Phosphotyrosyl Protein Research Articles

Structural and Functional Characterization of Novel Phosphotyrosine Phosphatase Protein from Drosophila melanogaster (Pupal Retina).

A novel pair of protein tyrosine phosphatases in Drosophila melanogaster (pupal retina) has been identified. Phosphotyrosyl protein phosphatases (PTPs) are structurally diverse enzymes increasingly recognized as having a fundamental role in cellular processes including effects on metabolism, cell proliferation, and differentiation. This study presents identification of novel sequences of PTPs and their comparative homology modeling from Drosophila melanogaster (Dr-PTPs) and complexation with the potent inhibitor HEPES. The 3D structure was predicted based on sequence homology with bovine heart low molecular weight PTPs (Bh-PTPs). The sequence homologies are approximately 50% identical to each other and to low molecular weight protein tyrosine phosphatases (PTPs) in other species. Comparison of the 3D structures of Bh-PTPs and Dr-PTPs (primo-2) reveals a remarkable similarity having a four stranded central parallel β sheet with flanking α helices on both sides, showing two right handed β-α-β motifs. The inhibitor shows similar binding features as seen in other PTPs. The study also highlights the key catalytic residues important for target recognition and PTPs' activation. The structure guided studies of both proteins clearly reveal a common mechanism of action and inhibitor binding at the active site and will be expected to contribute toward the basic understanding of functional association of this enzyme with other molecules.

Renal FGF23 signaling depends on redox protein Memo1 and promotes orthovanadate-sensitive protein phosphotyrosyl phosphatase activity.

Memo1 deletion in mice causes premature aging and an unbalanced metabolism partially resembling Fgf23 and Klotho loss-of-function animals. We report a role for Memo's redox function in renal FGF23-Klotho signaling using mice with postnatally induced Memo deficiency in the whole body (cKO). Memo cKO mice showed impaired FGF23-driven renal ERK phosphorylation and transcriptional responses. FGF23 actions involved activation of oxidation-sensitive protein phosphotyrosyl phosphatases in the kidney. Redox proteomics revealed excessive thiols of Rho-GDP dissociation inhibitor 1 (Rho-GDI1) in Memo cKO, and we detected a functional interaction between Memo's redox function and oxidation at Rho-GDI1 Cys79. In isolated cellular systems, Rho-GDI1 did not directly affect FGF23-driven cell signaling, but we detected disturbed Rho-GDI1 dependent small Rho-GTPase protein abundance and activity in the kidney of Memo cKO mice. Collectively, this study reveals previously unknown layers in the regulation of renal FGF23 signaling and connects Memo with the network of small Rho-GTPases.

Unbalanced Expression of Glutathione Peroxidase 4 and Arachidonate 15-Lipoxygenase Affects Acrosome Reaction and In Vitro Fertilization

Glutathione peroxidase 4 (Gpx4) and arachidonic acid 15 lipoxygenase (Alox15) are counterplayers in oxidative lipid metabolism and both enzymes have been implicated in spermatogenesis. However, the roles of the two proteins in acrosomal exocytosis have not been explored in detail. Here we characterized Gpx4 distribution in mouse sperm and detected the enzyme not only in the midpiece of the resting sperm but also at the anterior region of the head, where the acrosome is localized. During sperm capacitation, Gpx4 translocated to the post-acrosomal compartment. Sperm from Gpx4+/Sec46Ala mice heterozygously expressing a catalytically silent enzyme displayed an increased expression of phosphotyrosyl proteins, impaired acrosomal exocytosis after in vitro capacitation and were not suitable for in vitro fertilization. Alox15-deficient sperm showed normal acrosome reactions but when crossed into a Gpx4-deficient background spontaneous acrosomal exocytosis was observed during capacitation and these cells were even less suitable for in vitro fertilization. Taken together, our data indicate that heterozygous expression of a catalytically silent Gpx4 variant impairs acrosomal exocytosis and in vitro fertilization. Alox15 deficiency hardly impacted the acrosome reaction but when crossed into the Gpx4-deficient background spontaneous acrosomal exocytosis was induced. The detailed molecular mechanisms for the observed effects may be related to the compromised redox homeostasis.

A tyrosine phosphatase SHP2 gain-of-function mutation enhances malignancy of breast carcinoma.

