To determine the function of E5 open reading frame (ORF) human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with ORF HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts transformed to colony formation in soft agar, transformation frequency was increased by epidermal growth factor (EGF) but not platelet-derived factor. In a transitory assay, ORFs both HPV-6 mitogenic primary foreskin epithelial cells (keratinocytes) acted...

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in keratinocytes and inhibits the degradation of EGF receptor endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is acidification endosomes. This may be mediated through 16-kDa component vacuolar proton-ATPase, since animal proteins bind this subunit protein. Using a ratio-imaging technique to...

In Caenorhabditis elegans, lin-2, lin-7, and lin-10 genetically interact to control the trafficking of Let-23 growth factor receptor basolateral surface body epithelia. The human homologue gene has recently been identified as a member X11 family. proteins contain one phosphotyrosine binding (PTB) two PSD-95.Dlg.ZO-1 (PDZ) domains well an extended amino terminus. We have previously shown that PTB domain X11alpha (also known Mint1) can bind amyloid precursor protein (APP) in...

Prior work in our laboratory established a connection between the PALS1/PATJ/CRB3 and Par6/Par3/aPKC protein complexes at tight junction of mammalian epithelial cells. Utilizing stable small interfering RNA expression system, we have markedly reduced junction-associated PALS1 MDCKII The loss resulted corresponding PATJ, known binding partner PALS1, but had no effect on CRB3. However, absence PATJ did result decreased association CRB3 with members complex. consequences were exhibited by delay...

The Type VI Secretion System (T6SS) functions in bacteria as a contractile nanomachine that punctures and delivers lethal effectors to target cell. Virtually nothing is known about the lifestyle or physiology dictates when normally produce their T6SS, which prevents clear understanding of how benefit from its action natural habitat. Proteus mirabilis undergoes characteristic developmental process coordinate multicellular swarming behavior will discriminate itself another isolate during...

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of Let-23 growth factor receptor to basolateral surface epithelial cells. It has been demonstrated that homologues Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins can bind amino terminus mLin-7 cloned genes encoding these using bacterial expression cloning. We call Pals, associated with Lin-7....

Mammalian Lin-2 (mLin-2)/CASK is a membrane-associated guanylate kinase (MAGUK) and contains multidomain modules that mediate protein-protein interactions important for the establishment maintenance of neuronal epithelial cell polarization.The importance mLin-2/CASK in mammalian development demonstrated by fact mutations or SAP97, another MAGUK protein, lead to cleft palate mice.We recently identified new interaction domain, called L27 which present twice mLin-2/CASK.In this report, we...

Human papillomavirus type 16 (HPV-16) is the most common genome found in human cervical cancers and its DNA capable of immortalizing primary keratinocytesin vitro.When expressed by their native promoter, two separate HPV-16 oncogenes, E6 E7, cooperate to immortalize keratinocytes. Early HPV lesions express abundant amounts E5-specific RNA, using a quantitative keratinocyte immortalization assay, we demonstrate here that E5 can actin cisto increase (4–10 fold) efficiency cellular E6/E7. These...

The GTPase Rab5a regulates the homotypic and heterotypic fusion of membranous organelles during early stages endocytosis. Many molecules which regulate cycle association with membranes, activation, deactivation dissociation are known. However, extent to these molecular scale activities coordinated on membranes affect behavior individual has not been determined. This study used novel Förster resonance energy transfer (FRET) microscopic methods analyze macropinosomes, large endocytic vesicles...

Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET was extended imaging of multiple donor-acceptor pairs, thereby enabling visualization biochemical events within single living cell. These methods require numerous equations that must be defined on case-by-case basis. Here, we present universal multispectral method...

Mammalian Lin-7 forms a complex with several proteins, including PALS1, that have role in polarity determination epithelial cells. In this study we found loss of protein from the polarized cell line Madin-Darby canine kidney II by small hairpin RNA results defects tight junction formation as indicated lowered transepithelial electrical resistance and mislocalization ZO-1 after calcium switch. The knock down also resulted expression binding partners, PALS1 PATJ. effects were rescued exogenous...

In Caenorhabditis elegans, the basolateral localization of Let-23 growth factor receptor tyrosine kinase requires expression three genes: lin-2, lin-7, and lin-10. Mammalian homologs these genes have been identified, a complex their protein products exists in mammalian neurons. this paper, we examine interaction proteins renal epithelia. Coprecipitation experiments demonstrated that mLin-2/CASK binds to mLin-7, immunofluorescent labeling showed colocalized at surface Madin-Darby canine...

To investigate the targeting mechanism for proteins bound to mammalian Lin-7 (mLin-7) PDZ domain, we created receptor protein chimeras composed of carboxyl-terminal amino acids LET-23 fused truncated nerve growth factor receptor/P75. mLin-7 chimera with a wild-type tail (P75t-Let23WT), but not mutant (P75t-Let23MUT). In Madin-Darby canine kidney (MDCK) cells, P75t-Let23WT localized basolateral plasma membrane whereas P75t-Let23MUT remained apical. Furthermore, constructs acted as dominant...

The transmembrane sialoglycoprotein podocalyxin is thought to be essential in the fine interdigitating foot process structure of podocyte. intracellular COOH-terminal amino acids Asp-Thr-His-Leu (DTHL) comprise a putative ligand for type I PSD95-Dlg-zona occludens-1 (PDZ) domain. A 20-amino acid synthetic peptide containing this motif was used screen cDNA library, and clones rabbit Na(+)/H(+) exchange regulatory factor-2 (NHERF-2) were obtained. In vitro analysis demonstrated that each PDZ...

Cyclic AMP (cAMP) regulates many biological processes and cellular functions. The importance of spatially localized intracellular gradients cAMP is increasingly appreciated. Previous work in macrophages has shown that produced during phagocytosis elevated levels suppress host defense functions, including generation proinflammatory mediators, killing. However, the spatial kinetic characteristics phagocytosing have yet to be examined. Using a Förster resonance energy transfer (FRET)-based...

Protein kinase C ε (PKC ) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKC was defined. Phosphopeptide mapping revealed that phosphorylates at T127 S188. Recombinant bound recombinant in absence of ATP indicating association does not require an active ATP-docked conformation. Activation resulted a dramatic coordinated translocation from cytoplasm membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis...

Fluorescence Resonance Energy Transfer (FRET) microscopy is a commonly-used technique to study problems in biophysics that range from uncovering cellular signaling pathways detecting conformational changes single biomolecules. Unfortunately, excitation and emission spectral overlap between the fluorophores create challenges quantitative FRET studies. It has been shown previously stoichiometry can be performed by selective of donor acceptor fluorophores. Extending this approach two-photon...

Abstract Accumulating evidence has clearly defined the importance of PKCε and small Rho GTPases in tumor metastasis. Previous studies our laboratory have demonstrated that signals through RhoA to modulate cell invasion motility head neck squamous carcinoma. In this study, we further delineated link between RhoA. Recombinant directly phosphorylated recombinant using an vitro kinase assay. Phosphopeptide mapping with LC-MSE identified T127 S188 as confident phosphorylation sites T19 S26...

We present quantitative pulse-shaping-based two-photon fluorescence resonance energy transfer microscopy. tailor the spectral phase of excitation pulses to achieve selective donor and acceptor, demonstrating method in live cells.