title: Identification of a functional nuclear translocation sequence in hPPIP5K2.
abstract: BACKGROUND
  Cells contain several inositol pyrophosphates (PP-InsPs; also known as diphosphoinositol polyphosphates), which play pivotal roles in cellular and organismic homeostasis. It has been proposed that determining mechanisms of compartmentation of the synthesis of a particular PP-InsP is key to understanding how each of them may exert a specific function. Human PPIP5K2 (hPPIP5K2), one of the key enzymes that synthesizes PP-InsPs, contains a putative consensus sequence for a nuclear localization signal (NLS). However, such in silico analysis has limited predictive power, and may be complicated by phosphorylation events that can dynamically modulate NLS function. We investigated if this candidate NLS is functional and regulated, using the techniques of cell biology, mutagenesis and mass spectrometry.
  
  
  RESULTS
  Multiple sequence alignments revealed that the metazoan PPIP5K2 family contains a candidate NLS within a strikingly well-conserved 63 amino-acid domain. By analyzing the distribution of hPPIP5K2-GFP in HEK293T cells with the techniques of confocal microscopy and imaging flow cytometry, we found that a distinct pool of hPPIP5K2 is present in the nucleus. Imaging flow cytometry yielded particular insight into the characteristics of the nuclear hPPIP5K2 sub-pool, through a high-throughput, statistically-robust analysis of many hundreds of cells. Mutagenic disruption of the candidate NLS in hPPIP5K2 reduced its degree of nuclear localization. Proximal to the NLS is a Ser residue (S1006) that mass spectrometry data indicate is phosphorylated inside cells. The degree of nuclear localization of hPPIP5K2 was increased when S1006 was rendered non-phosphorylatable by its mutation to Ala. Conversely, a S1006D phosphomimetic mutant of hPPIP5K2 exhibited a lower degree of nuclear localization.
  
  
  CONCLUSIONS
  The current study describes for the first time the functional significance of an NLS in the conserved PPIP5K2 family. We have further demonstrated that there is phosphorylation of a Ser residue that is proximal to the NLS of hPPIP5K2. These conclusions draw attention to nuclear compartmentation of PPIP5K2 as being a physiologically relevant and covalently-regulated event. Our study also increases general insight into the consensus sequences of other NLSs, the functions of which might be similarly regulated.
authors.affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 101 T.W. Alexander Drive, Research Triangle Park, NC, 27709, USA. yongst.work@gmail.com.
authors.forename: Sheila T
authors.initials: ST
authors.lastname: Yong
authors.affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 101 T.W. Alexander Drive, Research Triangle Park, NC, 27709, USA. hoainghia.nguyen@nih.gov.
authors.forename: Hoai-Nghia
authors.initials: HN
authors.lastname: Nguyen
authors.affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 101 T.W. Alexander Drive, Research Triangle Park, NC, 27709, USA. jae.choi@thermofisher.com.|Current address: Thermo Fisher Scientific, LSG/Biosciences Division, 3747 N. Meridian Drive, Rockford, IL, 61101, USA. jae.choi@thermofisher.com.
authors.forename: Jae H
authors.initials: JH
authors.lastname: Choi
authors.affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 101 T.W. Alexander Drive, Research Triangle Park, NC, 27709, USA. bortner@niehs.nih.gov.
authors.forename: Carl D
authors.initials: CD
authors.lastname: Bortner
authors.affiliation: Protein Microcharacterization Core Facility, Mass Spectrometry Group, National Institute of Environmental Health Sciences, National Institutes of Health, 101 T.W. Alexander Drive, Research Triangle Park, NC, 27709, USA. willia56@niehs.nih.gov.
authors.forename: Jason
authors.initials: J
authors.lastname: Williams
authors.affiliation: Program on Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, 8 College Road, Singapore, 169857, Republic of Singapore. niyas@duke-nus.edu.sg.
authors.forename: Niyas K
authors.initials: NK
authors.lastname: Pulloor
authors.affiliation: Program on Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, 8 College Road, Singapore, 169857, Republic of Singapore. manoj.krishnan@duke-nus.edu.sg.
authors.forename: Manoj N
authors.initials: MN
authors.lastname: Krishnan
authors.affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, 101 T.W. Alexander Drive, Research Triangle Park, NC, 27709, USA. shears@niehs.nih.gov.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Shears
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017856:Phosphotransferases (Phosphate Group Acceptor); C562140:PPIP5K2 protein, human