Background: Evidence suggests that Src homologous protein phosphotyrosyl phosphatase 2 (SHP2) mutations promote cancer development in several solid tumours. In this study, we focused on the in vivo and in vitro effects of an SHP2 mutation on the breast cancer phenotype to determine whether this mutation is correlated with a malignant phenotype.Methods: Mutant PTPN11 cDNA (D61G) was transduced into MDA-MB231 and MCF-7 cells. The effects of the D61G mutation on tumourigenesis and malignant behaviours, such as cell adhesion, proliferation, migration and invasion, were examined. Potential underlying molecular mechanisms, i.e., activation of the Gab1-Ras-Erk axis, were also examined.Results: In vitro experiments revealed that tumour adhesion, proliferation, migration and invasion were significantly increased in the SHP2 D61G mutant groups. Consistently, in vivo experiments also showed that the tumour sizes and weights were increased significantly in the SHP2 D61G-MB231 group (p < 0.001) in association with tumour metastasis. Mechanistically, the PTPN11 mutation resulted in activation of the Ras-ErK pathway. The binding between Gab1 and mutant SHP2 was significantly increased.Conclusion: Mutant SHP2 significantly promotes tumour migration and invasion at least partially through activation of the Gab1-Ras-Erk axis. This finding could have direct implications for breast cancer therapy.

Abstract 3634: A gain-of-function mutation in tyrosine phosphatase SHP-2 enhances breast carcinoma malignant phenotypes

Abstract Breast cancer is the most common malignant tumor in women worldwide, and it is the second leading cause of cancer-related mortality in the US women. Evidences suggested that overexpression of Src homologous protein phosphotyrosyl phosphatase 2 (SHP2) has been observed in most of the breast cancer cell lines and in breast cancer tissues, and accompanied with lymph nodes metastasis. Inhibition of SHP2 in the breast cancer cell lines abolishes the growth and decrease survival of tumor cells, which leads to the differentiation of malignant cells into normal breast epithelial phenotype. These observations suggest that SHP2 promotes the tumor development though increase tumor formation and metastasis. However, the pathophysiologic role of SHP2 (overexpression or gain-of-function mutation) and its relevance to tumorigenesis are still largely unknow. In this study, we focused on the in vivo and in vitro effects of the gain-of-function mutant SHP-2 on phenotypes of breast cancer. Mutant PTPN11 cDNA (D61G) was transduced into MDA-MB231 and MCF-7 cells. The effects of D61G mutant on breast cancer tumorigenesis and malignant behaviours, such as cell adhesion, proliferation, migration and invasion were tested. The potential underlying molecular mechanisms, i.e., the activation of the Gab1-Ras-Erk axis were also examined. In vitro studies found that, in SHP2 D61G mutant groups, the tumour adhesion, proliferation, migration and invasion were increased significantly. Consistently, in vivo studies also found that the incidence of tumour and the tumour weights of the SHP-2 D61G-MB231 group were increased significantly (P&amp;lt;0.001), which is also associated with tumour metastasis. Mechanistically, the PTPN11 D61G mutation resulted in the activation of Ras-ErK pathway. And interestingly, the interaction between Gab1 and mutant SHP2 was also significantly increased. The mutant SHP-2 significantly promoted tumour migration and invasion at least partially through the activation of Gab1-Ras-Erk axis. In conclusion, our findings clearly establish a role for the SHP-2 GOF mutant in breast tumour growth and provide novel insight into the mechanisms of SHP-2 GOF mutant-mediated tumor promotion. This finding could have direct implications for breast cancer therapy. This study was supported by a grant from the National Natural Science Foundation of China (codes: 30873046, 30973424, and 81072663) and by the Fundamental Research Funds for the Central Universities (code:3290004402). Note: This abstract was not presented at the meeting. Citation Format: Zhongqian Hu, Haoshu Fang, Siying Wang. A gain-of-function mutation in tyrosine phosphatase SHP-2 enhances breast carcinoma malignant phenotypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3634. doi:10.1158/1538-7445.AM2015-3634

Adverse cardiac remodeling due to maternal low protein diet is associated with alterations in expression of genes regulating glucose metabolism

