The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR

TopBP1 serves as an activator of the ATR-ATRIP complex in response to the presence of incompletely replicated or damaged DNA. This process involves binding of ATR to the ATR-activating domain of TopBP1, which is located between BRCT domains VI and VII. TopBP1 displays increased binding to ATR-ATRIP in Xenopus egg extracts containing checkpoint-inducing DNA templates. We show that an N-terminal region of TopBP1 containing BRCT repeats I-II is essential for this checkpoint-stimulated binding of TopBP1 to ATR-ATRIP. The BRCT I-II region of TopBP1 also binds specifically to the Rad9-Hus1-Rad1 (9-1-1) complex in Xenopus egg extracts. This binding occurs via the C-terminal domain of Rad9 and depends upon phosphorylation of its Ser-373 residue. Egg extracts containing either a mutant of TopBP1 lacking the BRCT I-II repeats or a mutant of Rad9 with an alanine substitution at Ser-373 are defective in checkpoint regulation. Furthermore, an isolated C-terminal fragment from Rad9 is an effective inhibitor of checkpoint signaling in egg extracts. These findings suggest that interaction of the 9-1-1 complex with the BRCT I-II region of TopBP1 is necessary for binding of ATR-ATRIP to the ATR-activating domain of TopBP1 and the ensuing activation of ATR.     

The cytoplasmic domain of neuropilin-1 regulates focal adhesion turnover

Though the vascular endothelial growth factor coreceptor neuropilin-1 (Nrp1) plays a critical role in vascular development, its precise function is not fully understood. We identified a group of novel binding partners of the cytoplasmic domain of Nrp1 that includes the focal adhesion regulator, Filamin A (FlnA). Endothelial cells (ECs) expressing a Nrp1 mutant devoid of the cytoplasmic domain (nrp1^cytoΔ/Δ) migrated significantly slower in response to VEGF relative to the cells expressing wild-type Nrp1 (nrp1^+/+ cells). The rate of FA turnover in VEGF-treated nrp1^cytoΔ/Δ ECs was an order of magnitude lower in comparison to nrp1^+/+ ECs, thus accounting for the slower migration rate of the nrp1^cytoΔ/Δ ECs.     

Jab1 mediates protein degradation of the Rad9-Rad1-Hus1 checkpoint complex

The Rad1-Rad9-Hus1 (9-1-1) complex serves a dual role as a DNA-damage sensor in checkpoint signaling and as a mediator in the DNA repair pathway. However, the intercellular mechanisms that regulate the 9-1-1 complex are poorly understood. Jab1, the fifth component of the COP9 signalosome complex, has a central role in the degradation of multiple proteins and is emerging as an important regulator in cancer development. Here, we tested the hypothesis that Jab1 controls the protein stability of the 9-1-1 complex via the proteosome pathway. We provide evidence that Jab1 physically associates with the 9-1-1 complex, and show that this association is mediated through direct interaction between Jab1 and Rad1, one of the subunits of the 9-1-1 complex. Importantly, Jab1 causes translocation of the 9-1-1 complex from the nucleus to the cytoplasm, mediating rapid degradation of the 9-1-1 complex via the 26 S proteasome. Furthermore, Jab1 significantly suppresses checkpoint signaling activation, DNA synthesis recovery from blockage and cell viability after replication stresses such as UV exposure, gamma radiation and treatment with hydroxyurea. These results suggest that Jab1 is an important regulator for the stability of protein 9-1-1 control in cells, which may provide novel information on the involvement of Jab1 in the checkpoint and DNA repair signaling in response to DNA damage.     

The PAF1 complex differentially regulates cardiomyocyte specification

The specification of an appropriate number of cardiomyocytes from the lateral plate mesoderm requires a careful balance of both positive and negative regulatory signals. To identify new regulators of cardiac specification, we performed a phenotype-driven ENU mutagenesis forward genetic screen in zebrafish. In our genetic screen we identified a zebrafish ctr9 mutant with a dramatic reduction in myocardial cell number as well as later defects in primitive heart tube elongation and atrioventricular boundary patterning. Ctr9, together with Paf1, Cdc73, Rtf1 and Leo1, constitute the RNA polymerase II associated protein complex, PAF1. We demonstrate that the PAF1 complex (PAF1C) is structurally conserved among zebrafish and other metazoans and that loss of any one of the components of the PAF1C results in abnormal development of the atrioventricular boundary of the heart. However, Ctr9, Cdc73, Paf1 and Rtf1, but not Leo1, are required for the specification of an appropriate number of cardiomyocytes and elongation of the heart tube. Interestingly, loss of Rtf1 function produced the most severe defects, resulting in a nearly complete absence of cardiac precursors. Based on gene expression analyses and transplantation studies, we found that the PAF1C regulates the developmental potential of the lateral plate mesoderm and is required cell autonomously for the specification of cardiac precursors. Our findings demonstrate critical but differential requirements for PAF1C components in zebrafish cardiac specification and heart morphogenesis.     

