title: Phosphorylation of ORF1p is required for L1 retrotransposition.
abstract: Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for ∼100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host.
authors.affiliation: Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0850.
authors.forename: Pamela R
authors.initials: PR
authors.lastname: Cook
authors.affiliation: Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0850.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Jones
authors.affiliation: Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0850 avf@helix.nih.gov.
authors.forename: Anthony V
authors.initials: AV
authors.lastname: Furano
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002853:Chromatography, Liquid; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007313:Insecta; D020084:Long Interspersed Nucleotide Elements; D008969:Molecular Sequence Data; D009154:Mutation; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D016366:Open Reading Frames; D019696:Peptidylprolyl Isomerase / Q000737:chemistry*; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D018626:Retroelements / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000737:chemistry
chemical_list: D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D018626:Retroelements; D014764:Viral Proteins; D012313:RNA; D011392:Proline; D005982:Glutathione Transferase; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase
keywords: LINE-1; Pin1; peptidyl prolyl isomerase 1; proline-directed protein kinase; retrotransposon
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