Background and aimsWe have previously shown that a maternal low protein (LP) diet during pregnancy in the rat results in adverse ventricular remodeling and contractile deficiencies of the neonatal rat heart. Since pathological cardiac hypertrophy is associated with increased expression of genes involved in glucose handling, this study was undertaken to examine if maternal LP diet alters the expression of genes encoding for some key components of glucose metabolism and uptake, and of the insulin receptor (IR) signal transduction in the heart of male offspring. Methods and resultsWe determined the effect of maternal LP and normal diet (90 and 180 g/casein/kg respectively) on IR β-subunit, insulin receptor substrate (IRS)-1, phosphotyrosyl protein phosphatase (PTP) 1B, GLUT4 and phosphatidylinositol (PI) 3-kinase in male rat offspring at 24 h and at 1, 4 and 8 wks post-partum. Quantitative real-time RT-PCR revealed significant age-dependent increases in the expression of IR β-subunit, IRS-1, PTP1B, GLUT4 and PI3-kinase in the LP group with concomitant increases in corresponding protein abundance at 4 wks of age. These changes were associated with increases in left ventricular (LV) internal diameters as well as increases in LV wall thickness. ConclusionA maternal LP diet can induce increases in the gene expression and protein levels of key components of glucose metabolism and the IR signal transduction pathway in the neonatal rat heart, which may be related to accelerated energy supply, demand and utilization for ventricular remodeling due to compromised contractile performance during early life.

Proteomic analysis of phosphotyrosyl proteins in human embryonic stem cell-derived neural stem cells

Phosphorylation can reveal essential cell functions, such as cell differentiation, signal transduction, metabolic maintenance and cell division. The aim of this study was to investigate phosphorylated protein expression changes during neuronal lineage differentiation from hESCs. To measure the phosphorylated protein expression change during neuronal differentiation, we performed a comparative phosphoproteome analysis using 2-DE after MALDI-TOF MS and an MS/MS protein identification method, making a comparison between neural lineage differentiating cells and normal embryoid bodies (EBs) differentiated from human embryonic stem cells (hESCs) and profiling constituent phosphorylated proteins. Of 36 differentially expressed protein spots, 12 spots were shown to be up-regulated in differentiating neural cells. Specifically, the 7 up-regulated proteins of the 12 have potential roles in neuronal differentiation or neuronal damage recovery, including ACTB, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNP A2B1), heterogeneous nuclear ribonucleoprotein L (hnRNP L), SET, chaperonin-containing TCP-1, vimentin and voltage-dependent anion channel protein 1 (VDAC1). These proteins are discussed further below.

N-terminal and C-terminal heparin-binding domain polypeptides derived from fibronectin reduce adhesion and invasion of liver cancer cells

BackgroundFibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including N-terminal and C-terminal heparin-binding domains. We investigated the effects of highly purified rhFNHN29 and rhFNHC36 polypeptides originally cloned from the two heparin-binding domains on the adhesion and invasion of highly metastatic human hepatocellular carcinoma cells (MHCC97H) and analyzed the underlying mechanism involved.MethodsThe MHCC97H cells that adhered to FN in the presence of various concentrations of rhFNHN29 and rhFNHC36 polypeptides were stained with crystal violet and measured, and the effects of rhFNHN29 and rhFNHC36 on the invasion of the MHCC97H cells were then detected using the Matrigel invasion assay as well as a lung-metastasis mouse model. The expression level of integrins and focal adhesion kinase (FAK) phosphotyrosyl protein was examined by Western blot, and the activity of matrix metalloproteinases (MMPs) and activator protein 1 (AP-1) was analyzed by gelatin zymography and the electrophoretic mobility band-shift assay (EMSA), respectively.ResultsBoth of the polypeptides rhFNHN29 and rhFNHC36 inhibited adhesion and invasion of MHCC97H cells; however, rhFNHC36 exhibited inhibition at a lower dose than rhFNHN29. These inhibitory effects were mediated by integrin αvβ3 and reversed by a protein tyrosine phosphatase inhibitor. Polypeptides rhFNHN29 and rhFNHC36 abrogated the tyrosine phosphorylation of focal adhesion kinase (p-FAK) and activation of activator protein 1 (AP-1), resulting in the decrease of integrin αv, β3 and β1 expression as well as the reduction of MMP-9 activity.ConclusionsPolypeptides rhFNHN29 and rhFNHC36 could potentially be applicable to human liver cancer as anti-adhesive and anti-invasive agents.

Molecular Mechanism for SHP2 in Promoting HER2-induced Signaling and Transformation

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in HER2-induced signaling and transformation, but its mechanism of action is poorly understood. Given the significance of HER2 in breast cancer, defining a mechanism for SHP2 in the HER2 signaling pathway is of paramount importance. In the current report we show that SHP2 positively modulates the Ras-extracellular signal-regulated kinase 1 and 2 and the phospoinositide-3-kinase-Akt pathways downstream of HER2 by increasing the half-life the activated form of Ras. This is accomplished by dephosphorylating an autophosphorylation site on HER2 that serves as a docking platform for the SH2 domains of the Ras GTPase-activating protein (RasGAP). The net effect is an increase in the intensity and duration of GTP-Ras levels with the overall impact of enhanced HER2 signaling and cell transformation. In conformity to these findings, the HER2 mutant that lacks the SHP2 target site exhibits an enhanced signaling and cell transformation potential. Therefore, SHP2 promotes HER2-induced signaling and transformation at least in part by dephosphorylating a negative regulatory autophosphorylation site. These results suggest that SHP2 might serve as a therapeutic target against breast cancer and other cancers characterized by HER2 overexpression.

