Carl Smythe

Activation of mammalian Chk1 during DNA replication arrest a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

ATR is a caffeine-sensitive, DNA-activated protein kinase with a substrate specificity distinct from DNA-PK

ATR is a large, >300 kDa protein containing a carboxy-terminus kinase domain related to PI-3 kinase, and is homologous to the ATM gene product in human cells and the rad3/MEC1 proteins in yeast. These proteins, together with the DNA-PK, are part of a new family of PI-3 kinase related proteins. All members of this family play important roles in checkpoints which operate to permit cell survival following many forms of DNA damage. We have expressed ATR protein in HEK293 cells and purified the protein to near-homogeneity. We show that pure ATR is a protein kinase which is activated by circular single-stranded, double-stranded or linear DNA. Thus ATR is a new member of a sub-family of PIK related kinases, founded by the DNA-PK, which are activated in the presence of DNA. Unlike DNA-PK, ATR does not appear to require Ku proteins for its activation by DNA. We show directly that, like ATM and DNA-PK, ATR phosphorylates the genome surveillance protein p53 on serine 15, a site which is up-regulated in response to DNA damage. In addition, we find that ATR has a substrate specificity similar to, but unique from, the DNA-PK in vitro, suggesting that these proteins have overlapping but distinct functions in vivo. Finally, we find that the kinase activity of ATR in the presence and absence of DNA is suppressed by caffeine, a compound which is known to induce loss of checkpoint control. Our results are consistent with the notion that ATR plays a role in monitoring DNA structure and phosphorylation of proteins involved in the DNA damage response pathways.

Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2

In cell-free extracts derived from Xenopus eggs which oscillate between S phase and mitosis, incompletely replicated DNA blocks the activation of p34cdc2-cyclin by maintaining p34cdc2 in a tyrosine-phosphorylated form. We used a recombinant cyclin fusion protein to generate a substrate to measure the ability of the tyrosine kinase(s) to phosphorylate and inactivate p34cdc2 in the absence of tyrosine phosphatase activity. p34cdc2 tyrosine phosphorylation is highly regulated during the cell cycle, being elevated in S phase and attenuated in mitosis. The elevation in p34cdc2 tyrosine phosphorylation rate occurs in response to the presence of incompletely replicated DNA. Moreover, okadaic acid and caffeine, which uncouple the dependence of mitosis on the completion of S phase, increase unphosphorylated p34cdc2 by attenuating tyrosine kinase function. These data indicate that the control system, which monitors the state of DNA replication, modulates the function of the tyrosine kinase by a phosphorylation/dephosphorylation mechanism, ensuring that mitosis occurs only when S phase is complete.

Chapter 17 Systems for the Study of Nuclear Assembly, DNA Replication, and Nuclear Breakdown in Xenopus laevis Egg Extracts

Publisher Summary This chapter describes the conditions for preparing cell-free extracts from Xenopus eggs, which allows the study of nuclear dynamics and has proved fruitful in elucidating the structural and functional requirements for nuclear reassembly, DNA replication, and nuclear disassembly. In the amphibian Xenopus laevis , egg fertilization is followed by a period of rapid and synchronous cell division, which occurs in the complete absence of transcription or of any increase in total mass of the egg. During oogenesis, the egg must synthesize and store a large reservoir of nuclear components for use during the rapid proliferation that occurs during early development. The existence of such a reservoir of nuclear components permits the development of systems for the study of nuclear dynamics in vitro . Xenopus sperm provides an abundant source of chromatin, which after removal of plasma and nuclear membranes, may be easily purified. The chapter diagrammatically presents the preparation of the S phase extract and membrane and cytosol fractions for nuclear assembly. The inhibition of DNA replication by aphidicolin prevents the subsequent activation of maturation promoting factor (MPF) and entry into mitosis, indicating the presence of a control system that monitors the replication state of DNA and regulates the activation of MPF.

Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21WAF1

BACKGROUND A common event in the development of human neoplasia is the inactivation of a damage-inducible cell-cycle checkpoint pathway regulated by p53. One approach to the restoration of this pathway is to mimic the activity of key downstream effectors. The cyclin-dependent kinase (Cdk) inhibitor p21(WAF1) is one such molecule, as it is a major mediator of the p53-dependent growth-arrest pathway, and can, by itself, mediate growth suppression. The primary function of the p21(WAF1) protein appears to be the inhibition of G1 cyclin-Cdk complexes. Thus, if we can identify the region(s) of p21(WAF1) that contain its inhibitor activity they may provide a template from which to develop novel anti-proliferative drugs for use in tumours with a defective p53 pathway. RESULTS We report on the discovery of small synthetic peptides based on the sequence of p21(WAF1) that bind to and inhibit cyclin D1-Cdk4. The peptides and the full-length protein are inhibitory at similar concentrations. A 20 amino-acid peptide based on the carboxy-terminal domain of p21(WAF1) inhibits Cdk4 activity with a concentration for half-maximal inhibition (l0.5) of 46 nM, and it is only four-fold less active than the full-length protein. The length of the peptide has been minimized and key hydrophobic residues forming the inhibitory domain have been defined. When introduced into cells, both a 20 amino-acid and truncated eight amino-acid peptide blocked phosphorylation of the retinoblastoma protein (pRb) and induced a potent G1/S growth arrest. These data support a physiological role for the carboxyl terminus of p21(WAF1) in the inhibition of Cdk4 activity. CONCLUSIONS We have discovered that a small peptide is sufficient to mimic p21(WAF1) function and inhibit the activity of a critical G1 cyclin-Cdk complex, preventing pRb phosphorylation and producing a G1 cell-cycle arrest in tissue culture cell systems. This makes cyclin D1-Cdk4 a realistic and exciting target for the design of novel synthetic compounds that can act as anti-proliferative agents in human cells.

