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CDK-activating kinase (CAK) activates the cyclin-CDK complex by phosphorylating threonine residue 160 in the CDK activation loop. CAK itself is a member of the Cdk family and functions as a positive regulator of Cdk1, Cdk2, Cdk4, and Cdk6. [1]
Cdk activation requires two steps. First, cyclin must bind to the Cdk. In the second step, CAK must phosphorylate the cyclin-Cdk complex on the threonine residue 160, which is located in the Cdk activation segment. Since Cdks need to be free of Cdk inhibitor proteins (CKIs) and associated with cyclins in order to be activated, CAK activity is considered to be indirectly regulated by cyclins.[ by whom? ]
Phosphorylation is generally considered a reversible modification used to change enzyme activity in different conditions. However, activating phosphorylation of Cdk by CAK appears to be an exception to this trend. In fact, CAK activity remains high throughout the cell cycle and is not regulated by any known cell-cycle control mechanism. However compared to normal cells, CAK activity is reduced in quiescent G0 cells and slightly elevated in tumor cells. [1]
In mammals, activating phosphorylation by CAK can only occur once cyclin is bound. In budding yeast, activating phosphorylation by CAK can take place before cyclin binding. In both humans and yeast, cyclin binding is the rate limiting step in the activation of Cdk. Therefore, phosphorylation of Cdk by CAK is considered a post-translational modification that is necessary for enzyme activity. Although activating phosphorylation by CAK is not exploited for cell-cycle regulation purposes, it is a highly conserved process because CAK also regulates transcription.
CAK varies dramatically in different species. In vertebrates and Drosophila, CAK is a trimeric protein complex consisting of Cdk7 (a Cdk-related protein kinase), cyclin H, and Mat1. [2] The Cdk7 subunit is responsible for Cdk activation while the Mat1 subunit is responsible for transcription. The CAK trimer can be phosphorylated on the activation segment of Cdk7 subunit. However, unlike other Cdks, this phosphorylation is might not be essential for CAK activity. In the presence of Mat1, activation of CAK does not require phosphorylation of the activation segment. However, in the absence of Mat1, phosphorylation of the activation segment is required for CAK activity. [1]
In vertebrates, CAK localizes to the nucleus. This suggests that CAK is not only involved in cell-cycle regulation but is also involved in transcription. In fact, the Cdk7 subunit of vertebrate CAK phosphorylates several components of the transcriptional machinery.
In budding yeast, CAK is a monomeric protein kinase and is referred to as Cak1. [2] Cak1 is distantly homologous to Cdks. Cak1 localizes to the cytoplasm and is responsible for Cdk activation. Budding yeast Cdk7 homolog, Kin28, does not have CAK activity.
Fission yeasts have two CAKs with both overlapping and specialized functions. The first CAK is a complex of Msc6 and Msc2. The Msc6 and Msc2 complex is related to the vertebrate Cdk7-cyclinH complex. Msc6 and Msc2 complex not only activates cell cycle Cdks but also regulates gene expression because it is part of the transcription factor TFIIH. The second fission yeast CAK, Csk1, is an ortholog of budding yeast Cak1. Csk1 can activate Cdks but is not essential for Cdk activity. [2]
Table of Cdk-activating Kinases
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Credit to: Oxford University Press "Morgan: The Cell Cycle"
Cdkactivation
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Credit to: Oxford University Press "Morgan: The Cell Cycle"
The conformation of the Cdk2 active site changes dramatically upon cyclin binding and CAK phosphorylation. The active site of Cdk2 lies in a cleft between the two lobes of the kinase. ATP binds deep within the cleft and its phosphate is oriented outwards. Protein substrates bind to the entrance of the active site cleft.
In its inactive form, Cdk2 cannot bind substrate because the entrance of its active site is blocked by the T-loop. Inactive Cdk2 also has a misoriented ATP binding site. When Cdk2 is inactive, the small L12 helix pushes the large PSTAIRE helix outwards. The PSTAIRE helix contains a residue, glutamate 51, that is important for positioning the ATP phosphates. [2]
When cyclinA binds, several conformational changes take place. The T-loop moves out of active site entrance and no longer blocks the substrate binding site. The PSTAIRE helix moves in. The L12 helix becomes a beta strand. This allows glutamate 51 to interact with lysine 33. Aspartate 145 also changes position. Together these structural changes allow ATP phosphates to bind correctly. [2]
When CAK phosphorylates Cdk's threonine residue160, the T-loop flattens and interacts more closely with cyclin A. Phosphorylation also allows the Cdk to interact more effectively with substrates that contain the SPXK sequence. Phosphorylation also increases the activity of cyclinA-Cdk2 complex. Different cyclins produce different conformation changes in Cdk.
