Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators.

The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumors as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: (i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; (ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; (iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumor suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors.

CDKL5 kinase controls transcription-coupled responses to DNA damage.

Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP-ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double-strand breaks. Thus, CDKL5 is a DNA damage-sensing, PAR-controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5-related diseases.

DNA demethylase activity maintains intestinal cells in an undifferentiated state following loss of APC.

Although genome-wide hypomethylation is a hallmark of many cancers, roles for active DNA demethylation during tumorigenesis are unknown. Here, loss of the APC tumor suppressor gene causes upregulation of a DNA demethylase system and the concomitant hypomethylation of key intestinal cell fating genes. Notably, this hypomethylation maintained zebrafish intestinal cells in an undifferentiated state that was released upon knockdown of demethylase components. Mechanistically, the demethylase genes are directly activated by Pou5f1 and Cebpß and are indirectly repressed by retinoic acid, which antagonizes Pou5f1 and Cebpß. Apc mutants lack retinoic acid as a result of the transcriptional repression of retinol dehydrogenase l1 via a complex that includes Lef1, Groucho2, Ctbp1, Lsd1, and Corest. Our findings imply a model wherein APC controls intestinal cell fating through a switch in DNA methylation dynamics. Wild-type APC and retinoic acid downregulate demethylase components, thereby promoting DNA methylation of key genes and helping progenitors commit to differentiation.

RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health.

Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health.

Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in Fan1 nuclease-defective knock-in mice.

The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1(nd/nd)) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1(nd/nd) kidneys are polyploid, and fibroblasts from Fan1(nd/nd) mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4-Slx1 nuclease also involved in ICL repair.

K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of k29 polyubiquitin.

Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages.

Differential roles and regulation of the protein kinases PAK4, PAK5 and PAK6 in melanoma cells.

The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations; PAK6 and PAK4 have fewer; and PAK4 is often amplified. To help interpret these genomic data, initially we compared the cellular regulation of the sister kinases and their roles in melanoma cells. In common with many ohnologue protein kinases, PAK4, PAK5 and PAK6 each have two 14-3-3-binding phosphosites of which phosphoSer99 is conserved. PAK4 localises to the leading edge of cells in response to phorbol ester-stimulated binding of 14-3-3 to phosphoSer99 and phosphoSer181, which are phosphorylated by two different PKCs or PKDs. These phosphorylations of PAK4 are essential for its phorbol ester-stimulated phosphorylation of downstream substrates. In contrast, 14-3-3 interacts with PAK5 in response to phorbol ester-stimulated phosphorylation of Ser99 and epidermal growth factor-stimulated phosphorylation of Ser288; whereas PAK6 docks onto 14-3-3 and is prevented from localising to cell-cell junctions when Ser133 is phosphorylated in response to cAMP-elevating agents via PKA and insulin-like growth factor 1 via PKB/Akt. Silencing of PAK4 impairs viability, migration and invasive behaviour of melanoma cells carrying BRAFV600E or NRASQ61K mutations. These defects are rescued by ectopic expression of PAK4, more so by a 14-3-3-binding deficient PAK4, and barely by PAK5 or PAK6. Together these genomic, biochemical and cellular data suggest that the oncogenic properties of PAK4 are regulated by PKC-PKD signalling in melanoma, while PAK5 and PAK6 are dispensable in this cancer.

Phosphoproteomic screening identifies physiological substrates of the CDKL5 kinase.

Mutations in the gene encoding the protein kinase CDKL5 cause a debilitating neurodevelopmental disease termed CDKL5 disorder. The impact of these mutations on CDKL5 function is poorly understood because the substrates and cellular processes controlled by CDKL5 are unclear. Here, we describe a quantitative phosphoproteomic screening which identified MAP1S, CEP131 and DLG5-regulators of microtubule and centrosome function-as cellular substrates of CDKL5. Antibodies against MAP1S phospho-Ser900 and CEP131 phospho-Ser35 confirmed CDKL5-dependent phosphorylation of these targets in human cells. The phospho-acceptor serine residues in MAP1S, CEP131 and DLG5 lie in the motif RPXSA, although CDKL5 can tolerate residues other than Ala immediately C-terminal to the phospho-acceptor serine. We provide insight into the control of CDKL5 activity and show that pathogenic mutations in CDKL5 cause a major reduction in CDKL5 activity in vitro and in cells. These data reveal the first cellular substrates of CDKL5, which may represent important biomarkers in the diagnosis and treatment of CDKL5 disorder, and illuminate the functions of this poorly characterized kinase.

Allosteric mechanism for site-specific ubiquitination of FANCD2.

DNA-damage repair is implemented by proteins that are coordinated by specialized molecular signals. One such signal in the Fanconi anemia (FA) pathway for the repair of DNA interstrand crosslinks is the site-specific monoubiquitination of FANCD2 and FANCI. The signal is mediated by a multiprotein FA core complex (FA-CC) however, the mechanics for precise ubiquitination remain elusive. We show that FANCL, the RING-bearing module in FA-CC, allosterically activates its cognate ubiqutin-conjugating enzyme E2 UBE2T to drive site-specific FANCD2 ubiquitination. Unlike typical RING E3 ligases, FANCL catalyzes ubiquitination by rewiring the intraresidue network of UBE2T to influence the active site. Consequently, a basic triad unique to UBE2T engages a structured acidic patch near the target lysine on FANCD2. This three-dimensional complementarity, between the E2 active site and substrate surface, induced by FANCL is central to site-specific monoubiquitination in the FA pathway. Furthermore, the allosteric network of UBE2T can be engineered to enhance FANCL-catalyzed FANCD2-FANCI di-monoubiquitination without compromising site specificity.

