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Identification of a functional nuclear translocation sequence in hPPIP5K2.

Yong ST, Nguyen HN, Choi JH, Bortner CD, Williams J, Pulloor NK, Krishnan MN, Shears SB - BMC Cell Biol. (2015)

Bottom Line: 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.Mutagenic disruption of the candidate NLS in hPPIP5K2 reduced its degree of nuclear localization.These conclusions draw attention to nuclear compartmentation of PPIP5K2 as being a physiologically relevant and covalently-regulated event.

View Article: PubMed Central - PubMed

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.

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.

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Identification of phospho-S1006 in hPPIP5K2 by mass spectroscopy. Tandem MS spectrum of a doubly charged ion at m/z 698.7 corresponding to the phosphorylated form of the hPPIP5K2 peptide spanning residues 1006–1018 (SGEQITSSPVSPK). Extensive b- and y-ion series unambiguously identify this peptide and also localize the site of phosphorylation to the N-terminus of the peptide at S1006
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Fig6: Identification of phospho-S1006 in hPPIP5K2 by mass spectroscopy. Tandem MS spectrum of a doubly charged ion at m/z 698.7 corresponding to the phosphorylated form of the hPPIP5K2 peptide spanning residues 1006–1018 (SGEQITSSPVSPK). Extensive b- and y-ion series unambiguously identify this peptide and also localize the site of phosphorylation to the N-terminus of the peptide at S1006

Mentions: There are a number of examples of phosphorylation/dephosphorylation of proteins regulating their trafficking between the nucleus and cytoplasm [37]. For example, the addition of a negatively charged phosphate group close to an NLS can interfere with its electrostatic interactions with importins [38, 39]. In the case of the adenomatous polyposis coli protein [40], the function of its monpartite NLS is inhibited upon phosphorylation of a proximal Ser residue (i.e. in the context of PKKKRPS). A conserved Ser residue (S1006) is present immediately C-terminal of the NLS in hPPIP5K2 (i.e., RRRRRS; Fig. 1a). We therefore investigated if this Ser might be phosphorylated and impact the nuclear localization of hPPIP5K2. The FLAG-tagged PPIP5K2 that we expressed in HEK293T cells (see above), was pulled-down using anti-FLAG beads, purified by gel electrophoresis, and then interrogated for phosphorylated residues by nanoLC-ESI-MS and MS/MS. This approach identified phospho-Ser at position 1006 (Fig. 6).Fig. 6


Identification of a functional nuclear translocation sequence in hPPIP5K2.

Yong ST, Nguyen HN, Choi JH, Bortner CD, Williams J, Pulloor NK, Krishnan MN, Shears SB - BMC Cell Biol. (2015)

Identification of phospho-S1006 in hPPIP5K2 by mass spectroscopy. Tandem MS spectrum of a doubly charged ion at m/z 698.7 corresponding to the phosphorylated form of the hPPIP5K2 peptide spanning residues 1006–1018 (SGEQITSSPVSPK). Extensive b- and y-ion series unambiguously identify this peptide and also localize the site of phosphorylation to the N-terminus of the peptide at S1006
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4472268&req=5

Fig6: Identification of phospho-S1006 in hPPIP5K2 by mass spectroscopy. Tandem MS spectrum of a doubly charged ion at m/z 698.7 corresponding to the phosphorylated form of the hPPIP5K2 peptide spanning residues 1006–1018 (SGEQITSSPVSPK). Extensive b- and y-ion series unambiguously identify this peptide and also localize the site of phosphorylation to the N-terminus of the peptide at S1006
Mentions: There are a number of examples of phosphorylation/dephosphorylation of proteins regulating their trafficking between the nucleus and cytoplasm [37]. For example, the addition of a negatively charged phosphate group close to an NLS can interfere with its electrostatic interactions with importins [38, 39]. In the case of the adenomatous polyposis coli protein [40], the function of its monpartite NLS is inhibited upon phosphorylation of a proximal Ser residue (i.e. in the context of PKKKRPS). A conserved Ser residue (S1006) is present immediately C-terminal of the NLS in hPPIP5K2 (i.e., RRRRRS; Fig. 1a). We therefore investigated if this Ser might be phosphorylated and impact the nuclear localization of hPPIP5K2. The FLAG-tagged PPIP5K2 that we expressed in HEK293T cells (see above), was pulled-down using anti-FLAG beads, purified by gel electrophoresis, and then interrogated for phosphorylated residues by nanoLC-ESI-MS and MS/MS. This approach identified phospho-Ser at position 1006 (Fig. 6).Fig. 6

Bottom Line: 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.Mutagenic disruption of the candidate NLS in hPPIP5K2 reduced its degree of nuclear localization.These conclusions draw attention to nuclear compartmentation of PPIP5K2 as being a physiologically relevant and covalently-regulated event.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH