Phosphorylation of the PCNA Binding Domain of the Large Subunit of Replication Factor C by Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits DNA Synthesis

Maga, Giovanni; Mossi, Romina; Fischer, Roland; Berchtold, Martin W.; Hübscher, Ulrich

doi:10.1021/bi962809n

Cited by 24 publications

(22 citation statements)

References 38 publications

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“…What is not clear is if other kinases besides CDKs play additional roles in regulating RF-C activity. In this direction phosphorylation of RF-C by Ca 2ϩ /calmodulin dependent protein kinase II has been reported to inactivate RF-C activity in vitro (35). Our demonstration in this study that RF-C phosphorylation can be inhibited by roscovitine suggests that in vivo, kinases like Ca 2ϩ /calmodulin-dependent protein kinase II target RF-C subsequent to CDK phosphorylation.…”

Section: Discussionmentioning

confidence: 53%

Cell Cycle-dependent Phosphorylation of the Large Subunit of Replication Factor C (RF-C) Leads to Its Dissociation from the RF-C Complex

Munshi

Cannella

Brickner

et al. 2003

Journal of Biological Chemistry

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The five subunit replication factor C (RF-C) complex plays a critical role in DNA elongation. We find that the large subunit of RF-C (RF-Cp145) is phosphorylated in vivo whereas the smaller RF-C subunits are not phosphorylated. The phosphorylation of endogenous RFCp145 is modulated in a cell cycle-dependent manner. Phosphorylation is maximal in G 2 /M and is inhibited by an inhibitor of cyclin-dependent kinases. Phosphorylation of purified recombinant RF-C complex in vitro reveals that RF-Cp145 is preferentially phosphorylated by cdc2-cyclin B but not by cdk2-cyclin A or cdk2-cyclin E. In vitro phosphorylation of RF-C complex by cdc2-cyclin B kinases leads to dissociation of phosphorylated RFCp145 from the RF-C complex. Using different approaches we demonstrate that phosphorylated RFCp145 is indeed dissociated from RF-Cp40 and RF-Cp37 in vivo. These results suggest that destabilization of the RF-C complex by CDKs may inactivate the RF-C complex at the end of S phase.Cell cycle progression is regulated by distinct cyclin-dependent kinases (CDKs), 1 which activate at different times in the cell cycle. In mammalian cells, cyclin E-dependent kinase is activated at the G 1 to S phase transition, after the D-type cyclins but prior to A-type cyclins. The timing of activation of cyclin A kinase activity coincides with the onset of DNA synthesis (1). Cdc2-cyclin B kinase activation at the G 2 /M transition is required for cells to enter mitosis. Replication of DNA is a highly regulated process, which occurs during the S phase of the cell cycle. The DNA polymerase ␣-primase complex allows the generation of RNA-DNA primers for initiation of leading strand synthesis and synthesis of each Okazaki fragment during lagging strand replication. The polymerase switch from DNA polymerase ␣ to DNA polymerase ␦ then catalyzes the replication of the leading strand and for completion of the lagging strand. The replication factor C (RF-C) complex and PCNA are essential for processive DNA synthesis. RF-C loads PCNA onto DNA in an ATP-dependent process (2, 3). The PCNA clamp then recruits DNA polymerase ␦ for processive DNA synthesis (4).RF-C is a five subunit heterodimer, and all five subunits of RF-C from human and yeast have been cloned (5-13). RF-C activity can be reconstituted in vitro when all five subunits are expressed as recombinant proteins (14 -16).The Escherichia coli clamp loader, the ␥ complex, like the yeast and human RF-C complexes is comprised of five polypeptides. The DNA-dependent ATPase activity of the clamp in the ␥ complex is provided by three copies of the ␥ polypeptide whereas human (and yeast) utilize three different polypeptide chains hRF-Cp37 (yRF-C2), hRF-Cp36 (yRF-C3), and hRFCp40 (yRF-C4). The ␦ subunit (homologous to yRF-C1 in yeast and hRF-Cp145 in man) binds the clamp and has conserved hydrophobic residues required for clamp loading. Structural studies of the ␥ complex: ␦Ј-␥1-␥2-␥3-␦ reveals that the Cterminal domains of ␦Ј, ␥, and ␦ form a helical scaffold (circular collar) and the N-terminal ends appear ...