Image Link - Structural Basis of Cdk Activation
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Credit to: Oxford University Press "Morgan: The Cell Cycle"
In addition to activating Cdks, CAK also regulates transcription. Two forms of CAK have been identified: free CAK and TFIIH-associated CAK. Free CAK is more abundant than TFIIH-associated CAK. [1] Free CAK phosphorylates Cdks and is involved in cell cycle regulation. Associated CAK is part of the general transcription factor TFIIH. CAK associated with TFIIH phosphorylates proteins involved in transcription including RNA polymerase II. More specifically, associated CAK is involved in promoter clearance and progression of transcription from the preinitiation to the initiation stage.
In vertebrates, the trimeric CAK complex is responsible for transcription regulation. In budding yeast, the Cdk7 homolog, Kin28, regulates transcription. In fission yeast, the Msc6 Msc2 complex controls basal gene transcription. [2]
In addition to regulating transcription, CAK also enhances transcription by phosphorylating retinoic acid and estrogen receptors. Phosphorylation of these receptors leads to increased expression of target genes. In leukemic cells, where DNA is damaged, CAK’s ability to phosphorylate retinoic acid and estrogen receptors is decreased. Decreased CAK activity creates a feedback loop, which turns off TFIIH activity.
CAK also plays a role in DNA damage response. [1] The activity of CAK associated with TFIIH decreases when DNA is damaged by UV irradiation. Inhibition of CAK prevents cell cycle from progressing. This mechanism ensures the fidelity of chromosome transmission. [1]
The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA and some of its organelles, and subsequently the partitioning of its cytoplasm and other components into two daughter cells in a process called cell division.
Anaphase-promoting complex 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 (Apc2) and RING (Apc11) subunit much like SCF. Other parts of the APC/C have unknown functions but are highly conserved.
Cyclin-dependent kinases (CDKs) are the families of protein kinases first discovered for their role in regulating the cell cycle. They are also involved in regulating transcription, mRNA processing, and the differentiation of nerve cells. They are present in all known eukaryotes, and their regulatory function in the cell cycle has been evolutionarily conserved. In fact, yeast cells can proliferate normally when their CDK gene has been replaced with the homologous human gene. CDKs are relatively small proteins, with molecular weights ranging from 34 to 40 kDa, and contain little more than the kinase domain. By definition, a CDK binds a regulatory protein called a cyclin. Without cyclin, CDK has little kinase activity; only the cyclin-CDK complex is an active kinase but its activity can be typically further modulated by phosphorylation and other binding proteins, like p27. CDKs phosphorylate their substrates on serines and threonines, so they are serine-threonine kinases. The consensus sequence for the phosphorylation site in the amino acid sequence of a CDK substrate is [S/T*]PX[K/R], where S/T* is the phosphorylated serine or threonine, P is proline, X is any amino acid, K is lysine, and R is arginine.
A cyclin-dependent kinase complex is a protein complex formed by the association of an inactive catalytic subunit of a protein kinase, cyclin-dependent kinase (CDK), with a regulatory subunit, cyclin. Once cyclin-dependent kinases bind to cyclin, the formed complex is in an activated state. Substrate specificity of the activated complex is mainly established by the associated cyclin within the complex. Activity of CDKCs is controlled by phosphorylation of target proteins, as well as binding of inhibitory proteins.
Cyclin A is a member of the cyclin family, a group of proteins that function in regulating progression through the cell cycle. The stages that a cell passes through that culminate in its division and replication are collectively known as the cell cycle Since the successful division and replication of a cell is essential for its survival, the cell cycle is tightly regulated by several components to ensure the efficient and error-free progression through the cell cycle. One such regulatory component is cyclin A which plays a role in the regulation of two different cell cycle stages.
Cyclin D is a member of the cyclin protein family that is involved in regulating cell cycle progression. The synthesis of cyclin D is initiated during G1 and drives the G1/S phase transition. Cyclin D protein is anywhere from 155 to 477 amino acids in length.