Incorporation of the nuclear pore basket protein Nup153 into nuclear pore structures is dependent upon lamina assembly: evidence from cell‐free extracts of Xenopus eggs

In cell‐free extracts of Xenopus eggs that support the assembly of replication‐competent nuclei, we found that lamin B3 specifically associates with four polypeptides (termed SLAPs, soluble lamin associated proteins). Here, one SLAP is identified as the nuclear pore complex protein Nup153, one member of the F/GXFG motif‐containing nucleoporins. In vitro translated Nup153 and lamin B3 co‐immunoprecipitate, and lamin B3 interacts specifically with the C‐terminal domain of Nup153. During nuclear envelope assembly, other F/GXFG‐containing nucleoporins are incorporated into the nuclear envelope preceding lamina assembly. Incorporation of Nup153 occurs at the same time as lamina assembly. When lamina assembly is prevented using the dominant‐negative mutant XlaminBΔ2+, Nup153 does not appear at the nuclear envelope, while other F/GXFG‐containing nucleoporins and Nup93 are recruited normally. When the lamina of pre‐assembled nuclei is disrupted using the same dominant‐negative mutant, the distribution of other nucleoporins is unaffected. However, Nup153 recruitment at the nuclear envelope is lost. Our results indicate that both the recruitment and maintenance of Nup153 at the pore are dependent upon the integrity of the lamina.

Assembly/disassembly of the nuclear envelope membrane: Cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro

Dissociation and association of membranes with chromatin at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employing purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhibitors, that membrane-chromatin association is regulated by a phosphatase/kinase system. In interphase, the balance in this system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the membrane receptor is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatin. Rather, binding of membranes to chromatin at mitosis appears to be regulated indirectly by MPF through its action on a phosphatase/kinase system that directly modulates the phosphorylation state of a nuclear membrane component.

Ionizing radiation induces ataxia telangiectasia mutated kinase (ATM)-mediated phosphorylation of LKB1/STK11 at Thr-366

The serine/threonine protein kinase LKB1 functions as a tumour suppressor, and mutations in this enzyme lead to the inherited Peutz-Jeghers cancer syndrome. We previously found that LKB1 was phosphorylated at Thr-366 in vivo, a residue conserved in mammalian, Xenopus and Drosophila LKB1, located on a C-terminal non-catalytic moiety of the enzyme. Mutation of Thr-366 to Ala or Asp partially inhibited the ability of LKB1 to suppress growth of G361 melanoma cells, but did not affect LKB1 activity in vitro or LKB1 localization in vivo. As a first step in exploring the role of this phosphorylation further, we have generated a phosphospecific antibody specifically recognizing LKB1 phosphorylated at Thr-366 and demonstrate that exposure of cells to ionizing radiation (IR) induced a marked phosphorylation of LKB1 at Thr-366 in the nucleus. Thr-366 lies in an optimal phosphorylation motif for the phosphoinositide 3-kinase-like kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia-related kinase (ATR), which function as sensors for DNA damage in cells and mediate cellular responses to DNA damage. We demonstrate that both DNA-PK and ATM efficiently phosphorylate LKB1 at Thr-366 in vitro and provide evidence that ATM mediates this phosphorylation in vivo. This is based on the finding that LKB1 is not phosphorylated in a cell line lacking ATM in response to IR, and that agents which induce cellular responses via ATR in preference to ATM poorly induce phosphorylation of LKB1 at Thr-366. These observations provide the first link between ATM and LKB1 and suggest that ATM could regulate LKB1.

Isolation and structural analysis of a peptide containing the novel tyrosyl-glucose linkage in glycogenin

The glucosylation site on glycogenin, the protein primer required for de novo glycogen synthesis, has been identified. The glucose is attached at position C1 in a glycosidic linkage with a unique tyrosine, and the sequence surrounding this residue was found to be: His‐Leu‐Pro‐Phe‐Ile‐Tyr‐Asn‐Leu‐Ser‐Ser‐Ile‐Ser‐Ile‐Tyr(Glc)‐Ser‐Tyr‐Leu ‐Pro‐ Ala‐Phe‐Lys. The same tyrosine residue is glycosylated whether glycogenin is isolated as a complex with the catalytic subunit of glycogen synthase, or covalently attached to glycogen. The possibility that insulin and growth factors may enhance glycogen synthesis via stimulation of the priming reaction is discussed.

Dinuclear Ruthenium(II) Complexes as Two‐Photon, Time‐Resolved Emission Microscopy Probes for Cellular DNA

The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging.