Julian Gannon

CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair

Inherited mutations in BRCA2 are associated with a predisposition to early-onset breast cancers. The underlying basis of tumorigenesis is thought to be linked to defects in DNA double-strand break repair by homologous recombination. Here we show that the carboxy-terminal region of BRCA2, which interacts directly with the essential recombination protein RAD51, contains a site (serine 3291; S3291) that is phosphorylated by cyclin-dependent kinases. Phosphorylation of S3291 is low in S phase when recombination is active, but increases as cells progress towards mitosis. This modification blocks C-terminal interactions between BRCA2 and RAD51. However, DNA damage overcomes cell cycle regulation by decreasing S3291 phosphorylation and stimulating interactions with RAD51. These results indicate that S3291 phosphorylation might provide a molecular switch to regulate RAD51 recombination activity, providing new insight into why BRCA2 C-terminal deletions lead to radiation sensitivity and cancer predisposition.

Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts

Entry into mitosis depends on the activity of cyclin‐dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55δ subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle—high in interphase and suppressed during mitosis. Depletion of PP2A–B55δ (in interphase) from ‘cycling’ frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A–B55δ was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A–B55δ in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.

The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe.

A cell‐free system derived from Xenopus eggs was used to identify the ‘destruction box’ of the Schizosaccharomyces pombe B‐type cyclin, Cdc13, as residues 59–67: RHALDDVSN. Expression of indestructible Cdc13 from a regulated promoter in S.pombe blocked cells in anaphase and inhibited septation, showing that destruction of Cdc13 is necessary for exit from mitosis, but not for sister chromatid separation. In contrast, strong expression of a polypeptide comprising the N‐terminal 70 residues of Cdc13, which acts as a competitive inhibitor of destruction box‐mediated proteolysis, inhibited both sister chromatid separation and the destruction of Cdc13, whereas an equivalent construct with a mutated destruction box did not. Appropriately timed expression of this N‐terminal fragment of Cdc13 overcame the G1 arrest seen in cdc10 mutant strains, suggesting that proteins required for the initiation of S phase are subject to destruction by the same proteolytic machinery as cyclin.

Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation‐promoting factor (MPF) and mitogen‐activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone‐induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST‐p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant‐negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.

Cell Cycle-Regulated Recognition of the Destruction Box of Cyclin B by the APC/C in Xenopus Egg Extracts

Substrates for mitotic proteolysis such as cyclin B have a 9 residue destruction motif, the destruction box (D-box). To identify the receptor that specifically binds the D-box, we used affinity chromatography with immobilized D-box matrices. We find that the APC/C from Xenopus egg extracts binds to the D-box of cyclin B, whereas Fizzy (Cdc20) does not. Mutations in the D-box abolished this interaction. We show that this binding is regulated in the cell cycle, such that the APC/C from egg extracts in interphase does not bind to the D-box matrix. Our results suggest that the APC/C forms a stable interaction with the D-box of its substrates in a cell cycle-dependent manner.

The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin‐dependent proteolysis in fission yeast: defining the D‐box receptor

Programmed proteolysis of proteins such as mitotic cyclins and Cut2/Pds1p requires a 9‐residue conserved motif known as the destruction box (D‐box). Strong expression of protein fragments containing destruction boxes, such as the first 70 residues of Cdc13 (N70), inhibits the growth of Schizosaccharomyces pombe at metaphase. This inhibition can be overcome either by removal of all lysine residues from N70 using site‐directed mutagenesis (K0‐N70) or by raising the concentration of intracellular ubiquitin. Consistent with the idea that competition for ubiquitin accounts for some of its inhibitory effects, wild‐type N70 not only stabilized D‐box proteins, but also Rum1 and Cdc18, which are degraded by a different pathway. The K0‐N70 construct was neither polyubiquitinated nor degraded in vitro, but it blocked the growth of strains of yeast in which anaphase‐promoting complex/cyclosome (APC/C) function was compromised by mutation, and specifically inhibited proteolysis of APC/C substrates in vivo. Both K0‐N70 and 20‐residue D‐box peptides blocked polyubiquitination of other D‐box‐containing substrates in a cell‐free ubiquitination assay system. These data suggest the existence of a D‐box receptor protein that recognizes D‐boxes prior to ubiquitination.

