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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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The effects of mutating either the NLS or the adjacent S1006 upon the degree of nuclear localization of hPPIP5K2. Panel a shows Western analysis of extracts of HEK293T cells in which various hPPIP5K2-GFP constructs were expressed. For clarity, only four of the molecular markers are labeled. Each of the lanes transfected with a hPPIP5K-GFP construct showed a faint immunoreactive band that corresponded to the migration of GFP alone. ImageJ analysis indicated this represented <2% of the total signal, which is too small to compromise any of our conclusions. Panel b shows representative images of live HEK293T cells that expressed each of the various constructs. The size bar represents 10 μm. From each group, fifteen cells were randomly selected and FN / FC ratios were calculated (panel c); data represent means ± SEM (** p < 0.001, *p < 0.02 compared to wild-type (WT), determined using Student's t-test). Imaging flow cytometry was also used to analyze fixed and DRAQ5 stained HEK293T cells that were transfected with either d, hPPIP5K2-GFP (n = 2357; 57719 ± 1116 total fluorescence units); e, hPPIP5K2NLS-3A-GFP (n = 2179; 52386 ± 907 total fluorescence units); f, hPPIP5K2S1006A-GFP (n = 1686; 50383 ± 947 total fluorescence units); g, hPPIP5K2S1006D-GFP (n = 1696; 57991 ± 1162 total fluorescence units). The histograms depict the frequency of binned. Similarity Scores denoting the degree of co-localization of the GFP signal and nuclear stain, derived on a cell-by-cell basis. Best-fit Gaussian plots are also shown. The blue Gaussian plot for hPPIP5K2-GFP is superimposed upon the other plots for comparative purposes. Statistical differences in median similarity scores (“M”) compared to hPPIP5K2-GFP were determined using a Mann–Whitney Rank Sum Test. Note that the Western analysis includes an extract from cells transfected with a hPPIP5K2S1006E-GFP construct. The Similarity Score of the latter (not shown) was not significantly different from that of hPPIP5K2S1006D-GFP
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Fig7: The effects of mutating either the NLS or the adjacent S1006 upon the degree of nuclear localization of hPPIP5K2. Panel a shows Western analysis of extracts of HEK293T cells in which various hPPIP5K2-GFP constructs were expressed. For clarity, only four of the molecular markers are labeled. Each of the lanes transfected with a hPPIP5K-GFP construct showed a faint immunoreactive band that corresponded to the migration of GFP alone. ImageJ analysis indicated this represented <2% of the total signal, which is too small to compromise any of our conclusions. Panel b shows representative images of live HEK293T cells that expressed each of the various constructs. The size bar represents 10 μm. From each group, fifteen cells were randomly selected and FN / FC ratios were calculated (panel c); data represent means ± SEM (** p < 0.001, *p < 0.02 compared to wild-type (WT), determined using Student's t-test). Imaging flow cytometry was also used to analyze fixed and DRAQ5 stained HEK293T cells that were transfected with either d, hPPIP5K2-GFP (n = 2357; 57719 ± 1116 total fluorescence units); e, hPPIP5K2NLS-3A-GFP (n = 2179; 52386 ± 907 total fluorescence units); f, hPPIP5K2S1006A-GFP (n = 1686; 50383 ± 947 total fluorescence units); g, hPPIP5K2S1006D-GFP (n = 1696; 57991 ± 1162 total fluorescence units). The histograms depict the frequency of binned. Similarity Scores denoting the degree of co-localization of the GFP signal and nuclear stain, derived on a cell-by-cell basis. Best-fit Gaussian plots are also shown. The blue Gaussian plot for hPPIP5K2-GFP is superimposed upon the other plots for comparative purposes. Statistical differences in median similarity scores (“M”) compared to hPPIP5K2-GFP were determined using a Mann–Whitney Rank Sum Test. Note that the Western analysis includes an extract from cells transfected with a hPPIP5K2S1006E-GFP construct. The Similarity Score of the latter (not shown) was not significantly different from that of hPPIP5K2S1006D-GFP

Mentions: We used a mutagenic approach to investigate whether the phosphorylation status of S1006 impacts nuclear localization (Fig. 7a). For illustrative purposes, we also added to these experiments an analysis by traditional confocal microscopy. The small number of cells analyzed (n = 15 for each group; Fig. 7b,c) make these qualitative rather that quantitative observations. Nevertheless, these data show that the hPPIP5K2NLS-3A mutant exhibited significantly less nuclear localization than did wild-type enzyme. We also analyzed the distribution of a hPPIP5K2S1006A mutant in which S1006 was rendered non-phosphorylatable; this mutant showed a significantly higher degree of nuclear localization than did wild-type enzyme (Fig. 7b,c). Such data indicate that the phosphorylation of S1006 that we have observed (Fig. 6) normally restricts the degree of translocation of hPPIP5K into the nucleus. Nevertheless, as described above, a far more rigorous analysis can be accomplished by acquiring data from a large number of cells, in an automated manner that eliminates the possibility of investigator bias. We therefore quantified the nuclear localization of these mutants by Similarity Scores, obtained by imaging flow cytometry. We used fixed cells to facilitate larger sample sets.Fig. 7


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)

