CDK7 pathway

Last updated June 07, 2023

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.

Introduction

An intricate network of cyclin-dependent kinases (CDKs) is organized in a pathway to ensure that each cell accurately replicates its DNA and segregate s it equally between the two daughter cells.^[1] One CDK–the CDK7 complex–cannot be so easily classified. CDK7 is both a CDK-activating kinase (CAK), which phosphorylates cell-cycle CDKs within the activation segment (T-loop), and a component of the general transcription factor TFIIH, which phosphorylates the C-terminal domain (CTD) of the largest subunit of Pol II.^[2] A proposed mode of CDK7 inhibition is the phosphorylation of cyclin H by CDK7 itself^[3] or by another kinase.^[4]

CDK7 has been observed as a prerequisite to S phase entry and mitosis. CDK7 is activated by the binding of cyclin H and its substrate specificity is altered by the binding of MAT1.^[5] The free form of the complex formed, CDK7-cycH-MAT1, operates as CDK-activating kinase (CAK).^[6] In vivo, CDK7 forms a stable complex with cyclin H and MAT1 only when its T-loop is phosphorylated on either Ser164 or Thr170 residues.^[7]

The T-loop

To be active, most CDKs^{[ citation needed ]} require not only a cyclin partner but also phosphorylation at one particular site, which corresponds to Thr161 in human CDK1, and which is located within the so-called T-loop (or activation loop) of kinase subdomain VIII.^[8]^[9] CDKl, CDK2 and CDK4 all require T-loop phosphorylation for maximum activity.^[10]^[11]

The free form of CDK7-cycH-MAT1 phosphorylates the T-loops of CDK1, CDK2, CDK4 and CDK6.^[12] For all CDK substrates of CDK7, phosphorylation by CDK7 occurs following the binding of the substrate kinase to its associated cyclin.^[6] This two-step process has been observed in CDK2, where the association of CDK2 with cyclin A results in a conformational change that primes the catalytic site for binding of its ATP substrate and phosphorylation by CDK7 of Thr160 in its activation segment improves the substrate protein’s ability to bind. It’s been further observed that CDK1 is not phosphorylated by CDK7 in its monomeric form and that monomeric CDK2 and CDK6 are poorly phosphorylated by CDK7, since the activation segment threonine is inaccessible to CDK7 in monomeric CDKs.^[6]

While CDK7 is indeed responsible for the phosphorylation of CDK1, CDK2, CDK4 and CDK6 in vivo, it has been observed that they have varying levels of dependence on CDK7. CDK1 and CDK2 require phosphorylation by CDK7 in order to reach their active states, while CDK4 and CDK6 have been found to require consistent CDK7 activity in order to maintain their phosphorylation states. This discrepancy is likely because the phosphorylated T-loops on CDK2 and CDK1 are protected when they are bound to cyclin while the phosphorylated T-loops on CDK4 and CDK6 remain exposed and therefore are vulnerable to phosphatases. It is therefore proposed that phosphatases work to counter the phosphorylation of CDK4 and CDK6 by CDK7, creating a competition between CDK7 and phosphatases.^[13]

Dual activity

An entirely new perspective on CDK7 function was opened when CDK7 was identified as a subunit of transcription factor IIH (TFIIH) and shown to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII).^[14] TFIIH is a multiprotein complex required not only for class II transcription but also for nucleotide-excision repair.^[15] Its associated CTD-kinase activity is considered important for the promoter-clearance step of transcription, but the precise structural consequences of the phosphorylation of the CTD remain the subject of debate.^[16] Cyclin H and MAT1 are also present in TFIIH,^[17] and it is not known what, if anything, distinguishes the TFIIH-associated form of CDK7 from the quantitatively predominant free form. Whether CDK7 really displays dual-substrate specificity remains to be further explored, but there is no question that the CDK7-cyclin H-MAT1 complex is able to phosphorylate both the T-loop of CDKs and the YSPTSPS (single-letter code for amino acids) repeats of the RNAPII CTD in vitro.