Cyclin-dependent kinase 2, also known as cell division protein kinase 2, or Cdk2, is an enzyme that in humans is encoded by the CDK2 gene. The protein encoded by this gene is a member of the cyclin-dependent kinase family of Ser/Thr protein kinases. This protein kinase is highly similar to the gene products of S. cerevisiae cdc28, and S. pombe cdc2, also known as Cdk1 in humans. It is a catalytic subunit of the cyclin-dependent kinase complex, whose activity is restricted to the G1-S phase of the cell cycle, where cells make proteins necessary for mitosis and replicate their DNA. This protein associates with and is regulated by the regulatory subunits of the complex including cyclin E or A. Cyclin E binds G1 phase Cdk2, which is required for the transition from G1 to S phase while binding with Cyclin A is required to progress through the S phase. Its activity is also regulated by phosphorylation. Multiple alternatively spliced variants and multiple transcription initiation sites of this gene have been reported. The role of this protein in G1-S transition has been recently questioned as cells lacking Cdk2 are reported to have no problem during this transition.
Cell division protein kinase 6 (CDK6) is an enzyme encoded by the CDK6 gene. It is regulated by cyclins, more specifically by Cyclin D proteins and Cyclin-dependent kinase inhibitor proteins. The protein encoded by this gene is a member of the cyclin-dependent kinase, (CDK) family, which includes CDK4. CDK family members are highly similar to the gene products of Saccharomyces cerevisiae cdc28, and Schizosaccharomyces pombe cdc2, and are known to be important regulators of cell cycle progression in the point of regulation named R or restriction point.
The Cyclin D/Cdk4 complex is a multi-protein structure consisting of the proteins Cyclin D and cyclin-dependent kinase 4, or Cdk4, a serine-threonine kinase. This complex is one of many cyclin/cyclin-dependent kinase complexes that are the "hearts of the cell-cycle control system" and govern the cell cycle and its progression. As its name would suggest, the cyclin-dependent kinase is only active and able to phosphorylate its substrates when it is bound by the corresponding cyclin. The Cyclin D/Cdk4 complex is integral for the progression of the cell from the Growth 1 phase to the Synthesis phase of the cell cycle, for the Start or G1/S checkpoint.
CDK7 is a cyclin-dependent kinase shown to be not easily classified. CDK7 is both a CDK-activating kinase (CAK) and a component of the general transcription factor TFIIH.
Cyclin-dependent kinase 1 also known as CDK1 or cell division cycle protein 2 homolog is a highly conserved protein that functions as a serine/threonine kinase, and is a key player in cell cycle regulation. It has been highly studied in the budding yeast S. cerevisiae, and the fission yeast S. pombe, where it is encoded by genes cdc28 and cdc2, respectively. In humans, Cdk1 is encoded by the CDC2 gene. With its cyclin partners, Cdk1 forms complexes that phosphorylate a variety of target substrates ; phosphorylation of these proteins leads to cell cycle progression.
Polo-like kinases (Plks) are regulatory serine/threonin kinases of the cell cycle involved in mitotic entry, mitotic exit, spindle formation, cytokinesis, and meiosis. Only one Plk is found in the genomes of fruit flies (Polo), budding yeast (Cdc5) and fission yeast (Plo1). Vertebrates, however, have many Plk family members including Plk1, Plk2/Snk, Plk3/Prk/FnK, Plk4/Sak and Plk5. Of the vertebrate Plk family members, the mammalian Plk1 has been most extensively studied. During mitosis and cytokinesis, Plks associate with several structures including the centrosome, kinetochores, and the central spindle.
Cyclin-dependent kinase 7, or cell division protein kinase 7, is an enzyme that in humans is encoded by the CDK7 gene.
CDK-activating kinase assembly factor MAT1 is an enzyme that in humans is encoded by the MNAT1 gene.
Cyclin-H is a protein that in humans is encoded by the CCNH gene.
RNA polymerase II holoenzyme is a form of eukaryotic RNA polymerase II that is recruited to the promoters of protein-coding genes in living cells. It consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins.
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. Multisite phosphorylation of Sic1 is thought to time Sic1 ubiquitination and destruction, and by extension, the timing of S-phase entry.
Pho4 is a protein with a basic helix-loop-helix (bHLH) transcription factor. It is found in S. cerevisiae and other yeasts. It functions as a transcription factor to regulate phosphate responsive genes located in yeast cells. The Pho4 protein homodimer is able to do this by binding to DNA sequences containing the bHLH binding site 5'-CACGTG-3'. This sequence is found in the promoters of genes up-regulated in response to phosphate availability such as the PHO5 gene.
Wee1 is a nuclear kinase belonging to the Ser/Thr family of protein kinases in the fission yeast Schizosaccharomyces pombe. Wee1 has a molecular mass of 96 kDa and is a key regulator of cell cycle progression. It influences cell size by inhibiting the entry into mitosis, through inhibiting Cdk1. Wee1 has homologues in many other organisms, including mammals.
In cell biology, eukaryotes possess a regulatory system that ensures that DNA replication occurs only once per cell cycle.