Origins of Growth Factors: NGF and EGF

Growing up on the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures. I, therefore, became a double major in chemistry and biology (also with the hope of earning a living as a laboratory technician). My first job after graduating was working nights as a bacteriologist in a milk processing plant. One of my professors at Brooklyn College wrote asking whether I was interested in going to graduate school. The college had been sending one student a year to the Biology Department at Oberlin to work as a laboratory assistant while studying for a master's degree. Because Oberlin offered to pay my tuition plus some $300 a semester for living expenses, I jumped at the chance. School and science were both interesting and fun. Upon graduating, I continued my education toward a Ph.D. working as an assistant in the Biochemistry Department of the University of Michigan under Howard B. Lewis. My research involved studying the Krebs urea cycle in the common earthworms that I collected from the university campus. I had been told at Oberlin that the yellow cells surrounding the gut in the worm corresponded to the mammalian liver. I decided to check whether the enzymatic reactions in worms were similar to those in mammals. They were (1). My first “real job” was in the Pediatrics Department at the University of Colorado studying creatine metabolism in premature infants under Harry Gordon. I think he hired me because he was impressed by my ability to stomach tube earthworms. After several years, I decided I needed to learn the then new technique of using radioisotopes in metabolic studies, and I obtained an American Cancer Society fellowship to work in the Radiology Department of Washington University under Martin Kamen where I combined this new knowledge with my interest in embryology. Although fertilized frog eggs and early embryos are impermeable to small molecules (amino acids, phosphates, etc.), sufficient amounts of C14O2 were metabolized by intact eggs and embryos to permit the identification of the radioactive compounds present at various stages of development (2). Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the “growth factors,” nerve growth factor (NGF) and epidermal growth factor (EGF).

Identification of phosphotyrosyl proteins in vitreous humours of patients with vitreoretinal diseases by sodium dodecyl sulphate–polyacrylamide gel electrophoresis/Western blotting/matrix-assisted laser desorption time-of-flight mass spectrometry

It is important to explain target proteins for the understanding of the pathogenesis of vitreoretinal diseases. In a previous study, we identified more than 100 proteins including seven angiogenic-modulated factors in vitreous humours (VHs). Although there have been many reports of expressed protein profiles in VHs, only a few of these are modified proteins, such as those undergoing phosphorylation and oxidation. We applied Western blotting (WB), selective staining of phosphoproteins and mass spectrometry to detect and identify phosphoproteins in VHs of patients with vitreoretinal diseases. After the removal of albumin and immunoglobulins A/G in VHs, the proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reacted with anti-PY-20 monoclonal antibody on transfer membranes, and treated proteins were visualized with Phos-tag and identified by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS). WB analysis detected four positive bands, 20, 30, 35 and 55 kDa, in VHs of patients with vitreoretinal diseases. One of them, 55 kDa, was frequently detected in VHs of patients with macular hole (MH) and retinal detachment (RD), but the band was not found in patients with proliferative diabetic retinopathy (PDR). alpha-1 antitrypsin (alpha-1 AT) was identified in excised gel pieces of this band. We identified five phosphorylated proteins such as alpha-1 AT in VHs of patients with vitreoretinal diseases by MALDI-TOFMS and WB analysis. Phosphotyrosyl alpha-1 AT was neither detected in PDR patients nor in any plasma. Phosphotyrosyl alpha-1 AT may be a new biomarker of MH and RD.

Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets

Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src–GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src.

Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells

With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR. In addition, EGFR activity was shown to be necessary for EGF-induced localization of Endofin, an FYVE domain-containing protein regulated by phosphoinositol lipid and engaged in endosome-mediated receptor modulation. Although several groups have conducted phosphoproteomics of EGF signaling in recent years, our study is the first to identify and validate Endofin, DCBLD2, and KIAA0582 as part of a complex EGF phosphotyrosine signaling network. These novel data will provide new insights into the complex EGF signaling and may have implications on target-directed cancer therapeutics.