CDK7-cycH-MAT1 binds to TFIIH, which alters the substrate preference of CDK7. CDK7-cycH-MAT1 then preferentially phosphorylates the large subunit C-terminal domain of polymerase II instead of CDK2 when compared to the free-form complex.^[18] In addition, phosphorylation of the Thr170 residue on the T-loop of CDK7 has been found to greatly increase activity of the CDK7– cyclin H–MAT1 complex in favor of CTD phosphorylation. Phosphorylation of Thr170, then, is a proposed mechanism for regulating CTD phosphorylation when CDK7 is associated with TFIIH.^[7]

The role of CDK7 in transcription was confirmed in vivo in Caenorhabditis elegans embryos. Mutants with cdk-7(ax224) were both unable to synthesize most mRNAs and had greatly reduced CTD phosphorylation as well, indicating that CDK7 is required for both transcription and CTD phosphorylation.^[19] In addition, similar results have been found in human cells. An “analog sensitive” CDK7 mutant (CDK7as) was devised which operates normally but is inhibited by an ATP analog competitive inhibitor. Inhibition of CDK7as was correlated with a reduction in CTD phosphorylation, where high inhibition led to very little instances of phosphorylated CTD-Ser5 (the phosphorylation target of CDK7 on CTD).^[20]

HIV latency

It has been demonstrated that TFIIH is a rate-limiting factor for HIV transcription in unactivated T-cells by using a combination of in vivo ChIP experiments and cell-free transcription studies.^[21] The ability of NF-κB to rapidly recruit TFIIH during HIV activation in T-cells is an unexpected discovery; however, there are several precedents in the literature of cellular genes that are activated through the recruitment of TFIIH. In an early and influential paper,^[22] demonstrated that type I activators such as Sp1 and CTF, which were able to support initiation but were unable to support efficient elongation, were also unable to bind TFIIH. By contrast, type II activators such as VP16, p53 and E2F1, which supported both initiation and elongation, were able to bind to TFIIH. In one of the most thoroughly characterized transcription systems,^[23] have studied the temporal order of recruitment of transcription factors during the activation of the major histocompatibility class II (MHC II) DRA gene by IFN-gamma. Following induction of the CIITA transcription factor by IFN-gamma, there was recruitment of both CDK7 and CDK9 causing RNAP CTD phosphorylation and elongation. Finally, Nissen and Yamamoto (2000)^[24] in their studies of the activation of the IL-8 and ICAM-1 promoters observed enhanced CDK7 recruitment and RNAP II CTD phosphorylation in response to NF-κB activation by TNF.

Stem Cells

The CDK7-cycH-MAT1 complex has been found to play a role in the differentiation of embryonic stem cells. It has been observed that the depletion of Cyclin H leads to differentiation of embryonic stem cells. In addition, Spt5, which leads to the differentiation of stem cells upon down-regulation, is a phosphorylation target of CDK7 in vitro, suggesting a possible mechanism by which Cyclin H depletion leads to differentiation.^[5]

Role in Cancer Therapy

Given that CDK7 is involved in two important regulation roles, it’s expected that CDK7 regulation may play a role in cancerous cells. Cells from breast cancer tumors were found to have elevated levels of CDK7 and Cyclin H when compared to normal breast cells. It was also found that the higher levels were generally found in ER-positive breast cancer. Together, these findings indicate that CDK7 therapy might make sense for some breast cancer patients.^[25] Further confirming these findings, recent research indicates that inhibition of CDK7 may be an effective therapy for HER2-positive breast cancers, even overcoming therapeutic resistance. THZ1 was used as a treatment for HER2-positive breast cancer cells and exhibited high potency for the cells regardless of their sensitivity to HER2 inhibitors. This finding was demonstrated in vivo, where inhibition of HER2 and CDK7 resulted in tumor regression in therapeutically resistant HER2+ xenograft models.^[26]

Inhibitors

The growth suppressor p53 has been shown to interact with cyclin H both in vitro and in vivo. Addition of wild type p53 was found to heavily downregulated CAK activity, resulting in decreased phosphorylation of both CDK2 and CTD by CDK7. Mutant p53 was unable to downregulate CDK7 activity and mutant p21 had no effect on downregulation, indicating that p53 is responsible for negative regulation of CDK7.^[27]

THZ1 has recently been discovered to be an inhibitor for CDK7 that selectively forms a covalent bond with the CDK7-cycH-MAT1 complex. This selectivity stems from forming a bond at C312, which is unique to CDK7 within the CDK family. CDK12 and CDK13 could also be inhibited using THZ1 (but at higher concentrations) because they have similar structures in the region surrounding C312.^[28] It was found that treatment of 250 nM THZ1 was sufficient to inhibit global transcription and that cancer cell lines were sensitive to much lower concentrations, opening up further research into the efficacy of using THZ1 as a component of cancer therapy, as described above.

Related Research Articles

In biochemistry, a kinase is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the high-energy ATP molecule donates a phosphate group to the substrate molecule. This transesterification produces a phosphorylated substrate and ADP. Conversely, it is referred to as dephosphorylation when the phosphorylated substrate donates a phosphate group and ADP gains a phosphate group. These two processes, phosphorylation and dephosphorylation, occur four times during glycolysis.

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.

Maturation-promoting factor (abbreviated MPF, also called mitosis-promoting factor or M-Phase-promoting factor) is the cyclin-Cdk complex that was discovered first in frog eggs. It stimulates the mitotic and meiotic phases of the cell cycle. MPF promotes the entrance into mitosis (the M phase) from the G₂ phase by phosphorylating multiple proteins needed during mitosis. MPF is activated at the end of G₂ by a phosphatase, which removes an inhibitory phosphate group added earlier.

The restriction point (R), also known as the Start or G₁/S checkpoint, is a cell cycle checkpoint in the G₁ phase of the animal cell cycle at which the cell becomes "committed" to the cell cycle, and after which extracellular signals are no longer required to stimulate proliferation. The defining biochemical feature of the restriction point is the activation of G₁/S- and S-phase cyclin-CDK complexes, which in turn phosphorylate proteins that initiate DNA replication, centrosome duplication, and other early cell cycle events. It is one of three main cell cycle checkpoints, the other two being the G2-M DNA damage checkpoint and the spindle checkpoint.

Cell cycle checkpoints are control mechanisms in the eukaryotic cell cycle which ensure its proper progression. Each checkpoint serves as a potential termination point along the cell cycle, during which the conditions of the cell are assessed, with progression through the various phases of the cell cycle occurring only when favorable conditions are met. There are many checkpoints in the cell cycle, but the three major ones are: the G1 checkpoint, also known as the Start or restriction checkpoint or Major Checkpoint; the G2/M checkpoint; and the metaphase-to-anaphase transition, also known as the spindle checkpoint. Progression through these checkpoints is largely determined by the activation of cyclin-dependent kinases by regulatory protein subunits called cyclins, different forms of which are produced at each stage of the cell cycle to control the specific events that occur therein.

Cyclin E is a member of the cyclin family.

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.

<span class="mw-page-title-main">Cyclin D</span>

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.

Transcription factor II H (TF_IIH) is an important protein complex, having roles in transcription of various protein-coding genes and DNA nucleotide excision repair (NER) pathways. TF_IIH first came to light in 1989 when general transcription factor-δ or basic transcription factor 2 was characterized as an indispensable transcription factor in vitro. This factor was also isolated from yeast and finally named TF_IIH in 1992.

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.

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 protein 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. With its cyclin partners, Cdk1 forms complexes that phosphorylate a variety of target substrates ; phosphorylation of these proteins leads to cell cycle progression.

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.

General transcription factor IIH subunit 1 is a protein that in humans is encoded by the GTF2H1 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.

A series of biochemical switches control transitions between and within the various phases of the cell cycle. The cell cycle is a series of complex, ordered, sequential events that control how a single cell divides into two cells, and involves several different phases. The phases include the G1 and G2 phases, DNA replication or S phase, and the actual process of cell division, mitosis or M phase. During the M phase, the chromosomes separate and cytokinesis occurs.

References

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