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12.13: Regulation of the Cell Cycle by Protein Kinases

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    15122
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    This section is an integration of materials as referenced, with significant modification and additions.

    Aleem and Arceci . Targeting cell cycle regulators in hematologic malignancies. Article in Frontiers in Cell and Developmental Biology 2015. DOI: 10.3389/fcell.2015.00016. Creative Commons Attribution 4.0 International

    Introduction

    In order for a cell to undergo successful division, it has to perform four key tasks in a highly ordered fashion. First, there is a preparatory synthetic phase (G1) that results in an increased cell size in anticipation of DNA replication (S phase). Cells then proceed through (G2-phase) to prepare to equally segregate duplicated DNA (M phase) and finally divide into two equal daughter cells. From G1 a cell can also exit the cell cycle and enter a state of qui-escence (G0), undergo differentiation or re-enter the cell cycle to proliferate in response to mitogenic signals.

    The core molecular machinery controlling the mammalian cell cycle consists of a family of serine/threonine protein kinases called cyclin-dependent kinases (CDKs). These are catalytic subunits, which are activated in most cases by association with cyclin regulatory subunits. The activity of CDK/cyclin complexes is further regulated by CDK-inhibitors (CKIs), phosphorylation and dephosphorylation, ubiquitin-mediated degradation, transcriptional regulation, substrate recognition, and subcellular localization. The family of CDKs/cyclins/CKIs contains more than 30 members. They are implicated in essential cellular functions such as transcription, DNA damage repair, epigenetic regulation, metabolism, proteolytic degradation, stem cell self renewal, neuronal functions, and spermatogenesis. Figure \(\PageIndex{1}\) below shows the cell cycle and the involvement of CDKs/cyclins at key points.

    CellCycleRegulatorsFig1.svg
    Figure \(\PageIndex{1}\): Cyclin-dependent kinases (CDKs) and their cyclin regulatory subunits. Aleem and Arceci. Frontiers in Cell and Developmental Biology 2015. DOI: 10.3389/fcell.2015.00016. Creative Commons Attribution 4.0 International

    CDKs with Direct Functions in Cell Cycle Regulation

    The classical CDKs that directly regulate the mammalian cell cycle in complexes with cyclin subunits include CDK3, CDK4, CDK6, CDK2, and CDK1. CDK3 promotes cell cycle entry from quiescence in association with cyclin C. CDK8 has also been suggested to play a role in cell cycle entry from G0 and in the G1/S transition. In its simplest model, the mammalian cell cycle proceeds as follows:

    • In early G1, CDK4/CDK6 in complex with cyclin D receive mitogenic signals that result in activation of cell cycle entry, as shown in Figure \(\PageIndex{1}\).. Key signaling events include initia-tion of retinoblastoma protein (pRb) phosphorylation and the sequestration of p21Cip1 and p27kip1, which are both inhibitors of CDK2, thus promoting the activation of CDK2/ cyclin E complex. In late G1, CDK2 in complex with cyclin E completes the phosphorylation and hence inactivation of pRb, which in turn releases the E2F transcription factors. E2F promotes transcription of cyclin E that is necessary for the G1/S transition.
    • Progression through S phase is mediated by CDK2/cyclin A complex.
    • Mitosis is then initiated by CDK1/cyclin B complexes. CDK1/cyclin A complexes contribute to the preparation for mitosis in G2 phase. The activity of CDK1/cyclin B is tightly regulated by activating phosphorylation by the CDK-activating kinase (CAK) (a heterodimer of cyclin H and CDK7) and inhibitory phosphorylations by WEE-1 and Myt1 on Tyr15 and Thr14. Mitosis starts after WEE-1 is degraded and CDC25C phosphatase releases the inhibitory phosphorylation on CDK1/cyclin B.

    The specific CDK4/CDK6 pharmacological inhibitors described in this study are shown.

    HVJ: The cyclins are also expressed in a coordinated fashion throughout the cell cycle. The cyclin expression cycle is shown in Figure \(\PageIndex{2}\) below. The timing of expression is consistent with the explanations above.

