Structure of monoubiquitinated PCNA

Ubiquitination of proliferating cell nuclear antigen (PCNA) to ub-PCNA is essential for DNA replication across bulky template lesions caused by UV radiation and alkylating agents, as ub-PCNA orchestrates the recruitment and switching of translesion synthesis (TLS) polymerases with replication polymerases. This allows replication to proceed, leaving the DNA to be repaired subsequently. Defects in a TLS polymerase, Pol η, lead to a form of Xeroderma pigmentosum, a disease characterized by severe skin sensitivity to sunlight damage and an increased incidence of skin cancer. Structurally, however, information on how ub-PCNA orchestrates the switching of these two classes of polymerases is lacking. We have solved the structure of ub-PCNA and demonstrate that the ubiquitin molecules in ub-PCNA are radially extended away from the PCNA without structural contact aside from the isopeptide bond linkage. This unique orientation provides an open platform for the recruitment of TLS polymerases through ubiquitin-interacting domains. However, the ubiquitin moieties, to the side of the equatorial PCNA plane, can place spatial constraints on the conformational flexibility of proteins bound to ub-PCNA. We show that ub-PCNA is impaired in its ability to support the coordinated actions of Fen1 and Pol δ in assays mimicking Okazaki fragment processing. This provides evidence for the novel concept that ub-PCNA may modulate additional DNA transactions other than TLS polymerase recruitment and switching.     

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

The calcium/calmodulin-dependent kinase II (CaMKII) participates with Ras to Raf-1 activation, and it is necessary for activation of the extracellular signal-regulated kinase (ERK) by different factors in epithelial and mesenchimal cells. Raf-1 activation is a complex multistep process, and its maximal activation is achieved by phosphorylation at Y341 by Src and at S338 by other kinase/s. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 remains to be definitively identified. In this study, we verified the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To do so, we determined the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors. Serum, fibronectin, Src^Y527 and Ras^V12 activated CaMKII and ERK, at different extents. The inhibition of CaMKII attenuated Raf-1 and ERK activation by all these factors. CaMKII was also necessary for the phosphorylation of Raf-1 at S338 by serum, fibronectin and Ras. Conversely, inhibition of Pak3 activation by blocking phosphatidylinositol 3-kinase was ineffective. The direct phosphorylation of S338 Raf-1 by CaMKII was demonstrated in vitro by interaction of purified kinases. These results demonstrate that Ras activates CaMKII, which, in turn, phosphorylates Raf-1 at S338 and participates in ERK activation upon different stimuli.     

Sister chromatid cohesion establishment occurs in concert with lagging strand synthesis

Cohesion establishment is central to sister chromatid tethering reactions and requires Ctf7/Eco1-dependent acetylation of the cohesin subunit Smc3. Ctf7/Eco1 is essential during S phase, and a number of replication proteins (RFC complexes, PCNA and the DNA helicase Chl1) all play individual roles in sister chromatid cohesion. While the mechanism of cohesion establishment is largely unknown, a popular model is that Ctf7/Eco1 acetylates cohesins encountered by and located in front of the fork. In turn, acetylation is posited both to allow fork passage past cohesin barriers and convert cohesins to a state competent to capture subsequent production of sister chromatids. Here, we report evidence that challenges this pre-replicative cohesion establishment model. Our genetic and biochemical studies link Ctf7/Eco1 to the Okazaki fragment flap endonuclease, Fen1. We further report genetic and biochemical interactions between Fen1 and the cohesion-associated DNA helicase, Chl1. These results raise a new model wherein cohesin deposition and establishment occur in concert with lagging strand-processing events and in the presence of both sister chromatids.     

The ubiquitin-specific protease USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer

The deubiquitinating enzyme USP2a has shown oncogenic properties in many cancer types by impairing ubiquitination of FASN, MDM2, MDMX or Aurora A. Aberrant expression of USP2a has been linked to progression of human tumors, particularly prostate cancer. However, little is known about the role of USP2a or its mechanism of action in bladder cancer. Here, we provide evidence that USP2a is an oncoprotein in bladder cancer cells. Enforced expression of USP2a caused enhanced proliferation, invasion, migration and resistance to several chemotherapeutic reagents, while USP2a loss resulted in slower proliferation, greater chemosensitivity and reduced migratory/invasive capability compared with control cells. USP2a, but not a catalytically inactive mutant, enhanced proliferation in immortalized TRT-HU1 normal human bladder epithelial cells. USP2a bound to cyclin A1 and prevented cyclin A1 ubiquitination, leading to accumulation of cyclin A1 by a block in degradation. Enforced expression of wild type USP2a, but not an inactive USP2a mutant, resulted in cyclin A1 accumulation and increased cell proliferation. We conclude that USP2a impairs ubiquitination and stabilizes an important cell cycle regulator, cyclin A1, raising the possibility of USP2a targeting as a therapeutic strategy against bladder tumors in combination with chemotherapy.     

Co-regulation of senescence-associated genes by oncogenic homeobox proteins and polycomb repressive complexes

Cellular senescence is a stable cell cycle arrest that can be induced by stresses such as telomere shortening, oncogene activation or DNA damage. Senescence is a potent anticancer barrier that needs to be circumvented during tumorigenesis. The cell cycle regulator p16^INK4a is a key effector upregulated during senescence. Polycomb repressive complexes (PRCs) play a crucial role in silencing the INK4/ARF locus, which encodes for p16^INK4a, but the mechanisms by which PRCs are recruited to this locus as well as to other targets remain poorly understood. Recently we discovered the ability of the homeobox proteins HLX1 (H2.0-like homeobox 1) and HOXA9 (Homeobox A9) to bypass senescence. We showed that HLX1 and HOXA9 recruit PRCs to repress INK4a, which constitutes a key mechanism explaining their effects on senescence. Here we provide evidence for the regulation of additional senescence-associated PRC target genes by HLX1 and HOXA9. As both HLX1 and HOXA9 are oncogenes implicated in leukemogenesis, we discuss the implications that the collaboration between Homeobox proteins and PRCs has for senescence and cancer.     

A role for protein phosphatase 4 in regulating non-homologous end-joining

Comment on: Liu J, et al. Cell Cycle 2012; 11:2643-9.     

Ketone bodies and two-compartment tumor metabolism: Stromal ketone production fuels mitochondrial biogenesis in epithelial cancer cells

We have previously suggested that ketone body metabolism is critical for tumor progression and metastasis. Here, using a co-culture system employing human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts, we provide new evidence to directly support this hypothesis. More specifically, we show that the enzymes required for ketone body production are highly upregulated within cancer-associated fibroblasts. This appears to be mechanistically controlled by the stromal expression of caveolin-1 (Cav-1) and/or serum starvation. In addition, treatment with ketone bodies (such as 3-hydroxy-butyrate, and/or butanediol) is sufficient to drive mitochondrial biogenesis in human breast cancer cells. This observation was also validated by unbiased proteomic analysis. Interestingly, an MCT1 inhibitor was sufficient to block the onset of mitochondrial biogenesis in human breast cancer cells, suggesting a possible avenue for anticancer therapy. Finally, using human breast cancer tumor samples, we directly confirmed that the enzymes associated with ketone body production (HMGCS2, HMGCL and BDH1) were preferentially expressed in the tumor stroma. Conversely, enzymes associated with ketone re-utilization (ACAT1) and mitochondrial biogenesis (HSP60) were selectively associated with the epithelial tumor cell compartment. Our current findings are consistent with the “two-compartment tumor metabolism” model. Furthermore, they suggest that we should target ketone body metabolism as a new area for drug discovery, for the prevention and treatment of human cancers.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.