The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure

To survive extreme environmental conditions, and in response to certain developmental and pathological situations, eukaryotic organisms employ the catabolic process of autophagy. Structures targeted for destruction are enwrapped by double-membrane vesicles, then delivered into the interior of the lysosome/vacuole. Despite the identification of many specific components, the molecular mechanism that directs formation of the sequestering vesicles remains largely unknown. We analyzed the trafficking of Atg23 and the integral membrane protein Atg9 in the yeast Saccharomyces cerevisiae. These components localize both to the pre-autophagosomal structure (PAS) and other cytosolic punctate compartments. We show that Atg9 and Atg23 cycle through the PAS in a process governed by the Atg1-Atg13 signaling complex. Atg1 kinase activity is essential only for retrograde transport of Atg23, while recycling of Atg9 requires additional factors including Atg18 and Atg2. We postulate that Atg9 employs a recycling system mechanistically similar to that used at yeast early and late endosomes.     

Structure-Function Analysis of Fission Yeast Hus1-Rad1-Rad9 Checkpoint Complex

Hus1, Rad1, and Rad9 are three evolutionarily conserved proteins required for checkpoint control in fission yeast. These proteins are known to form a stable complex in vivo. Recently, computational studies have predicted structural similarity between the individual proteins of Hus1-Rad1-Rad9 complex and the replication processivity factor proliferating cell nuclear antigen (PCNA). This has led to the proposal that the Hus1-Rad1-Rad9 complex may form a PCNA-like ring structure, and could function as a sliding clamp during checkpoint control. In the present study, we have attempted to test the predictions of this model by asking whether the PCNA alignment identifies functionally important residues or explains mutant phenotypes of hus1, rad1, or rad9 alleles. Although some of our results are consistent with the PCNA alignment, others indicate that the Hus1-Rad1-Rad9 complex possesses unique structural and functional features.     

Rad23 provides a link between the Png1 deglycosylating enzyme and the 26 S proteasome in yeast

In addition to a role in DNA repair events in yeast, several lines of evidence indicate that the Rad23 protein (Rad23p) may regulate the activity of the 26 S proteasome. We report evidence that a de-N-glycosylating enzyme, Png1p, may be involved in the proteasomal degradation pathway via its binding to Rad23p. Interaction of Rad23p and Png1p was first detected by two-hybrid screening, and this interaction in vivo was confirmed by biochemical analyses. The Png1p-Rad23p complex was shown to be distinct from the well established DNA repair complex, Rad4p-Rad23p. We propose a model in which Rad23p functions as an escort protein to link the 26 S proteasome with proteins such as Rad4p or Png1p to regulate their cellular activities.     

Mre11-Rad50 complex crystals suggest molecular calisthenics

Recently published crystal structures of different Mre11 and Rad50 complexes show the arrangement of these proteins and imply dramatic ligand-induced rearrangements with important functional consequences.     

Talin1 regulates integrin turnover to promote embryonic epithelial morphogenesis

Talin is a cytoskeletal protein that binds to integrin β cytoplasmic tails and regulates integrin activation. Talin1 ablation in mice disrupts gastrulation and causes embryonic lethality. However, the role of talin in mammalian epithelial morphogenesis is poorly understood. Here we demonstrate that embryoid bodies (EBs) differentiated from talin1-null embryonic stem cells are defective in integrin adhesion complex assembly, epiblast elongation, and lineage differentiation. These defects are accompanied by a significant reduction in integrin β1 protein levels due to accelerated degradation through an MG-132-sensitive proteasomal pathway. Overexpression of integrin β1 or MG-132 treatment in mutant EBs largely rescues the phenotype. In addition, epiblast cells isolated from talin1-null EBs exhibit impaired cell spreading and focal adhesion formation. Transfection of the mutant cells with green fluorescent protein (GFP)-tagged wild-type but not mutant talin1 that is defective in integrin binding normalizes integrin β1 protein levels and restores focal adhesion formation. Significantly, cell adhesion and spreading are also improved by overexpression of integrin β1. All together, these results suggest that talin1 binding to integrin promotes epiblast adhesion and morphogenesis in part by preventing integrin β1 degradation.     

Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex

DNA double-strand breaks (DSBs) trigger activation of the ATM protein kinase, which coordinates cell-cycle arrest, DNA repair and apoptosis. We propose that ATM activation by DSBs occurs in two steps. First, dimeric ATM is recruited to damaged DNA and dissociates into monomers. The Mre11-Rad50-Nbs1 complex (MRN) facilitates this process by tethering DNA, thereby increasing the local concentration of damaged DNA. Notably, increasing the concentration of damaged DNA bypasses the requirement for MRN, and ATM monomers generated in the absence of MRN are not phosphorylated on Ser1981. Second, the ATM-binding domain of Nbs1 is required and sufficient to convert unphosphorylated ATM monomers into enzymatically active monomers in the absence of DNA. This model clarifies the mechanism of ATM activation in normal cells and explains the phenotype of cells from patients with ataxia telangiectasia-like disorder and Nijmegen breakage syndrome.     

Serine phosphorylation regulates disabled-1 early isoform turnover independently of Reelin

The Reelin-Disabled 1 (Dab1) signaling pathway plays an important role in neuronal cell migration during brain development. Dab1, an intracellular adapter protein which is tyrosine phosphorylated upon Reelin stimulation, has been directly implicated in the transmission and termination of Reelin-mediated signaling. Two main forms of Dab1 have been identified in the developing chick retina, an early isoform (Dab1-E) expressed in progenitor cells and a late isoform (Dab1-L, a.k.a. Dab1) expressed in differentiated cells. Dab1-E is missing two Src family kinase (SFK) phosphorylation sites that are critical for Reelin-Dab1 signaling and is not tyrosine phosphorylated. We have recently demonstrated a role for Dab1-E in the maintenance of retinal progenitor cells. Here, we report that Dab1-E is phosphorylated at serine/threonine residues independent of Reelin. Cdk2, highly expressed in retinal progenitor cells, mediates Dab1-E phosphorylation at serine 475 which in turn promotes ubiquitination-triggered proteasome degradation of Dab1-E. Inhibition of protein phosphatase 1 and/or protein phosphatase 2A leads to increased Dab1-E instability. We propose that Dab1 turnover is regulated by both Reelin-independent serine/threonine phosphorylation and Reelin-dependent tyrosine phosphorylation.     

Phosphorylation of SNAP-23 by the novel kinase SNAK regulates t-SNARE complex assembly

The docking and fusion of cargo-containing vesicles with target membranes of eukaryotic cells is mediated by the interaction of SNARE proteins present on both vesicle and target membranes. In many cases, the target membrane SNARE, or t-SNARE, exists as a complex of syntaxin with a member of the SNAP-25 family of palmitoylated proteins. We have identified a novel human kinase SNAK (SNARE kinase) that specifically phosphorylates the nonneuronal t-SNARE SNAP-23 in vivo. Interestingly, only SNAP-23 that is not assembled into t-SNARE complexes is phosphorylated by SNAK, and phosphorylated SNAP-23 resides exclusively in the cytosol. Coexpression with SNAK significantly enhances the stability of unassembled SNAP-23, and as a consequence, the assembly of newly synthesized SNAP-23 with syntaxin is augmented. These data demonstrate that phosphorylation of SNAP-23 by SNAK enhances the kinetics of t-SNARE assembly in vivo.     

The Rad52-Rad59 complex interacts with Rad51 and replication protein A

The RAD52 gene is essential for homology-dependent repair of double-strand breaks in Saccharomyces cerevisiae. Rad52 forms complexes with Rad51, replication protein A (RPA) or Rad59 and its presence is essential for the formation of Rad51-Rad52-Rad59 and RPA-Rad52-Rad59 complexes. The N-terminal region of Rad52, which is required for self-interaction to form a ring structure, is required for interaction with Rad59. Rad59 also shows self-interaction suggesting the formation of heteromeric and homomeric rings of Rad52 and Rad59. In wild-type cells, we propose the Rad51-Rad52-Rad59 complex is involved in conservative recombination events, including gene conversion and reciprocal recombination, whereas the Rad52-Rad59 complex participates in single-strand annealing.     

The Rad1-Rad10 complex promotes the production of gross chromosomal rearrangements from spontaneous DNA damage in Saccharomyces cerevisiae

Gross chromosomal rearrangements (GCRs) have been observed in many cancers. Previously, we have demonstrated many mechanisms for suppression of GCR formation in yeast. However, pathways that promote the formation of GCRs are not as well understood. Here, we present evidence that the Rad1-Rad10 endonuclease, which plays an important role in nucleotide excision and recombination repairs, has a novel role to produce GCRs. A mutation of either the RAD1 or the RAD10 gene reduced GCR rates in many GCR mutator strains. The inactivation of Rad1 or Rad10 in GCR mutator strains also slightly enhanced methyl methanesulfonate sensitivity. Although the GCRs induced by treatment with DNA-damaging agents were not reduced by rad1 or rad10 mutations, the translocation- and deletion-type GCRs created by a single double-strand break are mostly replaced by de novo telomere-addition-type GCR. Results presented here suggest that Rad1-Rad10 functions at different stages of GCR formation and that there is an alternative pathway for the GCR formation that is independent of Rad1-Rad10.