    Cyclin_Expression.svg
    Figure \(\PageIndex{2}\): Cyclin expression cycle. https://commons.wikimedia.org/wiki/F...Expression.svg

    CDKs with Transcriptional and Other Functions

    In addition to their direct role in the mitotic cell cycle regulation, some classical CDK/cyclin complexes have essential functions in meiosis, such as CDK2, in transcription and/or DNA repair. Other CDKs act by activating the classical CDKs, such as CDK7/cyclin H (CAK) and the related CDK20, also known as cell cycle related kinase (CCRK). Some CDKs function mainly in influencing transcription by phosphorylating the carboxy-terminal domain (CTD) of ribonucleic acid (RNA) polymerase II (RNA pol II). This phosphorylation also serves as a platform for RNA processing and chromatin regulation.

    CDKs that have important transcriptional roles include CDK7/cyclin H/MAT1 complex, a component of the basal transcription factor, TFIIH and facilitates transcriptional initiation. CDK8/cyclin C, in addition to its role in transcription, is also involved in the Wnt/β-catenin pathway and in inhibition of lipogenesis. Cyclin C can recruit CDK8 or CDK19 to the CDK8 module of the Mediator complex, which can function as a positive or negative regulator of transcription by RNA pol II. CDK3/cyclin C also plays a role in NHEJ-mediated DNA damage repair. While CDK9 in complex with cyclin T forms the phospho-transcription elongation factor b (p-TEFb) and promotes transcriptional elongation, CDK9 also functions in the DNA damage response when complexed with cyclin K. CDK10/cyclin M phosphorylates the Ets2 transcription factor and positively controls its degradation by the proteasome. Ets2 plays key roles in cancer and development. CDK11/cyclin L controls the assembly of the RNA pol II mediator complex. CDK12 and CDK13 in complex with cyclin K control RNA pol II transcription, and CDK12/cyclin K controls DNA damage response.

    Structure of CDKs–cyclins

    Open Biology. Wood and Jane A. Endicott (2018) https://doi.org/10.1098/rsob.180112. Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/,

    The structures of inactive cyclin free kinases are very similar but vary at the N-terminal and C-terminal ends. Figure \(\PageIndex{3}\) below shows an interactive iCn3D model of the prototypical active human cyclin-dependent kinase 2 with a bound ATP (1HCK).

    human cyclin-dependent kinase 2 with a bound ATP (1HCK).png
    Figure \(\PageIndex{3}\): Human cyclin-dependent kinase 2 with a bound ATP analog (1HCK) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...3Sf81pwLKQZx9A

    The model shows that CDK2 has structural features shown in all the kinases we have studied previously:

    • a smaller N-terminal lobe (light cyan) and larger C-terminal lobe (light magenta) in between which ATP binds (along with Mg2+).
    • the C-helix (residues 45 – 55, purple), which contains a conserved Glu. It form an interaction with and helps position a key Lys in the active, that facilitates ATP binding and transition state stabilization;
    • hinge (residues 80 – 84, yellow),
    • activation loop (residues 145– 172, red), which contains T160 (sticks, CPK colors, labeled) that becomes phosphorylated on activation by yet another kinase called CDK-activation kinase (CAK). When T160 is phosphorylated, the kinase binds to cyclin A. The loop starts and ends with the conserved residue DFG and APE, repsectively.
    • Not highlighted in the model is a conserved conformationally flexible glycine-rich region (residue 12-16) with a the motif GXGXXG

    In the inactive form, the N-terminal end of activation loop has a short alpha helix the prevents the C-helix from adopting the correct position for catalysis. Activation requires movement of the C-helix allowing the Glu in the C-helix to position the active site Lys.

    CDK2–cyclin A activation

    Binding of cyclin A to CD2K activates it through repositioning of the C-helix and the activation loop. When CDK2 is phosphorylated and bound to cyclin A, there is a large shift in the C-helix allowing the interaction of the C-helix Glu with the active site Lys.

    First let's look at the structure of cyclins. Each cyclin has unique sequence and structural features that allow them to interact with specific CDKs and associated proteins. However they all a conserved "cyclin box" structure containing about 100 amino acids.

    Figure \(\PageIndex{4}\) below shows an interactive iCn3D model of bovine cyclin A (1VIN).

