Roles of Human AND-1 in Chromosome Transactions in S Phase

Coordinated execution of DNA replication, checkpoint activation, and postreplicative chromatid cohesion is intimately related to the replication fork machinery. Human AND-1/chromosome transmission fidelity 4 is localized adjacent to replication foci and is required for efficient DNA synthesis. In S phase, AND-1 is phosphorylated in response to replication arrest in a manner dependent on checkpoint kinase, ataxia telangiectasia-mutated, ataxia telangiectasia-mutated and Rad3-related protein, and Cdc7 kinase but not on Chk1. Depletion of AND-1 increases DNA damage, delays progression of S phase, leads to accumulation of late S and/or G₂ phase cells, and induces cell death in cancer cells. It also elevated UV-radioresistant DNA synthesis and caused premature recovery of replication after hydroxyurea arrest, indicating that lack of AND-1 compromises checkpoint activation. This may be partly due to the decreased levels of Chk1 protein in AND-1-depleted cells. Furthermore, AND-1 interacts with cohesin proteins Smc1, Smc3, and Rad21/Scc1, consistent with proposed roles of yeast counterparts of AND-1 in sister chromatid cohesion. Depletion of AND-1 leads to significant inhibition of homologous recombination repair of an I-SceI-driven double strand break. Based on these data, we propose that AND-1 coordinates multiple cellular events in S phase and G₂ phase, such as DNA replication, checkpoint activation, sister chromatid cohesion, and DNA damage repair, thus playing a pivotal role in maintenance of genome integrity.Replication fork is not only the site of DNA synthesis but also the center for coordinated execution of various chromosome transactions. The preparation for replication forks starts in the G₁ phase, when the prereplicative complex composed of origin recognition and minichromosome maintenance assembles on the chromosome. At the G₁-S boundary, Cdc45, GINS complex, and other factors join the prereplicative complex to generate a complex capable of initiating DNA replication. A series of phosphorylation events mediated by cyclin-dependent kinase and Cdc7 kinase play crucial roles in this process and facilitate the generation of active replication forks (1–6). Purification of the putative replisome complex in yeast indicated the presence of the checkpoint mediator Mrc1 and fork protection complex proteins Tof1 and Csm3 in the replication fork machinery (7), consistent with a previous report on the genome-wide analyses with chromatin immunoprecipitation analyses on chip (microarray) (8). Mcm10 is another factor present in the isolated complex, required for loading of replication protein A (RPA)² and primase-DNA polymerase α onto the replisome complex (7, 9, 10).Replication fork machinery can cope with various stresses, including shortage of the cellular nucleotide pool and replication fork blockages that interfere with its progression. Stalled replication forks activate checkpoint pathways, leading to cell cycle arrest, DNA repair, restart of DNA replication, or cell death in some cases (11–14). Single-stranded DNAs coated with RPA at the stalled replication forks are recognized by the ATR-ATR-interacting protein kinase complex and Rad17 for loading of the Rad9-Rad1-Hus1 checkpoint clamp (14–16). Factors present in the replisome complex are also known to be required for checkpoint activation. Claspin, Tim, and Tipin functionally and physically associate with sensor and effector kinases and serve as mediator/adaptors (17–23). Mcm7, a component of the replicative DNA helicase in eukaryotes, was reported to associate with the checkpoint clamp loader Rad17 (24) and to have a distinct function in checkpoint (24, 25). We recently reported that Cdc7 kinase, known to be required for DNA replication initiation, plays a role in activation of DNA replication checkpoint possibly through regulating Claspin phosphorylation (26). Thus, it appears that DNA replication and checkpoint activation functionally and physically interact with each other.Another crucial cellular event for maintenance of genome stability is sister chromatid cohesion. The cohesin complex, a conserved apparatus required for sister chromatid cohesion, contains Smc1, Smc3, and Rad21/Scc1/Mcd1 proteins. The assembled cohesin complexes are loaded onto chromatin prior to DNA replication in G₁ phase and link the sister chromosomes during S and G₂ phase until mitosis when they separate (27, 28). The mitotic cohesion defects are not rescued by supplementing cohesin in G₂ phase, and it has been suggested that establishment of sister chromatid cohesion is coupled with DNA replication (29, 30). Indeed, yeast mutants in some replisome components show defect in sister chromosome cohesion or undergo chromosome loss (31–33). Cdc7 kinase is also required for efficient mitotic chromosome cohesion (34, 35).Human AND-1 is the putative homolog of budding yeast CTF4/Pob1/CHL15 and fission yeast Mcl1/Slr3. The budding yeast counterpart was identified as a replisome component described above (7), which travels along with the replication fork (29). CTF4 is nonessential for viability, but its interactions with primase, Rad2 (FEN1 family of nuclease), and Dna2 have implicated CTF4 in lagging strand synthesis and/or Okazaki fragment processing (36–39). Yeast CTF4 and Mcl1 are involved in chromosome cohesion (33, 40, 41) and genetically interact with a cohesin, Mcd1/Rad21 (40, 42). Recently, it was reported that human AND-1 protein interacts with human primase-DNA polymerase α and Mcm10 and is required for DNA synthesis (43).Here we confirm that human AND-1 protein is required for DNA replication and efficient progression of S phase, and we further show that it facilitates replication checkpoint. Depletion of AND-1 causes accumulation of DNA damage and cell cycle arrest at late S to G₂ phase, ultimately leading to cell death. Furthermore, we also show that human AND-1 physically interacts with cohesin proteins Smc1, Smc3, Rad21/Scc1, suggesting a possibility that AND-1 may physically and functionally link replisome and cohesin complexes in vivo. Recent studies indicate that sister chromatid cohesion is required for recombinational DNA repair (44–47). Thus, we examined the requirement of AND-1 for repair of artificially induced double-stranded DNA breaks and showed that AND-1 depletion leads to significant reduction of the double strand break repair. Possible roles of AND-1 in coordination of various chromosome transactions at a replication fork and in maintenance of genome integrity during S phase will be discussed.     

Differential Regulation of Glycogenolysis by Mutant Protein Phosphatase-1 Glycogen-targeting Subunits

PTG and G_L are hepatic protein phosphatase-1 (PP1) glycogen-targeting subunits, which direct PP1 activity against glycogen synthase (GS) and/or phosphorylase (GP). The C-terminal 16 amino residues of G_L comprise a high affinity binding site for GP that regulates bound PP1 activity against GS. In this study, a truncated G_L construct lacking the GP-binding site (G_Ltr) and a chimeric PTG molecule containing the C-terminal site (PTG-G_L) were generated. As expected, GP binding to glutathione S-transferase (GST)-G_Ltr was reduced, whereas GP binding to GST-PTG-G_L was increased 2- to 3-fold versus GST-PTG. In contrast, PP1 binding to all proteins was equivalent. Primary mouse hepatocytes were infected with adenoviral constructs for each subunit, and their effects on glycogen metabolism were investigated. G_Ltr expression was more effective at promoting GP inactivation, GS activation, and glycogen accumulation than G_L. Removal of the regulatory GP-binding site from G_Ltr completely blocked the inactivation of GS seen in G_L-expressing cells following a drop in extracellular glucose. As a result, G_Ltr expression prevented glycogen mobilization under 5 mm glucose conditions. In contrast, equivalent overexpression of PTG or PTG-G_L caused a similar increase in glycogen-targeted PP1 levels and GS dephosphorylation. Surprisingly, GP dephosphorylation was significantly reduced in PTG-G_L-overexpressing cells. As a result, PTG-G_L expression permitted glycogenolysis under 5 mm glucose conditions that was prevented in PTG-expressing cells. Thus, expression of constructs that contained the high affinity GP-binding site (G_L and PTG-G_L) displayed reduced glycogen accumulation and enhanced glycogenolysis compared with their respective controls, albeit via different mechanisms.Hepatic glycogen metabolism plays a central role in the maintenance of circulating plasma glucose levels under various physiological conditions. The rate-controlling enzymes in glycogen metabolism, glycogen synthase (GS)² and glycogen phosphorylase (GP), are subject to multiple levels of regulation, including allosteric binding of activators and inhibitors, protein phosphorylation, and changes in subcellular localization. GS is phosphorylated on up to 9 residues by a variety of kinases, although site 2 appears to be the most important regulator of hepatic GS (1). In contrast, GP is phosphorylated on a single N-terminal serine residue by phosphorylase kinase, which increases GP activity and its sensitivity to allosteric activators. Both GS and GP are in turn also regulated by protein phosphatases, most notably PP1. Although PP1 is a cytosolic protein, a family of five molecules has been reported that targets the enzyme to glycogen particles (2–7), whereas another two glycogen-targeting subunits have been putatively identified based on sequence homology (8). Published work has indicated that each targeting subunit confers differential regulation of PP1 activity by extracellular hormonal signals and/or intracellular changes in metabolites (9–11).Four PP1-glycogen-targeting proteins are expressed in rodent liver, although G_L and PTG/R5 have been most extensively studied (9, 12–15). G_L is present at higher levels in rat liver than PTG (12), but the expression of both proteins is subject to coordinate regulation by fasting/refeeding and insulin (12, 13). Previous studies indicated that the PTG-PP1 complex is primarily responsible for GP dephosphorylation and regulation of glycogenolysis (13, 16), whereas the G_L-PP1 complex preferentially mediates the activation of GS upon elevation of extracellular glucose (9, 13). However, the molecular mechanisms underlying these differential properties of PTG and G_L have not been completely defined.Both PTG and G_L directly bind to specific PP1 substrates involved in glycogen metabolism, albeit for different physiological reasons. The extreme C-terminal 16 amino acids of G_L comprises a unique, high affinity binding site for phosphorylated GP (GPa (17)), which has been further delineated to two critical tyrosine residues (18, 37). Interaction of PP1 with G_L reduces phosphatase activity against GPa (3). In turn, GPa binding to the G_L-PP1 complex potently inhibits phosphatase activity against GS in vitro (3, 19) and regulates glycogen-targeted PP1 activity in liver cells and extracts (20–22). PTG contains a single substrate-binding site that interacts with GS and GP (5, 23). In contrast to the regulatory role of the GPa binding to G_L, interaction of substrates with PTG increases PP1 activity against these proteins (24). Indeed, disruption of the substrate-binding site by point mutagenesis abrogated the ability of mutant PTG expression to increase cellular glycogen levels (23), indicating an important role for substrate binding to the PTG-PP1 complex.Previous work has comprehensively compared the metabolic impact of PTG versus G_L overexpression in hepatocytes and thus was not the goal of this study (9, 10). Instead, two novel PP1 targeting constructs were generated in which the high affinity GPa-binding site was removed from G_L or added to the C terminus of PTG. The effects of expressing wild-type and mutant constructs on GS and GP activities and on the regulation of glycogen metabolism by extracellular glucose were investigated using primary mouse hepatocytes.     

Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.Tumor-associated microtubule-associated protein (TMAP),³ also known as cytoskeleton-associated protein 2 (CKAP2), LB-1, and se20-10, is frequently up-regulated in various malignancies, including gastric adenocarcinoma, diffuse B-cell lymphoma, and cutaneous T-cell lymphoma (1–3), and detected in various cancer cell lines (1, 4). Knockdown of TMAP significantly reduces the rate of cell growth (5, 6), indicating that it is essential for normal cell growth. However, the cellular functions of TMAP remain largely unknown. Recent findings indicate that TMAP plays an essential role in mitosis. Expression of TMAP changes in a cell cycle-dependent manner; its expression is relatively low during G₁, starts to incline during G₁/S transition, and peaks at G₂/M phases of the cell cycle (5, 7). TMAP primarily localizes at mitotic spindle and spindle poles during mitosis (1, 4, 8, 9). During late stages of mitosis, however, TMAP localizes near the chromatin region and to the midbody microtubules (8). TMAP has microtubule-stabilizing properties (4, 8, 9), and its overexpression induces mitotic spindle defects, including monopolar spindle formation, and arrests cells at mitosis as a result (8). Similar to other mitotic regulators, TMAP is a substrate of the anaphase-promoting complex (8). TMAP is degraded during mitotic exit by the anaphase-promoting complex-Cdh1 in a KEN box-dependent manner. Results of the experiments using a nondegradable mutant of TMAP suggested that proper regulation of the TMAP protein level is functionally important for establishment of bipolar spindles and completion of cytokinesis. Recently, we also have shown that siRNA-mediated depletion of TMAP in mammalian cells results in chromosome missegregation, characterized by chromatin bridge formation and malformation of interphase nuclei, and such phenotype was associated with a reduction in the spindle assembly checkpoint activity (6). These findings suggest that TMAP is a potential regulator of mitotic spindle function and dynamics and that proper regulation of its protein level and functions is necessary for establishment of bipolar spindles as well as for maintaining the fidelity of the chromosome segregation process.At the onset of mitosis, the microtubule network undergoes extensive rearrangements to form a unique bipolar structure, called the mitotic spindle. Multiple factors have been shown to associate with the mitotic spindle and regulate its function by influencing its assembly and dynamics (10, 11). Establishment of a functional bipolar mitotic spindle is critical for faithful segregation of sister chromatids and maintenance of genomic stability. In support of this notion, disruption or depletion of factors involved in regulation of the spindle microtubule dynamics or establishment of spindle bipolarity have been shown to induce spindle dysfunction and ultimately chromosome missegregation (12–14).The cyclin-dependent kinase 1 (Cdk1) in complex with cyclin B1 (Cdk1-cyclin B1) is one of the key mitotic kinases. The kinase activity of Cdk1-cyclin B1 governs the entry into mitosis from G₂ phase of the cell cycle (15, 16). Through mediating phosphorylation of a variety of substrates, Cdk1-cyclin B1 also plays an important role in multiple processes during mitosis, including chromosome condensation, nuclear envelope breakdown, centrosome separation, regulation of spindle microtubule dynamics, and metaphase to anaphase transition (17–20). In particular, a number of regulators of microtubules are among Cdk1-cyclin B1 substrates (21). For instance, phosphorylation of a kinesin-related motor protein, Eg5, by Cdk1-cyclin B1 is necessary for its centrosomal localization and ultimately for the centrosome separation process to occur properly (18). Also, Cdk1-cyclin B1-mediated phosphorylation of some of the effectors of microtubule dynamics has been shown to regulate their microtubule-stabilizing or -destabilizing activities during mitosis (22, 23). These suggest that the assembly and maintenance of bipolar spindles during mitosis are under regulation of Cdk1-cyclin B1.We have recently reported that TMAP is phosphorylated specifically during mitosis (24), which led us to hypothesize that the mitotic functions of TMAP are regulated by timely phosphorylation. In the present study, we identified multiple, mitosis-specific phosphorylation sites on TMAP, one of which is phosphorylated by Cdk1-cyclin B1, and investigated the functional importance of Cdk1-cyclin B1-mediated phosphorylation of TMAP during mitosis.