An ERK5-KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells.

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5 signalling and ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern 2C gene transcription and a key mESC rejuvenation process.

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

Cooperative control of holliday junction resolution and DNA repair by the SLX1 and MUS81-EME1 nucleases.

Holliday junctions (HJs) are X-shaped DNA structures that arise during homologous recombination, which must be removed to enable chromosome segregation. The SLX1 and MUS81-EME1 nucleases can both process HJs in vitro, and they bind in close proximity on the SLX4 scaffold, hinting at possible cooperation. However, the cellular roles of mammalian SLX1 are not yet known. Here, we use mouse genetics and structure function analysis to investigate SLX1 function. Disrupting the murine Slx1 and Slx4 genes revealed that they are essential for HJ resolution in mitotic cells. Moreover, SLX1 and MUS81-EME1 act together to resolve HJs in a manner that requires tethering to SLX4. We also show that SLX1, like MUS81-EME1, is required for repair of DNA interstrand crosslinks, but this role appears to be independent of HJ cleavage, at least in mouse cells. These findings shed light on HJ resolution in mammals and on maintenance of genome stability.

A complex comprising C15ORF41 and Codanin-1: the products of two genes mutated in congenital dyserythropoietic anaemia type I (CDA-I).

Congenital dyserythropoietic anaemia (CDA) type I is a rare blood disorder characterised by moderate to severe macrocytic anaemia and hepatomegaly, with spongy heterochromatin and inter-nuclear bridges seen in bone marrow erythroblasts. The vast majority of cases of CDA type I are caused by mutations in the CDAN1 gene. The product of CDAN1 is Codanin-1, which interacts the histone chaperone ASF1 in the cytoplasm. Codanin-1 is a negative regulator of chromatin replication, sequestering ASF1 in the cytoplasm, restraining histone deposition and thereby limiting DNA replication. The remainder of CDA-I cases are caused by mutations in the C15ORF41 gene, but very little is known about the product of this gene. Here, we report that C15ORF41 forms a tight, near-stoichiometric complex with Codanin1 in human cells, interacting with the C-terminal region of Codanin-1. We present the characterisation of the C15ORF41-Codanin-1 complex in humans in cells and in vitro, and demonstrate that Codanin-1 appears to sequester C15ORF41 in the cytoplasm as previously shown for ASF1. The findings in this study have major implications for understanding the functions of C15ORF41 and Codanin-1, and the aetiology of CDA-I.

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2-mediated phosphorylation at Threonine72.

Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.

The kinases MSK1 and MSK2 act as negative regulators of Toll-like receptor signaling.

The kinases MSK1 and MSK2 are activated 'downstream' of the p38 and Erk1/2 mitogen-activated protein kinases. Here we found that MSK1 and MSK2 were needed to limit the production of proinflammatory cytokines in response to stimulation of primary macrophages with lipopolysaccharide. By inducing transcription of the mitogen-activated protein kinase phosphatase DUSP1 and the anti-inflammatory cytokine interleukin 10, MSK1 and MSK2 exerted many negative feedback mechanisms. Deficiency in MSK1 and MSK2 prevented the binding of phosphorylated transcription factors CREB and ATF1 to the promoters of the genes encoding interleukin 10 and DUSP1. Mice doubly deficient in MSK1 and MSK2 were hypersensitive to lipopolysaccharide-induced endotoxic shock and showed prolonged inflammation in a model of toxic contact eczema induced by phorbol 12-myristate 13-acetate. Our results establish MSK1 and MSK2 as key components of negative feedback mechanisms needed to limit Toll-like receptor-driven inflammation.

mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival.

Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis.

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin-parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2~Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2~Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin.

Identification of the MMS22L-TONSL complex that promotes homologous recombination.

Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NFκBIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are required for the HR-mediated repair of replication fork-associated DSBs. In cells depleted of either protein, DSBs induced by the TOP1 inhibitor camptothecin are resected normally, but the loading of the RAD51 recombinase is defective. Therefore, MMS22L and TONSL are required for the maintenance of genome stability when unscheduled DSBs occur in the vicinity of DNA replication forks.

Inhibition of SIK2 and SIK3 during differentiation enhances the anti-inflammatory phenotype of macrophages.

The salt-inducible kinases (SIKs) control a novel molecular switch regulating macrophage polarization. Pharmacological inhibition of the SIKs induces a macrophage phenotype characterized by the secretion of high levels of anti-inflammatory cytokines, including interleukin (IL)-10, and the secretion of very low levels of pro-inflammatory cytokines, such as tumour necrosis factor α. The SIKs, therefore, represent attractive new drug targets for the treatment of macrophage-driven diseases, but which of the three isoforms, SIK1, SIK2 or SIK3, would be appropriate to target remains unknown. To address this question, we developed knock-in (KI) mice for SIK1, SIK2 and SIK3, in which we introduced a mutation that renders the enzymes catalytically inactive. Characterization of primary macrophages from the single and double KI mice established that all three SIK isoforms, and in particular SIK2 and SIK3, contribute to macrophage polarization. Moreover, we discovered that inhibition of SIK2 and SIK3 during macrophage differentiation greatly enhanced the production of IL-10 compared with their inhibition in mature macrophages. Interestingly, macrophages differentiated in the presence of SIK inhibitors, MRT199665 and HG-9-91-01, still produced very large amounts of IL-10, but very low levels of pro-inflammatory cytokines, even after the SIKs had been reactivated by removal of the drugs. Our data highlight an integral role for SIK2 and SIK3 in innate immunity by preventing the differentiation of macrophages into a potent and stable anti-inflammatory phenotype.