Comprehensive phosphoproteome analysis unravels the core signaling network that initiates the earliest synapse pathology in preclinical Alzheimer's disease brain.

Using a high-end mass spectrometry, we screened phosphoproteins and phosphopeptides in four types of Alzheimer's disease (AD) mouse models and human AD postmortem brains. We identified commonly changed phosphoproteins in multiple models and also determined phosphoproteins related to initiation of amyloid beta (Aß) deposition in the mouse brain. After confirming these proteins were also changed in and human AD brains, we put the proteins on experimentally verified protein-protein interaction databases. Surprisingly, most of the core phosphoproteins were directly connected, and they formed a functional network linked to synaptic spine formation. The change of the core network started at a preclinical stage even before histological Aß deposition. Systems biology analyses suggested that phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) by overactivated kinases including protein kinases C and calmodulin-dependent kinases initiates synapse pathology. Two-photon microscopic observation revealed recovery of abnormal spine formation in the AD model mice by targeting a core protein MARCKS or by inhibiting candidate kinases, supporting our hypothesis formulated based on phosphoproteome analysis.

Ataxin-7 associates with microtubules and stabilizes the cytoskeletal network.

The spinocerebellar ataxia type 7 (SCA7) gene product, Ataxin-7 (ATXN7), localizes to the nucleus and has been shown to function as a component of the TATA-binding protein-free TAF-containing-SPT3-TAF9-GCN5-acetyltransferase transcription complex, although cytoplasmic localization of ATXN7 in affected neurons of human SCA7 patients has also been detected. Here, we define a physiological function for cytoplasmic ATXN7. Live imaging reveals that the intracellular distribution of ATXN7 dynamically changes and that ATXN7 distribution frequently shifts from the nucleus to the cytoplasm. Immunocytochemistry and immunoprecipitation demonstrate that cytoplasmic ATXN7 associates with microtubules (MTs), and expression of ATXN7 stabilizes MTs against nocodazole treatment, while ATXN7 knockdown enhances MT degradation. Interestingly, normal and mutant ATXN7 similarly associate with and equally stabilize MTs. Taken together, these findings provide a novel physiological function of ATXN7 in the regulation of cytoskeletal dynamics, and suggest that abnormal cytoskeletal regulation may contribute to SCA7 disease pathology.

Y65C missense mutation in the WW domain of the Golabi-Ito-Hall syndrome protein PQBP1 affects its binding activity and deregulates pre-mRNA splicing.

The PQBP1 (polyglutamine tract-binding protein 1) gene encodes a nuclear protein that regulates pre-mRNA splicing and transcription. Mutations in the PQBP1 gene were reported in several X chromosome-linked mental retardation disorders including Golabi-Ito-Hall syndrome. The missense mutation that causes this syndrome is unique among other PQBP1 mutations reported to date because it maps within a functional domain of PQBP1, known as the WW domain. The mutation substitutes tyrosine 65 with cysteine and is located within the conserved core of aromatic amino acids of the domain. We show here that the binding property of the Y65C-mutated WW domain and the full-length mutant protein toward its cognate proline-rich ligands was diminished. Furthermore, in Golabi-Ito-Hall-derived lymphoblasts we showed that the complex between PQBP1-Y65C and WBP11 (WW domain-binding protein 11) splicing factor was compromised. In these cells a substantial decrease in pre-mRNA splicing efficiency was detected. Our study points to the critical role of the WW domain in the function of the PQBP1 protein and provides an insight into the molecular mechanism that underlies the X chromosome-linked mental retardation entities classified globally as Renpenning syndrome.

Functional complexes between YAP2 and ZO-2 are PDZ domain-dependent, and regulate YAP2 nuclear localization and signalling.

