Anthony J. Davis

Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits

Individual subunits of protein phosphatase 2A (PP2A), protein phosphatase 4, and protein phosphatase 5 were knocked out in Drosophila Schneider 2 cells by using RNA interference. Ablation of either the scaffold (A) or catalytic (C) subunits of PP2A caused the disappearance of all PP2A subunits. Treating cells with double-stranded RNA targeting all four of the Drosophila PP2A regulatory subunits caused the disappearance of both the A and C subunits. The loss of PP2A subunits was associated with decreased protein stability indicating that only the heterotrimeric forms of PP2A are stable in intact cells. Ablation of total PP2A by using double-stranded RNA against either the A or C subunit, or specific ablation of the R2/B regulatory subunit, enhanced insulin-induced ERK activation. These results indicated that the R2/B subunit targets PP2A to the mitogen-activated protein (MAP) kinase cascade in Schneider 2 cells, where it acts as a negative regulator. A severe loss of viability occurred in cells in which total PP2A or both isoforms of the Drosophila R5/B56 subunit had been ablated. The reduced viability of these cells correlated with the induction of markers of apoptosis including membrane blebbing and stimulation of caspase-3-like activity. These observations indicated that PP2A has a powerful antiapoptotic activity that is specifically mediated by the R5/B56 regulatory subunits. In contrast to PP2A, ablation of protein phosphatase 4 caused only a slight reduction in cell growth but had no effect on MAP kinase signaling or apoptosis. Depletion of protein phosphatase 5 had no effects on MAP kinase, cell growth, or apoptosis.

Autophosphorylation at serine 1981 stabilizes ATM at DNA damage sites

Initial localization of ATM to double-strand breaks requires the MRE11–RAD50–NBS1 complex, but prolonged association requires ATM autophosphorylation.

DNA-PK: A dynamic enzyme in a versatile DSB repair pathway

DNA double stranded breaks (DSBs) are the most cytoxic DNA lesion as the inability to properly repair them can lead to genomic instability and tumorigenesis. The prominent DSB repair pathway in humans is non-homologous end-joining (NHEJ). In the simplest sense, NHEJ mediates the direct re-ligation of the broken DNA molecule. However, NHEJ is a complex and versatile process that can repair DSBs with a variety of damages and ends via the utilization of a significant number of proteins. In this review we will describe the important factors and mechanisms modulating NHEJ with emphasis given to the versatility of this repair process and the DNA-PK complex.

Human Ku70/80 Protein Blocks Exonuclease 1-mediated DNA Resection in the Presence of Human Mre11 or Mre11/Rad50 Protein Complex

Background: Pathway choice for the repair of double strand breaks is not fully understood. Results: Human Ku blocks exonuclease 1-mediated DNA processing in the presence of Mre11 or Mre11/Rad50. Conclusion: Unlike in yeast, the displacement of Ku from DNA ends is not mediated by Mre11 or the Mre11/Rad50 complex. Significance: Pathway choice between nonhomologous end-joining and homologous recombination is likely more complex than simple competition between the two pathways. DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.

Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination.

DNA double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The DNA cell cycle stage and resection of the DSB ends are two key mechanisms which are believed to push DSB repair to the HR pathway. Here, we show that the NHEJ factor Ku80 associates with DSBs in S phase, when HR is thought to be the preferred repair pathway, and its dynamics/kinetics at DSBs is similar to those observed for Ku80 in non-S phase in mammalian cells. A Ku homolog from Mycobacterium tuberculosis binds to and is retained at DSBs in S phase and was used as a tool to determine if blocking DNA ends affects end resection and HR in mammalian cells. A decrease in DNA end resection, as marked by IR-induced RPA, BrdU, and Rad51 focus formation, and HR are observed when Ku deficient rodent cells are complemented with Mt-Ku. Together, this data suggests that Ku70/80 binds to DSBs in all cell cycle stages and is likely actively displaced from DSB ends to free the DNA ends for DNA end resection and thus HR to occur.

Protein phosphatase 2a

Publisher Summary Protein phosphatase 2 A (PP2A) is a ubiquitously expressed member of the PPP gene family that accounts for a substantial portion of the total serine/threonine phosphatase activity in many cell types. PP2A is an essential enzyme that functions in fundamental cellular processes, including metabolism and the cell cycle. Regulatory subunits play a primary role in specifying the proximity interactions of PP2A. The R2 family comprises a set of proteins present in a form of PP2A originally designated PP2A 1 . This family currently contains four known isoforms that are 79–87 percent identical. R2α mRNA is ubiquitously expressed and is the most abundant PP2A regulatory subunit in many cells and tissues. The R2β and R2γ isoforms are only expressed at high levels in brain and testis. The R2 subunit targets PP2A to pathways that regulate MAP kinase activity. Overexpression of the small-t antigen of SV40 virus disrupts endogenous PP2A complexes containing the R2 subunit. This leads to enhanced activation of MAP kinase in response to growth factors in some but not all cell types. The association of PP2A with microtubules in brain is specific for R2α- and R2β-containing isoforms, and can be enhanced by a heat-labile anchoring factor. The R5 regulatory subunits are a complex family of proteins that are components of a PP2A holoenzyme originally termed PP2A 0 . There are at least five isoforms that have distinct patterns of expression. The α and γ isoforms are expressed predominantly in muscle, the β and δ isoforms in brain, and the ɛ isoform in brain and testis.

Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks.

The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs)—a highly cytotoxic DNA lesion—activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair.

The N-terminal region of the DNA-dependent protein kinase catalytic subunit is required for its DNA double-stranded break-mediated activation.

Background: The Ku70/80-DNA complex recruits DNA-PKcs to DSBs and results in activation of DNA-PKcs kinase activity. Results: Truncation fragments of DNA-PKcs show that different regions of the protein are required for complete functionality of DNA-PKcs. Conclusion: The N-terminal region of DNA-PKcs is required for its ability to interact with the Ku-DNA complex and its full activation. Significance: We provide insights into the biochemical mechanism required for DNA-PKcs activation. DNA-dependent protein kinase (DNA-PK) plays an essential role in the repair of DNA double-stranded breaks (DSBs) mediated by the nonhomologous end-joining pathway. DNA-PK is a holoenzyme consisting of a DNA-binding (Ku70/Ku80) and catalytic (DNA-PKcs) subunit. DNA-PKcs is a serine/threonine protein kinase that is recruited to DSBs via Ku70/80 and is activated once the kinase is bound to the DSB ends. In this study, two large, distinct fragments of DNA-PKcs, consisting of the N terminus (amino acids 1–2713), termed N-PKcs, and the C terminus (amino acids 2714–4128), termed C-PKcs, were produced to determine the role of each terminal region in regulating the activity of DNA-PKcs. N-PKcs but not C-PKcs interacts with the Ku-DNA complex and is required for the ability of DNA-PKcs to localize to DSBs. C-PKcs has increased basal kinase activity compared with DNA-PKcs, suggesting that the N-terminal region of DNA-PKcs keeps basal activity low. The kinase activity of C-PKcs is not stimulated by Ku70/80 and DNA, further supporting that the N-terminal region is required for binding to the Ku-DNA complex and full activation of kinase activity. Collectively, the results show the N-terminal region mediates the interaction between DNA-PKcs and the Ku-DNA complex and is required for its DSB-induced enzymatic activity.

Protein Phosphatase 2A Is Targeted to Cell Division Control Protein 6 by a Calcium-binding Regulatory Subunit

The cell division control protein 6 (Cdc6) is essential for formation of pre-replication complexes at origins of DNA replication. Phosphorylation of Cdc6 by cyclin-dependent kinases inhibits ubiquitination of Cdc6 by APC/Ccdh1 and degradation by the proteasome. Experiments described here show that the PR70 member of the PPP2R3 family of regulatory subunits targets protein phosphatase 2A (PP2A) to Cdc6. Interaction with Cdc6 is mediated by residues within the C terminus of PR70, whereas interaction with PP2A requires N-terminal sequences conserved within the PPP2R3 family. Two functional EF-hand calcium-binding motifs mediate a calcium-enhanced interaction of PR70 with PP2A. Calcium has no effect on the interaction of PR70 with Cdc6 but enhances the association of PP2A with Cdc6 through its effects on PR70. Knockdown of PR70 by RNA interference results in an accumulation of endogenous and expressed Cdc6 protein that is dependent on the cyclin-dependent protein kinase phosphorylation sites on Cdc6. Knockdown of PR70 also causes G1 arrest, suggesting that PR70 function is critical for progression into S phase. These observations indicate that PP2A can be targeted in a calcium-regulated manner to Cdc6 via the PR70 subunit, where it plays a role in regulating protein phosphorylation and stability.

B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

Pocket proteins negatively regulate transcription of E2F-dependent genes and progression through the G0/G1 transition and the cell cycle restriction point in G1. Pocket protein repressor activities are inactivated via phosphorylation at multiple Pro-directed Ser/Thr sites by the coordinated action of G1 and G1/S cyclin-dependent kinases. These phosphorylations are reversed by the action of two families of Ser/Thr phosphatases: PP1, which has been implicated in abrupt dephosphorylation of retinoblastoma protein (pRB) in mitosis, and PP2A, which plays a role in an equilibrium that counteracts cyclin-dependent kinase (CDK) action throughout the cell cycle. However, the identity of the trimeric PP2A holoenzyme(s) functioning in this process is unknown. Here we report the identification of a PP2A trimeric holoenzyme containing B55α, which plays a major role in restricting the phosphorylation state of p107 and inducing its activation in human cells. Our data also suggest targeted selectivity in the interaction of pocket proteins with distinct PP2A holoenzymes, which is likely necessary for simultaneous pocket protein activation.