The Hippo pathway in Drosophila controls the size and shape of organs. In the fly, activation of this pathway conveys growthinhibitory 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.

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.

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 tumorsuppression. 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.

YAP (Yes-associated protein) oncogene has been found to form a stable complex with members of the Angiomotin (Amot) family of proteins, which bind WW domains of YAP and sequester the protein in the cytoplasm and junctional complexes. The Amot-mediated retention of YAP in the cytoplasm results in the inhibition of its proliferative function. Using apoptotic 'read-out' of YAP in HEK293 cells, we confirmed the molecular mode by which Amot regulates YAP. We showed that a representative member of the Amot family, AmotL1 (Angiomotinlike-1), uses its PPxY motifs to bind WW domains of YAP and inhibit YAP's nuclear translocation and pro-apoptotic function. Recently we also showed that YAP uses its PDZbinding motif to interact with zona occludens-2 (ZO-2) protein, which promotes YAP's translocation to the nucleus. We also asked if AmotL1, YAP and ZO-2 signal together. We report here that AmotL1 and ZO-2 form a tripartite complex with YAP and regulate its function in HEK293 cells in opposite directions. AmotL1 inhibits proapoptotic function of YAP, whereas ZO-2 enhances it. As YAP is a potent oncogene, the identification and characterization of its regulators is important. AmotL1 and ZO-2 are two candidates that could be harnessed to control the oncogenic function of YAP.

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 YAP1 gene, which should aid in the characterization of signaling differences among the potential protein products of the YAP1 gene.

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 (Aβ) 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 Aβ 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.

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.

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.