The Hippo pathway regulates the size of organs by controlling two opposing processes: proliferation and apoptosis. YAP2 (Yes kinase-associated protein 2), one of the three isoforms of YAP, is a WW domain-containing transcriptional co-activator that acts as the effector of the Hippo pathway in mammalian cells. In addition to WW domains, YAP2 has a PDZ-binding motif at its C-terminus. We reported previously that this motif was necessary for YAP2 localization in the nucleus and for promoting cell detachment and apoptosis. In the present study, we show that the tight junction protein ZO (zonula occludens)-2 uses its first PDZ domain to form a complex with YAP2. The endogenous ZO-2 and YAP2 proteins co-localize in the nucleus. We also found that ZO-2 facilitates the nuclear localization and pro-apoptotic function of YAP2, and that this activity of ZO-2 is PDZ-domain-dependent. The present paper is the first report on a PDZ-based nuclear translocation mechanism. Moreover, since the Hippo pathway acts as a tumour suppressor pathway, the YAP2-ZO-2 complex could represent a target for cancer therapy.

Nuclear localization and pro-apoptotic signaling of YAP2 require intact PDZ-binding motif.

The Hippo signaling pathway regulates the intrinsic size of organs by controlling two opposing processes, proliferation and apoptosis. The nuclear effector of this pathway is Yes kinase-associated protein (YAP) which is a WW domain-containing transcriptional co-activator. In addition to WW domains, YAP2 has a Post-synaptic density, Discs large, Zonula occludens-1 (PDZ)-binding motif that is located at its COOH terminus. To determine whether the localization of YAP2 in cells is PDZ-binding motif dependent, we generated a delta C mutant of YAP2 lacking the five most COOH terminal amino acids, -FLTWL, which constitute a well-conserved PDZ-binding motif. We report here that the PDZ-binding motif is necessary for YAP2 localization in the nucleus, for the stabilization of p73, and for promoting apoptosis of HEK293 cells maintained at low concentration of serum. We suggest that an unknown PDZ domain-containing protein (or proteins) functions as a shuttle, facilitating YAP2 translocation from the cytoplasm to the nucleus. Since the Hippo pathway acts as a tumor suppressor pathway, the PDZ complex of YAP represents a potential target of cancer therapy.

Kpm/Lats2 is linked to chemosensitivity of leukemic cells through the stabilization of p73.

Down-regulation of the Kpm/Lats2 tumor suppressor is observed in various malignancies and associated with poor prognosis in acute lymphoblastic leukemia. We documented that Kpm/Lats2 was markedly decreased in several leukemias that were highly resistant to conventional chemotherapy. Silencing of Kpm/Lats2 expression in leukemic cells did not change the rate of cell growth but rendered the cells more resistant to DNA damage-inducing agents. Expression of p21 and PUMA was strongly induced by these agents in control cells, despite defective p53, but was only slightly induced in Kpm/Lats2-knockdown cells. DNA damage-induced nuclear accumulation of p73 was clearly observed in control cells but hardly detected in Kpm/Lats2-knockdown cells. Chromatin immunoprecipitation (ChIP) assay showed that p73 was recruited to the PUMA gene promoter in control cells but not in Kpm/Lats2-knockdown cells after DNA damage. The analyses with transient coexpression of Kpm/Lats2, YAP2, and p73 showed that Kpm/Lats2 contributed the stability of YAP2 and p73, which was dependent on the kinase function of Kpm/Lats2 and YAP2 phosphorylation at serine 127. Our results suggest that Kpm/Lats2 is involved in the fate of p73 through the phosphorylation of YAP2 by Kpm/Lats2 and the induction of p73 target genes that underlie chemosensitivity of leukemic cells.

WW domains of the yes-kinase-associated-protein (YAP) transcriptional regulator behave as independent units with different binding preferences for PPxY motif-containing ligands.

YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling.

A functional deficiency of TERA/VCP/p97 contributes to impaired DNA repair in multiple polyglutamine diseases.

It is hypothesized that a common underlying mechanism links multiple neurodegenerative disorders. Here we show that transitional endoplasmic reticulum ATPase (TERA)/valosin-containing protein (VCP)/p97 directly binds to multiple polyglutamine disease proteins (huntingtin, ataxin-1, ataxin-7 and androgen receptor) via polyglutamine sequence. Although normal and mutant polyglutamine proteins interact with TERA/VCP/p97, only mutant proteins affect dynamism of TERA/VCP/p97. Among multiple functions of TERA/VCP/p97, we reveal that functional defect of TERA/VCP/p97 in DNA double-stranded break repair is critical for the pathology of neurons in which TERA/VCP/p97 is located dominantly in the nucleus in vivo. Mutant polyglutamine proteins impair accumulation of TERA/VCP/p97 and interaction of related double-stranded break repair proteins, finally causing the increase of unrepaired double-stranded break. Consistently, the recovery of lifespan in polyglutamine disease fly models by TERA/VCP/p97 corresponds well to the improvement of double-stranded break in neurons. Taken together, our results provide a novel common pathomechanism in multiple polyglutamine diseases that is mediated by DNA repair function of TERA/VCP/p97.

