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Human Replication Protein A (RPA) was historically discovered as one of the six components needed to reconstitute simian virus 40 DNA replication from purified components. RPA is now known to be involved in all DNA metabolism pathways that involve single-stranded DNA (ssDNA). Heterotrimeric RPA comprises several domains connected by flexible linkers and is heavily regulated by post-translational modifications (PTMs). The structure of RPA has been challenging to obtain. Various structural methods have been applied, but a complete understanding of RPA's flexible structure, its function, and how it is regulated by PTMs has yet to be obtained. This review will summarize recent literature concerning how RPA is phosphorylated in the cell cycle, the structural analysis of RPA, DNA and protein interactions involving RPA, and how PTMs regulate RPA activity and complex formation in double-strand break repair. There are many holes in our understanding of this research area. We will conclude with perspectives for future research on how RPA PTMs control double-strand break repair in the cell cycle. Show less

The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD⁺ leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD⁺ AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1-knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD⁺ AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation-dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD⁺ AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD⁺ AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD⁺ AML cells. Show less

AbstractVariable, diversity and joining (V(D)J) recombination and immunoglobulin class switch recombination (CSR) are key processes in adaptive immune responses that naturally generate DNA double-strand breaks (DSBs) and trigger a DNA repair response. It is unclear whether this response is associated with distinct survival signals that protect T and B cells. Glycogen synthase kinase 3β (GSK3β) is a constitutively active kinase known to promote cell death. Here we show that phosphorylation of GSK3β on Ser389 by p38 MAPK (mitogen-activated protein kinase) is induced selectively by DSBs through ATM (ataxia telangiectasia mutated) as a unique mechanism to attenuate the activity of nuclear GSK3β and promote survival of cells undergoing DSBs. Inability to inactivate GSK3β through Ser389 phosphorylation in Ser389Ala knockin mice causes a decrease in the fitness of cells undergoing V(D)J recombination and CSR. Preselection-Tcrβ repertoire is impaired and antigen-specific IgG antibody responses following immunization are blunted in Ser389GSK3β knockin mice. Thus, GSK3β emerges as an important modulator of the adaptive immune response. Show less

Chemokine receptor CCR7 directs mature dendritic cells (mDCs) to secondary lymph nodes where these cells regulate the activation of T cells. CCR7 also promotes survival in mDCs, which is believed to take place largely through Akt-dependent signaling mechanisms. We have analyzed the involvement of the AMP-dependent kinase (AMPK) in the control of CCR7-dependent survival. A pro-apoptotic role for AMPK is suggested by the finding that pharmacological activators induce apoptosis, whereas knocking down of AMPK with siRNA extends mDC survival. Pharmacological activation of AMPK also induces apoptosis of mDCs in the lymph nodes. Stimulation of CCR7 leads to inhibition of AMPK, through phosphorylation of Ser-485, which was mediated by G(i)/Gβγ, but not by Akt or S6K, two kinases that control the phosphorylation of AMPK on Ser-485 in other settings. Using selective pharmacological inhibitors, we show that CCR7-induced phosphorylation of AMPK on Ser-485 is mediated by MEK and ERK. Coimmunoprecipitation analysis and proximity ligation assays indicate that AMPK associates with ERK, but not with MEK. These results suggest that in addition to Akt-dependent signaling mechanisms, CCR7 can also promote survival of mDCs through a novel MEK1/2-ERK1/2-AMPK signaling axis. The data also suggest that AMPK may be a potential target to modulate mDC lifespan and the immune response. Show less