The effects of mutating either the NLS or the adjacent S1006 upon the degree of nuclear localization of hPPIP5K2. Panel a shows Western analysis of extracts of HEK293T cells in which various hPPIP5K2-GFP constructs were expressed. For clarity, only four of the molecular markers are labeled. Each of the lanes transfected with a hPPIP5K-GFP construct showed a faint immunoreactive band that corresponded to the migration of GFP alone. ImageJ analysis indicated this represented <2% of the total signal, which is too small to compromise any of our conclusions. Panel b shows representative images of live HEK293T cells that expressed each of the various constructs. The size bar represents 10 μm. From each group, fifteen cells were randomly selected and FN / FC ratios were calculated (panel c); data represent means ± SEM (** p < 0.001, *p < 0.02 compared to wild-type (WT), determined using Student's t-test). Imaging flow cytometry was also used to analyze fixed and DRAQ5 stained HEK293T cells that were transfected with either d, hPPIP5K2-GFP (n = 2357; 57719 ± 1116 total fluorescence units); e, hPPIP5K2NLS-3A-GFP (n = 2179; 52386 ± 907 total fluorescence units); f, hPPIP5K2S1006A-GFP (n = 1686; 50383 ± 947 total fluorescence units); g, hPPIP5K2S1006D-GFP (n = 1696; 57991 ± 1162 total fluorescence units). The histograms depict the frequency of binned. Similarity Scores denoting the degree of co-localization of the GFP signal and nuclear stain, derived on a cell-by-cell basis. Best-fit Gaussian plots are also shown. The blue Gaussian plot for hPPIP5K2-GFP is superimposed upon the other plots for comparative purposes. Statistical differences in median similarity scores (“M”) compared to hPPIP5K2-GFP were determined using a Mann–Whitney Rank Sum Test. Note that the Western analysis includes an extract from cells transfected with a hPPIP5K2S1006E-GFP construct. The Similarity Score of the latter (not shown) was not significantly different from that of hPPIP5K2S1006D-GFP
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: The effects of mutating either the NLS or the adjacent S1006 upon the degree of nuclear localization of hPPIP5K2. Panel a shows Western analysis of extracts of HEK293T cells in which various hPPIP5K2-GFP constructs were expressed. For clarity, only four of the molecular markers are labeled. Each of the lanes transfected with a hPPIP5K-GFP construct showed a faint immunoreactive band that corresponded to the migration of GFP alone. ImageJ analysis indicated this represented <2% of the total signal, which is too small to compromise any of our conclusions. Panel b shows representative images of live HEK293T cells that expressed each of the various constructs. The size bar represents 10 μm. From each group, fifteen cells were randomly selected and FN / FC ratios were calculated (panel c); data represent means ± SEM (** p < 0.001, *p < 0.02 compared to wild-type (WT), determined using Student's t-test). Imaging flow cytometry was also used to analyze fixed and DRAQ5 stained HEK293T cells that were transfected with either d, hPPIP5K2-GFP (n = 2357; 57719 ± 1116 total fluorescence units); e, hPPIP5K2NLS-3A-GFP (n = 2179; 52386 ± 907 total fluorescence units); f, hPPIP5K2S1006A-GFP (n = 1686; 50383 ± 947 total fluorescence units); g, hPPIP5K2S1006D-GFP (n = 1696; 57991 ± 1162 total fluorescence units). The histograms depict the frequency of binned. Similarity Scores denoting the degree of co-localization of the GFP signal and nuclear stain, derived on a cell-by-cell basis. Best-fit Gaussian plots are also shown. The blue Gaussian plot for hPPIP5K2-GFP is superimposed upon the other plots for comparative purposes. Statistical differences in median similarity scores (“M”) compared to hPPIP5K2-GFP were determined using a Mann–Whitney Rank Sum Test. Note that the Western analysis includes an extract from cells transfected with a hPPIP5K2S1006E-GFP construct. The Similarity Score of the latter (not shown) was not significantly different from that of hPPIP5K2S1006D-GFP
Mentions: We used a mutagenic approach to investigate whether the phosphorylation status of S1006 impacts nuclear localization (Fig. 7a). For illustrative purposes, we also added to these experiments an analysis by traditional confocal microscopy. The small number of cells analyzed (n = 15 for each group; Fig. 7b,c) make these qualitative rather that quantitative observations. Nevertheless, these data show that the hPPIP5K2NLS-3A mutant exhibited significantly less nuclear localization than did wild-type enzyme. We also analyzed the distribution of a hPPIP5K2S1006A mutant in which S1006 was rendered non-phosphorylatable; this mutant showed a significantly higher degree of nuclear localization than did wild-type enzyme (Fig. 7b,c). Such data indicate that the phosphorylation of S1006 that we have observed (Fig. 6) normally restricts the degree of translocation of hPPIP5K into the nucleus. Nevertheless, as described above, a far more rigorous analysis can be accomplished by acquiring data from a large number of cells, in an automated manner that eliminates the possibility of investigator bias. We therefore quantified the nuclear localization of these mutants by Similarity Scores, obtained by imaging flow cytometry. We used fixed cells to facilitate larger sample sets.Fig. 7

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
Related in: MedlinePlus