Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides

204

192

Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression. In vitro, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C␣ phosphorylated at T320. Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 was reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1͞PP-2A inhibitor, calyculin A. Immunof luorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase. Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions. Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.Protein phosphorylation is widely recognized as the major mechanism that controls cell cycle progression. A family of cyclin-dependent protein kinases (CDKs) have been identified and found to be enzymes critical for the initiation and completion of DNA replication and cell division from yeast to mammals (1-5). The activity of CDKs is modulated through phosphorylation of their catalytic subunits and by association with activating or inhibiting proteins. For example, mitotic transition is mediated by the cdc2 kinase͞cyclin B complex, and activated cdc2 kinase has been found to drive dramatic structural reorganizations in the nuclear envelope, spindle apparatus, and chromosomal DNA by phosphorylation of a variety of substrates including histone, Eg5, lamin, vimentin, and plectin (6-12).Evidence suggests that serine͞threonine protein phosphatases function as crucial regulators of cell proliferation (4, 13-15). In particular, protein phosphatase 1 (PP-1), which is highly conserved in all eukaryotes, has been found to play a pivotal role in the cell cycle. Genetic studies have indicated that mutation of the enzyme in yeast, Aspergillus, and in Drosophila leads to a variety of defects in mitosis (16-18). Microinjection of anti-PP-1 antibody into B cells before cell division arrests cells at metaphase, whereas injection of PP-1 into anaphase cells accelerates cytokinesis (19). Expression of inhibitor-2, a specific PP-1 inhibitor, changes during the cell cycle, peaking during S phase and mitosis (20). Thus, the tightly balanced activity of CDKs and PP-1 appears to be required for normal cell division. However, the substrates that are targets f...

Section: Methodsmentioning

confidence: 99%

Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase

Kwon

Lee

Choi

et al. 1997

204

192

“…To improve the efficiency of purification, we used an epitope tag that has been successfully used to purify a variety of transmembrane proteins, the 1D4 epitope from the C terminus of the G protein-coupled receptor rhodopsin (23,24). The nonapeptide epitope tag incorporated into the C terminus of TRPV1 allowed us to purify TRPV1 in a single step using an immobilized monoclonal antibody, 1-D4 (Fig.…”

Section: Overexpression and Purification Of Tetrameric Trpv1mentioning

confidence: 99%

Structure of TRPV1 channel revealed by electron cryomicroscopy

Moiseenkova-Bell

Stanciu

Serysheva

et al. 2008

189

176

The transient receptor potential (TRP) family of ion channels participate in many signaling pathways. TRPV1 functions as a molecular integrator of noxious stimuli, including heat, low pH, and chemical ligands. Here, we report the 3D structure of fulllength rat TRPV1 channel expressed in the yeast Saccharomyces cerevisiae and purified by immunoaffinity chromatography. We demonstrate that the recombinant purified TRPV1 channel retains its structural and functional integrity and is suitable for structural analysis. The 19-Å structure of TRPV1 determined by using singleparticle electron cryomicroscopy exhibits fourfold symmetry and comprises two distinct regions: a large open basket-like domain, likely corresponding to the cytoplasmic N-and C-terminal portions, and a more compact domain, corresponding to the transmembrane portion. The assignment of transmembrane and cytoplasmic regions was supported by fitting crystal structures of the structurally homologous Kv1.2 channel and isolated TRPV1 ankyrin repeats into the TRPV1 structure.cryoelectron microscopy ͉ ion channels ͉ TRP channels

“…The endocytic turnover of Rh1 can be followed using an inducible and uniquely tagged Rh1. Rh1-1D4 transgenic flies express a heat-shock inducible Drosophila Rh1 carrying a specific tag, 1D4, derived from the C terminus of bovine Rh1 (19). Rh1-1D4 transgene is functional, because it rescues the phenotypic defects in ninaE I17 (20,21).…”

Section: Ceramidase Facilitates Turnover Of Rh1 a Major Component Ofmentioning

confidence: 99%

Ceramidase expression facilitates membrane turnover and endocytosis of rhodopsin in photoreceptors

Acharya

Mowen²,

Nagashima³

et al. 2004

Transgenic expression of ceramidase suppresses retinal degeneration in Drosophila arrestin and phospholipase C mutants. Here, we show that expression of ceramidase facilitates the dissolution of incompletely formed and inappropriately located elements of rhabdomeric membranes in ninaE I17 mutants lacking the G protein receptor Rh1 in R1-R6 photoreceptor cells. Ceramidase expression facilitates the endocytic turnover of Rh1. Although ceramidase expression aids the removal of internalized rhodopsin, it does not affect the turnover of Rh1 in photoreceptors maintained in dark, where Rh1 is not activated and thus has a slower turnover and a long half-life. Therefore, the phenotypic consequence of ceramidase expression in photoreceptors is caused by facilitation of endocytosis. This study provides mechanistic insight into the sphingolipid biosynthetic pathway-mediated modulation of endocytosis and suppression of retinal degeneration.S phingolipids are integral components of eukaryotic cell membranes. They are essential for survival of yeast, Drosophila, and mammals. Sphingolipids have a hydrophobic backbone moiety, ceramide, linked to a variable polar head group, e.g., phosphorylcholine in sphingomyelin and a saccharide in glycosphingolipids. Ceramide itself consists of a long chain base linked to a fatty acid. Ceramide, sphingosine, and sphingosine-1-P are lipid second messengers modulating a variety of cellular functions (1-3). Ceramide is at the branch point of the sphingolipid biosynthetic pathway and serves as a substrate for several enzymes, e.g., sphingomyelin synthase, ceramidase, ceramide kinase, and glucosylceramide synthase. In Drosophila, transgenic expression of a neutral ceramidase is accompanied by a decrease in the levels of ceramide (4). We have shown that modulation of enzymes of the sphingolipid biosynthetic pathway suppressed retinal degeneration in certain phototransduction mutants (4). Transgenic expression of ceramidase or a loss of one copy of the rate-limiting enzyme in the sphingolipid biosynthetic pathway, namely, Serine Palmitoyl CoA transferase, suppressed retinal degeneration in arrestin and phospholipase C (norpA) mutants. These backgrounds also suppressed degeneration in a dynamin mutant, suggesting that ceramidase was modulating the endocytic pathway. To gain further insight into the mechanism of ceramidase action, we have investigated the effects of ceramidase expression in the rhodopsin null mutant, ninaE I17 . Rhodopsin, the G protein-coupled receptor, transduces light signal in Drosophila photoreceptors R1-R6 (5, 6). Rhodopsin is also involved in the formation of a rhabdomere terminal web (RTW), an actin-based cytoskeletal scaffold implicated in rhabdomere biogenesis (7,8). Rhabdomeres are densely organized, specialized areas of plasma membrane that house the signal transduction machinery of photoreceptors. In ninaE I17 mutants, the RTW is not organized during morphogenesis and thus photoreceptors have improperly formed rhabdomeres (8). In addition, in 3-dayold flies, rhabdomeric e...