domain is built on WD40 domains, which use repeats of 40 amino acids), each forming four anti-parallel beta-strands, to assemble the blades of a so-called beta-propeller. Beta-propellers are a quite frequent form of adaptable surface for interaction between different proteins. This substrate interaction region is located C-terminally.
There are three isoforms of Cdc4 in mammals: α, ß, and γ. These are produced via alternative splicing
RNA splicing
In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...
of 3 unique 5’ exons to 10 common 3’ exons. This results in proteins that differ only at their N-termini.
Cdc4 protein interacts with Cdc34
CDC34
Ubiquitin-conjugating enzyme E2 R1 is a protein that in humans is encoded by the CDC34 gene.-Interactions:CDC34 has been shown to interact with CSNK2B, BTRC and CDK9.-Further reading:...
, an ubiquitin-conjugating enzyme, and Cdc53 in vivo. (There is a Cdc4p/Cdc53p-binding region on Cdc34p.) All three proteins are stable throughout 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...
.
Function
Various cellular regulatory mechanisms heavily depend on ubiquitin-dependent degradation. The SCF (Cdc4) complex has a regulatory function in cell cycle progression, signal transduction, and transcriptionTranscription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
.
In order for the cell cycle to proceed, several inhibitory proteins, as well as cyclins, have to be eliminated at given time points. Cdc4 assists there by recruiting target molecules via its C-terminal substrate interaction domain (WD40 repeat domain) to the ubiquitination machinery. This causes transfer of ubiquitin molecules to the target, hence marks it for degradation.
Cdc4 recognizes and binds to phosphorylated target proteins.
Cdc4 can be essential
Essential (biology)
An essential part of an organism is something that the organism cannot continue to be alive or reproduce without. For instance, mitochondria are essential to most eukaryotic cells. Genes can also be considered with regards to their essentiality...
, or nonessential, depending on the organism. For instance, it is essential in S. cerevisiae, while it is non-essential in C. albicans.
It is essential for initiation of DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
and separation of spindle pole bodies, hence for the formation of the poles of the mitotic spindle
Mitotic spindle
In cell biology, the spindle fibers are the structure that separates the chromosomes into the daughter cells during cell division. It is part of the cytoskeleton in eukaryotic cells...
. In budding yeast it is also involved in bud development, fusion of zygotic nuclei (karyogamy
Karyogamy
Karyogamy is the fusion of pronuclei of two cells, as part of syngamy, fertilization, or true bacterial conjugation.It is one of the two major modes of reproduction in fungi...
) after conjugation, and several aspects of sporulation.
Roughly speaking, in the cell cycle Cdc4 function is required for G1/S and G2/M transition.
Some important interactions in which Cdc4 is involved are:
- ubiquitination of the phosphorylated form of the cell cycle kinase inhibitor (CKICyclin-dependent kinase inhibitor proteinA cyclin-dependent kinase inhibitor protein is a protein which inhibits cyclin-dependent kinase. Several function as tumor suppressor genes. Cell cycle progression is negatively controlled by cyclin-dependent kinases inhibitors . CDIs are involved in cell cycle arrest at the G1 phase.-Examples:...
) SIC1Sic1Sic1, a protein, is a stoichiometric inhibitor of Cdk1-Clb complexes in the budding yeast Saccharomyces cerevisiae. Because B-type cyclin-Cdk1 complexes are the drivers of S-phase initiation, Sic1 prevents premature S-phase entry... - degradation of the CKI FAR1 in absence of pheromone; restriction of FAR1 degradation to the nucleus (since Cdc4 is exclusively nuclear)
- transcription activation of the HTA1-HTB1 locus
- degradation of the phosphorylated form of Cdc6
Ultrasensitivity+in+the+mitogen-activated+protein+kinase+cascade
Onset of S-phase
Swi5 is a transcriptional activator of Sic1, which inhibits S-phase CDKCyclin-dependent kinase
thumb|350px|Schematic of the cell cycle. outer ring: I=[[Interphase]], M=[[Mitosis]]; inner ring: M=Mitosis; G1=[[G1 phase|Gap phase 1]]; S=[[S phase|Synthesis]]; G2=[[G2 phase|Gap phase 2]]...
s. Thus, Sic1
Sic1
Sic1, a protein, is a stoichiometric inhibitor of Cdk1-Clb complexes in the budding yeast Saccharomyces cerevisiae. Because B-type cyclin-Cdk1 complexes are the drivers of S-phase initiation, Sic1 prevents premature S-phase entry...
protein degradation is necessary to enter S-phase. SCF (Cdc4) complex’s regulatory function concerning S-phase entry comprises not only degradation
Chemical decomposition
Chemical decomposition, analysis or breakdown is the separation of a chemical compound into elements or simpler compounds. It is sometimes defined as the exact opposite of a chemical synthesis. Chemical decomposition is often an undesired chemical reaction...
of Sic1, but also degradation of Swi5.