Substrate Profiling of Protein Tyrosine Phosphatase PTP1B by Screening a Combinatorial Peptide Library

Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. To date, the in vivo substrates and physiological functions of PTPs remain poorly defined. In this work, we have developed a novel combinatorial library method to systematically determine the substrate specificity of PTPs. A one-bead-one-compound peptide library containing five randomized residues, Fmoc-XXXXXpYAA (where X is norleucine or 17 proteinogenic amino acids excluding Tyr, Cys, and Met), was chemically synthesized on 90-μm TentaGel resin by the split-and-pool method. Limited treatment of the library with a PTP removed the phosphoryl group from beads that carry the most preferred substrates. The exposed tyrosine side chain was selectively oxidized into an orthoquinone by the treatment with tyrosinase in the presence of atmospheric oxygen. The orthoquinone was then selectively derivatized with biotin-hydrazide, followed by on-bead colorim...

A Chemical Approach to the Identification of Tensin-Binding Proteins

Many protein-protein interactions are mediated by small modular domains, which recognize short peptide motifs in their partner proteins. However, for the great majority of these domains, the identity of their partner proteins remains unknown. In this work, a chemical/bioinformatics approach has been developed to identify phosphotyrosyl (pY) proteins that bind to tensin, a protein involved in the formation of actin cytoskeleton and signal transduction. A pY peptide library was chemically synthesized and screened against the Src homology 2 (SH2) domain of tensin to identify the peptide motifs that bind to the SH2 domain. Next, protein databases were searched for proteins containing the SH2 domain-binding peptide motifs. Finally, the potential tensin-binding proteins were confirmed (or disproved) by in vitro pull-down and coimmunoprecipitation assays. This procedure identified phosphoinositide-dependent kinase-1 and downstream of tyrosine kinase 2 as novel tensin-binding proteins. In addition, a cell-permeable pY peptide was designed as tensin SH2 domain inhibitor, which caused the disruption of actin filaments in NIH 3T3 cells. This method should be generally applicable to other modular domains that recognize small peptide motifs.

Interaction of Phospholipase C-γ1 with Villin Regulates Epithelial Cell Migration

Interaction of Phospholipase C-γ1 with Villin Regulates Epithelial Cell Migration

Reishi Polysaccharides Induce Immunoglobulin Production through the TLR4/TLR2-mediated Induction of Transcription Factor Blimp-1

The polysaccharides of Ganoderma lucidum (Reishi) possess immunomodulation activities; however, their mode of molecular action in regulating each cellular subset in the immune system is still not clear. Here, we investigate the function of the main polysaccharide fraction of Reishi (Reishi-F3) in B lymphocyte activation/differentiation. We find that Reishi-F3 causes mouse splenic B cell activation and differentiation to IgM-secreting plasma cells, and the process depends on Reishi-F3-mediated induction of Blimp-1, a master regulator capable of triggering the changes of a cascade of gene expression during plasmacytic differentiation. In human peripheral B lymphocytes, although Reishi-F3 fails to induce their activation, it is able to enhance antibody secretion, which is associated with Blimp-1 mRNA induction. The function of Reishi-F3 depends on the Toll-like receptors TLR4/TLR2 as neutralizing antibodies against TLR4/TLR2 block Reishi-F3-mediated induction of Blimp-1 mRNA and Ig secretion. We have shown that interaction of Reishi-F3 with TLR4/TLR2 followed by signaling through p38 MAPK is involved in the induction of Blimp-1 mRNA, whereas signaling through ERK, p38 MAPK, JNK, and IKK complex is involved in Reishi-F3-mediated Ig secretion. Furthermore, the differential mechanism of Reishi-F3 in mouse and human B cell activation is probably due to the presence of Blimp-1 regulatory site in human CD86 promoter. These results establish the signaling and molecular mechanisms of Reishi-F3 on promoting antibody secretion.