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Section: Discussionmentioning

confidence: 53%

Cell Cycle-dependent Phosphorylation of the Large Subunit of Replication Factor C (RF-C) Leads to Its Dissociation from the RF-C Complex

Munshi

Cannella

Brickner

et al. 2003

Journal of Biological Chemistry

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“…Furthermore, CaMKII has been demonstrated to impair nuclear translocation of nuclear factor of activated T cells (NFAT) by directly phosphorylating calcineurin [56], and to induce a G 2 /M cell cycle arrest when transiently expressed in a constitutively active form [57]. Moreover, CaMKII may inhibit DNA replication in the S phase of the cell cycle by phosphorylation of the PCNA binding domain of the large subunit of replication factor C [58]. Thus, CaMKII isoforms appear to interfere with cell cycle control in a complex manner at multiple pathways.…”

Section: Discussionmentioning

confidence: 99%

Non-Selective Cation Channel-Mediated Ca2+-Entry and Activation of Ca2+/Calmodulin-Dependent Kinase II Contribute to G2/M Cell Cycle Arrest and Survival of Irradiated Leukemia Cells

Heise

Palme

Misovic

et al. 2010

Cell Physiol Biochem

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Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca²⁺ signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured by fura-2 Ca²⁺ imaging. Nuclear activity of Ca²⁺/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G₂/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G₂/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca²⁺ entry as evident from fura-2 Ca²⁺ imaging. Similarly, IR stimulated a Ca²⁺-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca²⁺ entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G₂ phase was delayed upon buffering [Ca²⁺]_i with the Ca²⁺ chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca²⁺ entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.

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“…p40 markedly phorylation, suggesting a possible role of calmodulin-dependent decreases the synthesis of full-length M13 DNA, without a reprotein kinase II in regulating the dynamics of interaction be-duction of nucleotide incorporation. An interaction between p40 tween PCNA and RF-C [51].…”

Section: Rf-c As a Clamp Loader And Unloadermentioning

confidence: 99%

Clamping down on clamps and clamp loaders

Mossi

Hübscher

1998

European Journal of Biochemistry

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124

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DNA transactions such as DNA replication and DNA repair require the concerted action of many enzymes, together with other proteins and non-protein cofactors. Among them three main accessory proteins, replication factor C (RF-C), proliferating-cell nuclear antigen (PCNA) and replication protein A (RP-A), are essential for accurate and processive DNA synthesis by DNA polymerases. RF-C is a complex consisting of five polypeptides with distinct functions. RF-C can bind to a template-primer junction and, in the presence of ATP, load the PCNA clamp onto DNA, thereby recruiting DNA polymerases to the site of DNA synthesis. RF-C not only acts as a clamp loader in DNA replication and DNA repair, but there is some evidence that it could be involved in several other processes such as transcription, S-phase checkpoint regulation, apoptosis, differentiation and telomere-length regulation.Keywords : eukaryotic DNA replication; replication factor C; proliferating-cell nuclear antigen; clamp; clamp loader ; DNA polymerase δ ; S-phase checkpoint regulation ; apoptosis; differentiation; telomere. Of clamps and clamp loadersrespective clamps onto DNA. High sequence similarity between the clamp loaders suggest that their mechanisms of action may DNA replication, one of the most remarkable and challeng-be very similar [4]. The eukaryotic RF-C clamp loader consists ing cellular events, is the result of the collaboration of a formida-of five distinct subunits (Table 1), as does the E. coli γ complex. ble amount of proteins. The mechanisms at the basis of this By loading the homotrimer proliferating-cell nuclear antigen complicated event are similar in prokaryotes and eukaryotes, de-(PCNA) clamp onto DNA, it confers high processivity to polyspite the evolutionary distance. In each of these cell types, the merases δ and ε. Despite there being virtually no similarity in replicative polymerases are chaperoned by several accessory sequence between β and PCNA, the three-dimensional structures proteins that confer speed and high processivity.are almost identical [5]. The amino acid sequence of PCNA is Responsible for DNA replication in Escherichia coli is the only approximately 70% the length of that of β, so that three DNA polymerase III holoenzyme, a ten-subunit complex (Ta-monomers of PCNA are required to form the typical doughnutble 1) [1]. The DNA polymerase (polymerase III core) consists like structure, in contrast to β where only two monomers are of A (the polymerase), ε (the proofreading exonuclease) and θ enough. The T4 gene 44/62 protein complex contains five subsubunits. The clamp loader, called γ complex, contains five subunits, four protomers of the gene 44 protein and one copy of the units, γ, δ, δ′, ψ and χ, and is required for the ATP-dependent gene 62 protein [6]. It is required for the loading of the product assembly of the β clamp onto DNA. Contrary to all eukaryotes of gene 45, the T4 bacteriophage counterpart of PCNA and E. and the T4 phage, E. coli contains an additional connector procoli β. Despite the apparent similar...

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Phosphorylation of the PCNA Binding Domain of the Large Subunit of Replication Factor C by Ca²⁺/Calmodulin-Dependent Protein Kinase II Inhibits DNA Synthesis

Cited by 24 publications

References 38 publications

Cell Cycle-dependent Phosphorylation of the Large Subunit of Replication Factor C (RF-C) Leads to Its Dissociation from the RF-C Complex

Cell Cycle-dependent Phosphorylation of the Large Subunit of Replication Factor C (RF-C) Leads to Its Dissociation from the RF-C Complex

Non-Selective Cation Channel-Mediated Ca<sup>2+</sup>-Entry and Activation of Ca<sup>2+</sup>/Calmodulin-Dependent Kinase II Contribute to G<sub>2</sub>/M Cell Cycle Arrest and Survival of Irradiated Leukemia Cells

Clamping down on clamps and clamp loaders

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