Identification, basic characterization and evolutionary analysis of differentially spliced mRNA isoforms of human YAP1 gene.

The YAP1 gene encodes a potent new oncogene and stem cell factor. However, in some cancers, the YAP1 gene plays a role of tumor suppressor. At present, the gene and its products are intensely studied and its cDNAs are used as transgenes in cellular and animal models. Here, we report 4 new potential mRNA splicing isoforms of the YAP1 gene, bringing the total number of isoforms to 8. We detected all 8 YAP1 isoforms in a panel of human tissues and evaluated the expression of the longest isoform of YAP1 (YAP1-2δ) using Real Time PCR. All YAP1 isoforms are barely detectable in human leukocytes compared to fair levels of expression found in other human tissues. We analyzed the structure of the genomic region that gave rise to alternatively spliced YAP1 transcripts in different metazoans. We found that YAP1 isoforms, which utilize exon 6 emerged in evolution with the appearance of amniotes. Interestingly, 6 YAP1 isoforms, which contain the exon 5 extension, exon 6 or both would have their leucine zipper region disrupted in the predicted protein product, compared to the intact leucine zipper found in two YAP1 (α) isoforms. This observation has direct functional ramifications for YAP1 signaling. We also propose a normalized nomenclature for the mRNA splice variants of the YAP1 gene, which should aid in the characterization of signaling differences among the potential protein products of the YAP1 gene.

Dynamic changes of the phosphoproteome in postmortem mouse brains.

Protein phosphorylation is deeply involved in the pathological mechanism of various neurodegenerative disorders. However, in human pathological samples, phosphorylation can be modified during preservation by postmortem factors such as time and temperature. Postmortem changes may also differ among proteins. Unfortunately, there is no comprehensive database that could support the analysis of protein phosphorylation in human brain samples from the standpoint of postmortem changes. As a first step toward addressing the issue, we performed phosphoproteome analysis with brain tissue dissected from mouse bodies preserved under different conditions. Quantitative whole proteome mass analysis showed surprisingly diverse postmortem changes in phosphoproteins that were dependent on temperature, time and protein species. Twelve hrs postmortem was a critical time point for preservation at room temperature. At 4°C, after the body was cooled down, most phosphoproteins were stable for 72 hrs. At either temperature, increase greater than 2-fold was exceptional during this interval. We found several standard proteins by which we can calculate the postmortem time at room temperature. The information obtained in this study will be indispensable for evaluating experimental data with human as well as mouse brain samples.

TAZ interacts with zonula occludens-1 and -2 proteins in a PDZ-1 dependent manner.

The transcriptional coactivator TAZ recognizes L/PPxY motifs in transcription factors like Runx1/2 through its WW domain. We show that the first PDZ domain of zona occludens-1 (ZO-1) and 2 (ZO-2) interacts with the carboxy-terminal PDZ binding motif of TAZ. Deletion of this motif abrogates binding. ZO-2 colocalizes with TAZ in the nucleus of MDCK cells and ZO-2 expression alters TAZ localization in human embryonic kidney cells. Luciferase assays demonstrate ZO-2 inhibition of TAZ-mediated transactivation. We propose that zonula occludens is a negative regulator of TAZ and suggest that selected tight junction proteins control nuclear translocation and activity of TAZ.

YAP: at the crossroad between transformation and tumor suppression.

Yap is a small protein that binds to many transcription factors and modulates their activity. Yap was described to increase the ability of p73 in inducing apoptosis as a consequence of damage to the DNA, and therefore its activity was thought to favor tumor-suppression. However, other studies have recently shown a role for Yap in cell differentiation, cell transformation and in the regulation of organ size. It has been demonstrated that the Drosophila Hippo pathway has a mammalian equivalent, and that Yap is part of this pathway, where it might stimulate proliferation. In light of these new findings we ought to re-consider the role of Yap, which seems to be in service of several masters, and whose regulation--likely at the level of PTM--and cellular context might have a pivotal role in the choice of its partners and consequently on the final outcome.

Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP).

The Hippo pathway in Drosophila controls the size and shape of organs. In the fly, activation of this pathway conveys growth-inhibitory signals and promotes apoptosis in epithelial cells. We "reconstituted" the Hippo pathway in a human epithelial cell line and showed that, in contrast to flies, the activation of this pathway results in anti-apoptotic signals. We have shown that in human embryonic kidney (HEK) 293 cells, the complex formation between transcriptional co-activators YAPs (Yes kinase-associated proteins) and Lats kinases requires the intact WW domains of YAPs, as well as intact Pro-Pro-AA-Tyr (where AA is any amino acid) motifs in Lats kinases. These kinases cooperate with the upstream Mst2 kinase to phosphorylate YAPs at Ser-127. Overexpression of YAP2 in HEK293 cells promoted apoptosis, whereas the Mst2/Lats1-induced phosphorylation of YAP partially rescued the cells from apoptotic death. Apoptotic signaling of YAP2 was mediated via stabilization of p73, which formed a complex with YAP2. All components of the Hippo pathway that we studied were localized in the cytoplasm, with the exception of YAP, which also localized in the nucleus. The localization of YAP2 in the nucleus was negatively controlled by the Lats1 kinase. Our apoptotic "readout" of the Hippo pathway in embryonic kidney cells represents a useful experimental system for the identification of the putative upstream receptor, membrane protein, or extracellular factor that initiates an entire signaling cascade and ultimately controls the size of organs.

Cooperation of DEF6 with activated Rac in regulating cell morphology.

Rho-family GTPases have been implicated in actin remodeling and subsequent morphologic changes in various cells. DEF6, a pleckstrin homology domain-containing protein, has been reported to regulate Rho-family GTPases as a guanine nucleotide exchange factor. Here, we demonstrate that DEF6 also has the property of cooperating with activated Rac1. DEF6 bound selectively to Rac1 loaded with GTP. The interaction is mediated by the effector domain of Rac1. Overexpression of GFP-DEF6 together with constitutively active Rac1 in COS-7 cells significantly changed their cell shape; this was not seen in the absence of activated Rac1. This effect of DEF6 on cellular morphology was shown to be independent of its guanine nucleotide exchange activity. Because DEF6 does not contain any sequences previously known to interact with Rac, we explored the domain necessary for the binding. The amino-terminal portion and central parts of DEF6 were required for the binding. Finally, we succeeded in creating mutants of DEF6 with point mutations in the amino-terminal portion, which abrogate the binding to activated Rac1. These mutants did not exhibit the morphologic change in COS-7 cells when they were co-expressed with activated Rac1. These results suggest that DEF6 not only activates Rho-family GTPases but also cooperates with activated Rac1 to exert its cellular function.

Direct binding of SWAP-70 to non-muscle actin is required for membrane ruffling.

Membrane ruffling induced by growth factor stimulation is caused by actin remodeling, which is mediated by various signaling molecules including Rac. We have shown that SWAP-70, which binds phosphatidylinositol trisphosphate, is one such molecule required for membrane ruffling in mouse kidney cells. Here, we show that SWAP-70 directly binds to F-actin. The bacterially expressed C-terminal region of SWAP-70 co-sedimented with non-muscle F-actin, suggesting direct binding of SWAP-70 to F-actin. The binding was much weaker in muscle F-actin. A truncated mutant of SWAP-70 containing only the C-terminal region readily colocalizes with F-actin, supporting this idea. Full-length SWAP-70 does not colocalize with F-actin unless cells are stimulated with growth factors, suggesting the presence of a stimuli-dependent regulatory mechanism for actin-binding activity in vivo. Overexpression of the mutant SWAP-70 lacking this binding domain inhibits the membrane ruffling induced by epidermal growth factor stimulation in COS7 cells. This dominant-negative effect is also observed in membrane ruffling induced by a dominant-active Rac, suggesting that SWAP-70 cooperates with Rac. These results suggest that the binding activity of SWAP-70 to non-muscle F-actin is required for membrane ruffling.