Tyrosyl-DNA phosphodiesterase (Tdp1) participates in the repair of Top2-mediated DNA damage

Agents targeting topoisomerases are active against a wide range of human tumors. Stabilization of covalent complexes, converting topoisomerases into DNA-damaging agents, is an essential aspect of cell killing by these drugs. A unique aspect of the repair of topoisomerase-mediated DNA damage is the requirement for pathways that can remove protein covalently bound to DNA. Tyrosyl-DNA phosphodiesterase (Tdp1) is an enzyme that removes phosphotyrosyl moieties bound to the 3' end of DNA. Cells lacking Tdp1 are hypersensitive to camptothecin, consistent with a role for Tdp1 in processing 3' phosphotyrosyl protein-DNA covalent complexes. Because Top2p forms a 5' phosphotyrosyl linkage with DNA, previous work predicted that Tdp1p would not be active against lesions involving Top2p. We found that deletion of the TDP1 gene in yeast confers hypersensitivity to Top2 targeting agents. Combining tdp1 mutations with deletions of genes involved in nonhomologous end joining, excision repair, or postreplication repair enhanced sensitivity to Top2 targeting drugs over the level seen with single mutants, suggesting that Tdp1 may function in collaboration with multiple pathways involved in strand break repair. tdp1 mutations can sensitize yeast cells to drugs targeting Top2 even when TOP1 is deleted. Finally, bacterially expressed yeast Tdp1p is able to remove a peptide derived from yTop2 that is covalently bound to DNA by a 5' phosphotyrosyl linkage. Our results show that Tdp1 plays more general roles in DNA repair than repair of Top1 mediated DNA damage, and may participate in repairing many types of base damage to DNA.

Modulation of α-catenin Tyr phosphorylation by SHP2 positively effects cell transformation induced by the constitutively active FGFR3

The Src homology 2 phosphotyrosyl phosphatase (SHP2) is a nonreceptor-type phosphatase that acts as a positive transducer of receptor Tyr kinase (RTK) signaling, particularly the Ras-REK and PI3K-Akt pathways. Recently, we have demonstrated that SHP2 is required for cell transformation induced by the constitutively active fibroblast growth factor receptor 3 (K/E-FR3) (Oncogene, 22, 6909-6918). In that study, we had detected a phosphotyrosyl protein of approximately 100 KDa (p100) in cells expressing dominant-negative SHP2 (R/E-SHP2), but its identity and relevance in SHP2-meditaed transformation was not known. Here, we report the identification of p100 as alpha-catenin, a vinculin-related protein involved in adherens junction-mediated intercellular adhesion. We show that alpha-catenin becomes Tyr phosphorylated in intercellular adhesion-dependent manner and this event is counteracted by SHP2. Substrate trapping in intact cells and immunocomplex phosphatse assays confirmed that alpha-catenin is in deed an SHP2 substrate. Tyr phosphorylation of alpha-catenin enhances its translocation to the plasma membrane and its interaction with beta-catenin, leading to enhanced actin polymerization and stabilization of adherens junction-mediated intercellular adhesion, a phenomenon commensurate with loss of the transformation phenotype. Site-directed mutagenesis studies also suggested that Tyr phosphorylation of alpha-catenin enhances its inhibitory role on cell transformation. Based on our previous work and the current report, we demonstrate that mediation of cell transformation by SHP2 is a complex process that involves modulation of the Ras-ERK and PI3K-Akt signaling pathways, intercellular adhesion, focal adhesion and actin cytoskeletal reorganization. To our knowledge, this is the first report showing regulation of alpha-catenin function by Tyr phosphorylation and its inhibitory effect on cell transformation.

Phospholipase D1 Regulates Cell Migration in a Lipase Activity-independent Manner

Cell migration, a complex biological process, requires dynamic cytoskeletal remodeling. Phospholipase D (PLD) generates phosphatidic acid, a lipid second messenger. Although PLD activity has been proposed to play a role in cytoskeletal rearrangement, the manner in which PLD participates in the rearrangement process remains obscure. In this study, by silencing endogenous PLD isozymes using small interfering RNA in HeLa cells, we demonstrate that endogenous PLD1 is required for the normal organization of the actin cytoskeleton, and, more importantly, for cell motility. PLD1 silencing in HeLa cells resulted in dramatic changes in cellular morphology, including the accumulation of stress fibers, as well as cell elongation and flattening, which appeared to be caused by an increased number of focal adhesions, which ultimately culminated in enhanced cell-substratum interactions. Accordingly, serum-induced cell migration was profoundly inhibited by PLD1-silencing. Moreover, the augmented cell substratum interaction and retarded cell migration induced by PLD1-silencing could be restored by the adding back not only of wild type, but also of lipase-inactive PLD1 into knockdown cells. Taken together, our results strongly suggest that endogenous PLD1 is a critical factor in the organization of the actin-based cytoskeleton, with regard to cell adhesion and migration. These effects of PLD1 appear to operate in a lipase activity-independent manner. We also discuss the regulation of Src family kinases by PLD1, as related to the modulation of Pyk2 and cell migration.