A Mutual Inhibition between APC/C and Its Substrate Mes1 Required for Meiotic Progression in Fission Yeast

The anaphase-promoting complex/cyclosome (APC/C) is a cell-cycle-regulated essential E3 ubiquitin ligase; however, very little is known about its meiotic regulation. Here we show that fission yeast Mes1 is a substrate of the APC/C as well as an inhibitor, allowing autoregulation of the APC/C in meiosis. Both traits require a functional destruction box (D box) and KEN box. We show that Mes1 directly binds the WD40 domain of the Fizzy family of APC/C activators. Intriguingly, expression of nonubiquitylatable Mes1 blocks cells in metaphase I with high levels of APC/C substrates, suggesting that ubiquitylation of Mes1 is required for partial degradation of cyclin B in meiosis I by alleviating Mes1 inhibitory function. Consistently, a ternary complex, APC/C-Fizzy/Cdc20-Mes1, is stabilized by inhibiting Mes1 ubiquitylation. These results demonstrate that the fine-tuning of the APC/C activity, by a substrate that is also an inhibitor, is required for the precise coordination and transition through meiosis.

Dephosphorylation of Cdc20 is required for its C‐box‐dependent activation of the APC/C

The anaphase‐promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin‐dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co‐activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the ‘activation domain’ of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.

Cell cycle regulatory proteins—an overview with relevance to oral cancer

The cell cycle is controlled by a number of highly conserved proteins, found in species as diverse as yeast and mammals. The study of these proteins is a rapidly advancing field that is increasing our understanding of normal and abnormal cell division. Disruption of the cell cycle has been demonstrated in several different types of neoplasm, and there is increasing evidence that, in head and neck tumours, there is aberrant control of cyclins, cell cycle protein kinases and their inhibitors. Because of the phase specificity of some of the control proteins, antibodies to them are proving to be of value in studying cell kinetics of both normal tissues and malignant tumours.

Expression and subcellular localization of CDK2 and cdc2 kinases and their common partner cyclin A in thyroid epithelial cells: comparison of cyclic AMP-dependent and -independent cell cycles.

Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and GO‐S prereplicative phase progression by cyclic AMP as a second messenger for TSH. In its early steps, this mitogenic control is quite distinct from cyclic AMP‐independent mitogenic cascades elicited by growth factors. We demonstrate here that TSH (cyclic AMP) and EGF + serum (cyclic AMP‐independent) stimulations cooperate and finally converge on proteins that control the cell cycle machinery. This convergence included a common induction of the expression of cyclin A and p34cdc2, and to a lesser extent of p33/38cdk2, which was already expressed in quiescent thyroid cells, and common changes of cdc2 and CDK2 phosphorylations as evidenced by electrophoretic mobility shifts. Kinetic differences in these processes after stimulation by TSH or EGF + serum or by these factors in combination correlated with differences in cell cycle kinetics. Moreover, an immunofluorescence analysis of these proteins using the double labeling of PCNA as a marker of each cell cycle phase shows: (1) a previously undescribed nuclear translocation of CDK2 before S phase initiation; (2) a sudden increase of cdc2 nuclear immunoreactivity at G2/mitosis transition. These data support the roles of CDK2 and cdc2 at G1/S and G2/mitosis transitions, respectively. (3) We were unable to demonstrate in individual cells a strict association between the nuclear appearance of cyclin A and G1/S transition, and an association of cyclin A and CDK2 with PCNA‐stained DNA replication sites. On the other hand, the lengthening of G2 phase in the TSH/cyclic AMP‐dependent thyroid cell cycle was associated with a stabilization of Tyr15 inhibitory phosphorylation of cdc2 and an especially high nuclear concentration of cyclin A and CDK2. We hypothesize that high nuclear accumulation of cyclin A and CDK2 during G2 phase could be causative in the cyclic AMP‐dependent delay of mitosis onset.