    Bovine cyclin A (1VIN) .png
    Figure \(\PageIndex{4}\): Bovine cyclin A (1VIN) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...6j7kLcPzpRTAA9

    Cyclin A has two linked cyclin box folds, each containing around 100 amino acids and comprised of five helices. They inteeact with the more disorderd parts of unphosphorylated CDK2, which result is some low level of activity. Cyclin binding causes a large movement of the C-helix enabling the Glu -- Lys intereaction. Phosphorylation of T160 leads to reposition of the activation loop.

    Figure \(\PageIndex{5}\) below shows an interactive iCn3D model of Phosphorylated cyclin-dependent kinase 2 bound to cyclin A (1JST)

    CDK2_Cyclin_ATP_1JST.png
    Figure \(\PageIndex{5}\): Phosphorylated cyclin-dependent kinase 2 bound to cyclin A (1JST) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...cP6YsNcgUtGHW8

    CDK2 is shown in cyan and cyclin A in gray. Here are some structural features represented in the model.

    • the C-helix (residues 45 – 55, purple),
    • activation loop (residues 145– 172, red), which contains pT160 (sticks, CPK colors, labeled)
    • the catalytic "triad" Lys33, Glu51, and Asp145

    Figure \(\PageIndex{6}\) below shows an animation of structural changes in just CDK2 when "apo"-CDK2 (without bound cyclin A, pdbID 1HCK) binds cyclin A (1JST).

    1hckto1jst_CDK2_cyclinA.gif
    Figure \(\PageIndex{6}\): Structural changes CDK2 when it binds cyclin A

    Gray represents the structure of CDK2 in the absence of cyclin A. The structure of just CDK2 in the presence of cyclin A is shown in magenta. Note that large shifts in the C-helix (purple) and activation loop (red) on binding cyclin A.

    Cyclin partners of CDK1 and CDK2

    CDK1 is the closest member of the CDK family to CDK2 and for which structures of the cyclin-free and authentic cyclin-bound forms can also be compared .

    Depending on cyclin availability and concentration, CDK2 can binds cyclin A, B (if CDK1 expression is knocked down) and E (see Figure \(\PageIndex{1}\). The binding interface between CDK2 and the cyclins is quite large compared to the interface between CDK1 and cyclins. Three large aromatic side cyclin side chains (Y170, Y177 and Y258) are conserved in the binding interface. In cyclin E, the corresponding amino acids are smaller (N112, I119 and L202).

    The biding interface between CDK1 and the cyclins is smaller so it appears that might preferentially interact with cyclins A and B to gain binding affinity through the more robust interactions with the aromatic groups in the interface. In the CDK2: Cyclin A and CDK2:cyclin B complex,

    A comparison of the CDK1–cyclin B and CDK2–cyclin A/B/E structures also highlights the potential for these closely related CDKs to be differentially regulated by reversible phosphorylation. The antagonistic activities of Wee1/Myt1 kinases and Cdc25 phosphatases regulate the phosphorylation status of the CDK glycine-rich loop (defined by the GXGXXG motif, residues 11–16 in CDK2). The structure of CDK2–cyclin A phosphorylated on Y15 illustrates how phosphorylation promotes a glycine loop structure that antagonizes both peptide substrate binding and the ATP conformation required for catalysis . The flexibility of the glycine-rich loop is compatible with a model in which the phosphorylated Y15 side chain is solvent exposed and accessible to both kinases and phosphatases. CDK1 is also regulated by active-site phosphorylation, and the conserved nature of the structure in this region suggests that the mechanism of inhibition is also conserved.

    CDK substrate recognition

    Local and distal sequence motifs must be use to confer specificity to the binding of specific cyclins and other protein to specific CDKs. One interesting example is provide by examing the structure of a phospho-CDK2 Cyclin A in complex with a peptide substrate derived from the protein CDC6. Figure \(\PageIndex{7}\) below shows an interactive iCn3D model of Phospho-CDK2:Cyclin A complex with a peptide containing both the substrate and recruitment sites of CDC6 (2CCI)

    Phospho-CDK2 Cyclin A- peptide  CDC6 (2CCI).png
    Figure \(\PageIndex{7}\): Phospho-CDK2 Cyclin A in complex with a peptide containing both the substrate and recruitment sites of CDC6 (2CCI) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Ge8kdSBjxkaNd8

    The color coding is the same as the models above:

    • CDK2 is shown in cyan and cyclin A in gray.
    • the C-helix (residues 45 – 55, purple),
    • activation loop (residues 145– 172, red), which contains pT160 (sticks, CPK colors, labeled)
    • the catalytic "triad" Lys33, Glu51, and Asp145

    The 30 amino acids peptide (number 67-96) shown in gold is a substrate for phosphorylation by the CDK2:cyclin A complex. It derives from an actual biological substrate in the protein cell division control protein 6 homolog, also called CDC6-related protein. It is involved in a checkpoint control of the cycle cycle that "checks" that DNA replication is completed before mitosis. It is discontinuous in the model since part of the bound peptide is intrinsically disorder and not observed in the crystal structure.

    • The 1st fragment of the CDC6 peptide (67-73) contains the binding motif sequence S/T)PX(K/R) (the CDC6 substrate has the sequence 70Ser-Pro-Arg-Lys). Ser 70 is the target amino for phosphorylation by CDK2:cyclin A.
    • The second fragment seen in the model (amino acid 85-96) contains another binding motif, RXL (in this peptide RRL), which acts to recruit cyclin A. This binds to the sequence MRAIL (210-214) in cyclin A.

    What is so interesting is that this second binding site on cyclin A for its target protein is so far away through the active site of the CDK2:cyclin A. These kinds of interactions work to determine specificity for CDKs and the binding cyclin partners.

    CDKs 7, 9, 12 and 13 phosphorylate the RNA polymerase carboxy terminal domain (CTD). The sequence of the CTD is unusual, being composed of 52 heptad repeats in humans, with the consensus sequence YSPTSPS. Extracted from cells, CTD residues S2 and S5 are the most abundantly phosphorylated serine residues, while S7 is phosphorylated to a lesser extent. CDK7 has been shown to predominantly phosphorylate S5 and S7, CDK9 to have activity towards all three serines, and CDK12 and CDK13 to predominantly phosphorylate S2.

    CDKs form complex not only with target protein substrates but other protein which can serves as scaffolding anchors that bind both the CDK and the cyclin. Figure \(\PageIndex{8}\) below shows an interactive iCn3D model of human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and teh scaffolding protein MAT1 (6xbz)

    Human CDK-activating kinase (6xbz).png
    Figure \(\PageIndex{8}\): Human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1 (6xbz)(Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...3bdn39ARgLijB7

    The gray protein is CDK7, the cyan is cyclin H, and the orange MAT1. The purple again represents the C-helix of the CDK, which the red is the activation loop. The catalytic triad side chains in the active site of the CDK are shown in CPK-colored sticks. Also shown is phospho-Ser in the activation loop.

    The CDK activating kinase (CAK) shown above phosphorylates the target S/T in the activation loop (which is also called the T-loop) in CDKs, activating the kinase. In addition it regulates the initiation of transcription by phosphorylating the YSPTSPS repeats in C-terminus of RNA polymerase II subunit RPB1. There are 15 consecutive repeats in the sequence as well as others dispersed in the C-terminal domain.

    We already mentioned the motif RXL found in cyclin binding proteins that recruits them to cyclins (through, for example, their interaction with MRAIL (210-214) in cyclin A. Likewise short motifs in cyclins are used to bind to proteins that increase CDK activity or decrease it.

    A number of cyclin-encoded protein-binding sites or short peptide motifs have been structurally characterized. A well-characterized example is the recycling of the cyclin RXL recruitment site that is exploited to either enhance or inhibit CDK activity. Alternatively, short motifs encoded within the cyclin sequence can be used both to dock cyclins to substrates to enhance CDK activity and alternatively to localize them to CDK regulators frequently resulting in a loss of CDK activity. Members of the p27KIP1/p21CIP1 cyclin-dependent kinase inhibitor (CKI) family share an RXL motif with RXL-containing substrates and compete with them for CDK–cyclin association. The INK (inhibitors of CDK) family of CKIs selectively inhibits CDK4 or CDK6 and, through an allosteric mechanism, disfavours CDK–cyclin binding [15]. Their tandem ankyrin repeat structures exemplified by CDK6–p19INK4d bind in the vicinity of the CDK hinge on the interface opposite to the surface remodeled upon cyclin association.


    12.13: Regulation of the Cell Cycle by Protein Kinases is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski.

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