In order for the substrate adapter unit Cdc4 to bind to Sic1, a minimum of any six of the nine cyclin-dependent kinase
Cyclin-dependent kinase
thumb|350px|Schematic of the cell cycle. outer ring: I=[[Interphase]], M=[[Mitosis]]; inner ring: M=Mitosis; G1=[[G1 phase|Gap phase 1]]; S=[[S phase|Synthesis]]; G2=[[G2 phase|Gap phase 2]]...
sites on Sic1 have to be phosphorylated. In other words: There is a threshold number of phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
sites in order to achieve receptor-ligand binding. As recently stated, this “suggests that the ultrasensitivity in the Sic1-Cdc4 system may be driven at least in part by cumulative electrostatic interactions”. In general, an ultrasensitive enzyme requires less than 81-fold increase in stimulus to drive it from 10% to 90% activity. "Ultrasensitivity" highlights that the upstroke of the stimulus/response curve is steeper than the one that is obtained for a hyperbolic Michaelis-Menten enzyme. Thus, ultrasensitivity allows a highly sensitive response: A graded input can be transformed into a sharply thresholded output. The development of B-type cyclin–cyclin-dependent kinase activity, as well as the onset of DNA replication, requires degradation of Sic1 in the late G1 phase of the cell cycle. The WD domain of Cdc4 binds to the phosphorylated form of Sic1. Each bond to a Sic1-Phosphate is weak, but together the binding is strong enough to enable Sic1-degradation via the pathway described before. Hence, in this case ultrasensitivity allows precise definition ("fine tuning") of the time point in which destruction of Sic1 occurs, leading to initiation of the next step in the cell cycle (-> DNA replication).
G2/M transition
Up until now it is not satisfyingly understood how Cdc4 triggers G2-M transition. In general, the second degradation complex involved in cell cycle progression, APCAnaphase-promoting complex
Anaphase-Promoting Complex, also called cyclosome , is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation by the 26S proteasome. The APC/C is a large complex of 11–13 subunit proteins, including a cullin and RING subunit much like SCF...
, is responsible for proteolysis
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...
at that stage.
However, experimental data suggests that Cdc4 function in G2/M transition may be linked to the degradation of Pds1 (anaphase
Anaphase
Anaphase, from the ancient Greek ἀνά and φάσις , is the stage of mitosis or meiosis when chromosomes move to opposite poles of the cell....
inhibitor). And what is more, CDC4 and CDC20
CDC20
The cell-division cycle protein 20 is an essential regulator of cell division that is encoded by the CDC20 gene in humans. To the best of current knowledge its most important function is to activate the anaphase promoting complex , a large 11-13 subunit complex that initiates chromatid separation...
, an activator of APC, interact genetically.
Cdc4 recruits several other substrates than Sic1 to the SCF core complex, including the Cln-Cdc28 inhibitor / cytoskeletal scaffold protein
Scaffold protein
In biology, scaffold proteins are crucial regulators of many key signaling pathways. Although scaffolds are not strictly defined in function, they are known to interact and/or bind with multiple members of a signaling pathway, tethering them into complexes...
Far1, the transcription factor
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
Gcn4, and the replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
protein Cdc6.
In addition to those functions mentioned above, Cdc4 is involved in some other degradation-dependent events in S. cerevisiae like for instance unfolded protein response.
Cdc4 and diseases
In mammals, amongst others c-Myc, Src3, Cyclin ECyclin E
Cyclin E is a member of the cyclin family.Cyclin E binds to G1 phase Cdk2, which is required for the transition from G1 to S phase. The Cyclin E/CDK2 complex phosphorylates p27Kip1 , tagging it for degradation, thus promoting expression of Cyclin A, allowing progression to S phase....
, and the Notch
Notch signaling
The notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms.Notch is present in all metazoans, and mammals possess four different notch receptors, referred to as NOTCH1, NOTCH2, NOTCH3, and NOTCH4. The notch receptor is a single-pass...
intracellular domain are substrates of Cdc4. Due to its involvement in degradation of various cell cycle regulators, as well as several compounds of signaling pathways (e.g. Notch), Cdc4 is a highly sensible component of every organism in which it functions.
The cdc4 gene is a haplo-insufficient tumor suppressor gene. Knock-out of this gene in mice leads to an embryonic lethal phenotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
.
CDC4 mutations occur in a number of cancer types. They are described best in colorectal tumors, and also have been found to be a mutational target in pancreatic cancer.
Recently, it was found out that E3 has an additional function to its primary role in the degradation of certain cell cycle regulators: It is also involved in formation of the neural crest. Hence, Cdc4 is a protein “with separable but complementary functions in control of cell proliferation and differentiation” . This evokes the assumption -beyond regulating cell cycle progression- Cdc4 as a tumor suppressor protein may extend its ability to directly regulate tissue differentiation. However, its concrete role in diseases is still to be elucidated.