title: Phosphoinositide binding by the pleckstrin homology domains of Ipl and Tih1.
abstract: The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C delta1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the beta1-loop1-beta2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 are bona fide PH domain proteins, with broad specificity and moderate affinity for PIPs.
authors.affiliation: Institute for Cancer Genetics, Department of Pathology, Columbia University, 1150 St. Nicholas Avenue, New York, NY 10032, USA.
authors.forename: Anjana
authors.initials: A
authors.lastname: Saxena
authors.forename: Pavel
authors.initials: P
authors.lastname: Morozov
authors.forename: Dale
authors.initials: D
authors.lastname: Frank
authors.forename: Raymond
authors.initials: R
authors.lastname: Musalo
authors.forename: Mark A
authors.initials: MA
authors.lastname: Lemmon
authors.forename: Edward Y
authors.initials: EY
authors.lastname: Skolnik
authors.forename: Benjamin
authors.initials: B
authors.lastname: Tycko
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry*; D019556:COS Cells; D003593:Cytoplasm / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry*; D010802:Phylogeny; D010920:Placenta / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013696:Temperature; D020666:ras-GRF1 / Q000235:genetics
chemical_list: D001798:Blood Proteins; D004815:Epidermal Growth Factor; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D020666:ras-GRF1; C109277:TSSC3 protein; C039077:platelet protein P47