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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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Use of affinity pull-downs and mass spectroscopy to identify Importin-5 and Importin-8 as proteins interacting with hPPIP5K2. Panel a shows SDS-PAGE analysis of pull-downs of FLAG-beta-galactosidase (lane a) and FLAG-hPPIP5K2 (lane c), with molecular weight markers (lane b); see the Methods Section for further details. The entire gel lanes (a and c) were divided into 24 equal sections, each of which were then digested, lyophilized, and resuspended in 40 μl of 0.1% formic acid, prior to analysis. Panel b shows that importin-5 was identified in 13 spectra from 7 unique peptides (red font) and a distinct summed MS/MS search score of 91.06, yielding 8.2% sequence coverage. Panel c shows that importin-8 was identified by 6 spectra from 5 unique peptides (red font) and a distinct summed MS/MS search score of 68.63 with 5.9% sequence coverage. The individual spectra are provided in Additional file 2: Figures S1 and S2
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Fig3: Use of affinity pull-downs and mass spectroscopy to identify Importin-5 and Importin-8 as proteins interacting with hPPIP5K2. Panel a shows SDS-PAGE analysis of pull-downs of FLAG-beta-galactosidase (lane a) and FLAG-hPPIP5K2 (lane c), with molecular weight markers (lane b); see the Methods Section for further details. The entire gel lanes (a and c) were divided into 24 equal sections, each of which were then digested, lyophilized, and resuspended in 40 μl of 0.1% formic acid, prior to analysis. Panel b shows that importin-5 was identified in 13 spectra from 7 unique peptides (red font) and a distinct summed MS/MS search score of 91.06, yielding 8.2% sequence coverage. Panel c shows that importin-8 was identified by 6 spectra from 5 unique peptides (red font) and a distinct summed MS/MS search score of 68.63 with 5.9% sequence coverage. The individual spectra are provided in Additional file 2: Figures S1 and S2

Mentions: NLSs interact with α/β-importin complexes that shuttle many nuclear proteins into the nucleus immediately after their translation [31]. Other proteins - particularly those that participate in cell signaling - may not have a predominantly nuclear localization by default, but instead have their nuclear delivery regulated by certain β-importins that act independently of α-importin [32]. Thus, the identification of importins that associate with novel cargoes can be functionally illuminating [33]. We searched for importins that might associate with hPPIP5K2 by using a subtractive proteomic approach: either FLAG-tagged hPPIP5K2, or an epitope control, FLAG-tagged β-galactosidase, were stably expressed in HEK293T cells. Lysates were prepared, and incubated with immobilized anti-FLAG antibodies to pull-down the respective baits and associated proteins (Fig. 3a), which were then identified by mass-spectrometry (Fig. 3b,c; Additional file 2: Figures S1 and S2). Importin-5 was identified in 13 spectra from 7 unique peptides yielding 8.2% sequence coverage (Fig. 3b). Importin-8 was identified by 6 spectra from 5 unique peptides with 5.9% sequence coverage (Fig. 3c). Neither of these importins were identified in the control pull-downs with FLAG-galactosidase. Importin-5 and importin-8 are members of the β-importin family that are receiving increasing attention for regulating nuclear delivery of certain signaling proteins [32]. These data are consistent with the conclusion (see above) that hPPIP5K2 has some capacity to undergo nuclear translocation.Fig. 3


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)

Use of affinity pull-downs and mass spectroscopy to identify Importin-5 and Importin-8 as proteins interacting with hPPIP5K2. Panel a shows SDS-PAGE analysis of pull-downs of FLAG-beta-galactosidase (lane a) and FLAG-hPPIP5K2 (lane c), with molecular weight markers (lane b); see the Methods Section for further details. The entire gel lanes (a and c) were divided into 24 equal sections, each of which were then digested, lyophilized, and resuspended in 40 μl of 0.1% formic acid, prior to analysis. Panel b shows that importin-5 was identified in 13 spectra from 7 unique peptides (red font) and a distinct summed MS/MS search score of 91.06, yielding 8.2% sequence coverage. Panel c shows that importin-8 was identified by 6 spectra from 5 unique peptides (red font) and a distinct summed MS/MS search score of 68.63 with 5.9% sequence coverage. The individual spectra are provided in Additional file 2: Figures S1 and S2
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Related In: Results  -  Collection

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Fig3: Use of affinity pull-downs and mass spectroscopy to identify Importin-5 and Importin-8 as proteins interacting with hPPIP5K2. Panel a shows SDS-PAGE analysis of pull-downs of FLAG-beta-galactosidase (lane a) and FLAG-hPPIP5K2 (lane c), with molecular weight markers (lane b); see the Methods Section for further details. The entire gel lanes (a and c) were divided into 24 equal sections, each of which were then digested, lyophilized, and resuspended in 40 μl of 0.1% formic acid, prior to analysis. Panel b shows that importin-5 was identified in 13 spectra from 7 unique peptides (red font) and a distinct summed MS/MS search score of 91.06, yielding 8.2% sequence coverage. Panel c shows that importin-8 was identified by 6 spectra from 5 unique peptides (red font) and a distinct summed MS/MS search score of 68.63 with 5.9% sequence coverage. The individual spectra are provided in Additional file 2: Figures S1 and S2
Mentions: NLSs interact with α/β-importin complexes that shuttle many nuclear proteins into the nucleus immediately after their translation [31]. Other proteins - particularly those that participate in cell signaling - may not have a predominantly nuclear localization by default, but instead have their nuclear delivery regulated by certain β-importins that act independently of α-importin [32]. Thus, the identification of importins that associate with novel cargoes can be functionally illuminating [33]. We searched for importins that might associate with hPPIP5K2 by using a subtractive proteomic approach: either FLAG-tagged hPPIP5K2, or an epitope control, FLAG-tagged β-galactosidase, were stably expressed in HEK293T cells. Lysates were prepared, and incubated with immobilized anti-FLAG antibodies to pull-down the respective baits and associated proteins (Fig. 3a), which were then identified by mass-spectrometry (Fig. 3b,c; Additional file 2: Figures S1 and S2). Importin-5 was identified in 13 spectra from 7 unique peptides yielding 8.2% sequence coverage (Fig. 3b). Importin-8 was identified by 6 spectra from 5 unique peptides with 5.9% sequence coverage (Fig. 3c). Neither of these importins were identified in the control pull-downs with FLAG-galactosidase. Importin-5 and importin-8 are members of the β-importin family that are receiving increasing attention for regulating nuclear delivery of certain signaling proteins [32]. These data are consistent with the conclusion (see above) that hPPIP5K2 has some capacity to undergo nuclear translocation.Fig. 3

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