1. Volume 23, Issue 5, Pages 685-695 (September 2006)
Nuclear PtdIns5P as a Transducer of Stress Signaling: An In Vivo Role for PIP4Kbeta 
David R. Jones, Yvette Bultsma, Willem-Jan Keune, Jonathan R. Halstead, Dallila Elouarrat, Shabaz Mohammed, Albert J. Heck, Clive S. D'Santos, Nullin Divecha 
Molecular Cell 
Volume 23, Issue 5, Pages (September 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Nuclear PtdIns5P Increases in Response to Various Cellular Stressors
(A) Exponentially growing MEL cells were irradiated with UV light (200 J/m2) or with γ rays (10 Gy) and then analyzed 30 min later. Differentiated MEL cells were treated with H2O2 (500 μM) for 15 min or BSO (100 μM) or NAC (5 mM) for 4 hr prior to the isolation of cellular fractions, as indicated. A mass assay was used to determine the PtdIns5P content. The results are expressed as percent of control cells (16 pmol PtdIns5P/mg nuclear protein) and are calculated from three independent experiments, each performed in duplicate, and show the mean plus the standard deviation.
(B) Cells were treated as indicated. The CEF was then isolated, and its PtdIns5P content determined. The results are expressed as percent of control cells (4 pmol PtdIns5P/mg CEF protein) and are calculated from three independent experiments, each performed in duplicate, and show the mean plus the standard deviation. Inset, immunoblotting of subcellular fractions isolated from MEL cells. Correcting for cell equivalents reveals that the amount of actin in the mixed cytosol/membrane extract was nearly two orders of magnitude higher than that found in the nucleoplasm and CEF.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
PIP4K Activity Is Attenuated in Response to UV Irradiation in a p38-Dependent Manner
(A) MEL cells were subjected to UV irradiation before being returned to culture for the times indicated. Endogenous PIP4K was immunoprecipitated using a pan-PIP4K antibody, and PIP4K activity was determined in the immunoprecipitates. Data are triplicate samples from one experiment carried out at least three times.
(B) MEL cells were maintained as controls, subjected to UV irradiation, or treated with anisomycin (A, 10 μg/ml, 30 min). At the indicated times, cell lysates were prepared and subjected to immunoblotting using the indicated antibodies.
(C) MEL cells were either incubated with SB (1 μM) or DMSO (solvent control) for 30 min prior to being maintained either as controls or subjected to UV irradiation. Endogenous PIP4K was immunoprecipitated using a pan-PIP4K antibody, and PIP4K activity was determined in immunoprecipitates. The immunoblot shows endogenous PIP4K in total cell lysates. The results are calculated from three independent experiments, each performed in duplicate.
(D) MEL cells were either left untransfected or transfected with myc-PIP4Kbeta. After 40 hr, the cells were treated as indicated. Endogenous PIP4K and myc-PIP4Kbeta were immunoprecipitated from whole-cell lysates as indicated. PIP4K kinase activity in immunoprecipitates was determined. The results are calculated from three independent experiments, each performed in duplicate.
(E) MEL cells were transfected with myc-PIP4Kbeta before being treated as indicated. Myc-PIP4Kbeta activity was determined in immunoprecipitates. The immunoblot shows myc-PIP4Kbeta present in each immunoprecipitate. The results are calculated from three independent experiments, each performed in duplicate.
(F) HEK293 cells were transfected as indicated. Cell lysates were then subjected to immunoblotting using the antibodies indicated (left and right). Myc-PIP4Kbeta was immunoprecipitated, and its activity was determined (lower right) and quantitated in graphical form. Identical results were obtained in three independent experiments. In all cases, the graphical data show the mean plus the standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Ser326 of PIP4Kbeta Is Phosphorylated in Response to UV Irradiation
(A) HEK293 cells were transfected and irradiated with UV (100 J/m2) as indicated. Cell lysates were prepared 1 hr later. Purified HA-PIP4Kbeta was separated by SDS-PAGE and stained with Coomassie brilliant blue. Molecular weight markers are shown.
(B) Mass spectrometric analysis of HA-PIP4Kbeta from control cells. The triply charged [M+3H] peptide ion m/z corresponds to the phosphorylated peptide of residues 309–335 from HA-PIP4Kbeta. Y ions and b ions are shown. In addition, doubly charged fragment ions, when present, and the neutral loss of phosphate from the parent ion are indicated. The presence of a phosphate is confirmed by the identification of the fragment ion at m/z , the [M+3H] of the peptide minus the phosphate.
(C) Mass spectrometric analysis of HA-PIP4Kbeta from UV-irradiated cells. The mass spectrum shows the fragmentation of the triply charged, doubly phosphorylated peptide ion m/z 974.4, corresponding to residues 309–335 from HA-PIP4Kbeta. Y ions and b ions are indicated. In addition, doubly charged fragment ions, when present, and the neutral loss of phosphate from the parent ion are shown. Illustrated below (B) and (C) is the sequence of PIP4Kbeta that is phosphorylated at either Thr322 or Ser326. The sequence around Ser326 fits a consensus sequence for phosphorylation by a proline-directed MAPK. For a larger, color version of (B) and (C), see Figures S1 and S2.
(D) Purified HA-PIP4Kbeta from HEK293 cells was incubated for 30 min at 37°C in the absence or presence of CIP. Proteins were separated by SDS-PAGE and immunoblotted using the P-Ser326-PIP4K antibody (upper) and a total PIP4K antibody (lower).
(E) HEK293 cells were transduced with either pRetro (control cells) or pRetro containing RNAi sequences targeting the expression of p38 (pRetro-RNAi-p38). Cells were treated as indicated. Cell lysates were subjected to immunoblotting with the antibodies indicated.
(F) pRetro and pRetro-RNAi-p38 HEK293 cells were transiently transfected with HA-PIP4Kbeta or HA-S326A-PIP4Kbeta. After 36 hr, the cells were treated as indicated. HA-PIP4Kbeta was immunopurified, eluted, and subjected to immunoblotting with the antibodies indicated.
(G) pRetro and pRetro-RNAi-p38 HEK293 cells were transfected with myc-PIP4Kbeta. After 36 hr, the cells were treated as indicated. Myc-PIP4Kbeta was immunoprecipitated and was subjected to immunoblotting with the P-Ser326-PIP4K antibody and an antibody that recognizes total PIP4K. Densitometry was used to quantitate both Ser326 phosphorylation and total PIP4K. The data are plotted as the ratio of Ser326 phosphorylation to total PIP4Kbeta.
(H) Endogenous PIP4K was immunoprecipitated from control and UV-irradiated HEK293 cells. Ser326 phosphorylation was detected with the P-Ser326-PIP4K antibody (upper). Total PIP4K is also shown (lower).
(I) pRetro and pRetro-RNAi-p38 HEK293 cells were treated as indicated. After 30 min, cell lysates were prepared, which were subjected to immunoblotting with the antibodies indicated.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Ser326 Is Directly Phosphorylated by the Stress-Activated Kinase p38
(A) Purified recombinant proteins were used in an in vitro kinase assay as indicated. GST-p38 autophosphorylates and is apparent in the upper panel (∼65 kDa, running just below GST-PIP4Kbeta). Similar results were obtained in two identical experiments.
(B) GST-p38 preincubated with SB (1 μM) or DMSO was used to phosphorylate wild-type GST-PIP4Kbeta fusion protein with unlabeled ATP. A follow-on PtdIns5P 4-kinase assay was then performed. The upper panel shows the amount of labeled PtdIns(4,5)P2 generated, which was quantified graphically below.
(C) GST-PIP4Kbeta (wild-type and mutants as indicated) was incubated with GST-p38 and [32P]γATP. The panel shows the autorad (upper) and the Coomassie blue-stained dried gel (lower). The arrow in both panels indicates the position of GST-PIP4Kbeta (∼78 kDa). Similar results were obtained in two identical experiments.
(D) GST-p38 was preincubated with SB (1 μM) or DMSO before incubation with GST-PIP4Kbeta (wild-type or mutants as indicated) and unlabeled ATP. A follow-on PtdIns5P 4-kinase assay was performed and quantified as described in (B). The graphical results indicate the percent of PtdIns5P-kinase activity in DMSO compared to incubation with SB Similar results were found in an identical experiment performed in duplicate.
(E) Both GST-PIP4Kbeta and GST-S326A-PIP4Kbeta were incubated with recombinant p38 (as indicated) in the presence of unlabeled ATP for 30 min. Proteins were subjected to immunoblotting to detect Ser326 phosphorylation (upper), p38 (middle), and total PIP4K (lower). In all cases, the graphical data show the mean plus the standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 PIP4Kbeta Regulates the Level of Nuclear PtdIns5P
(A) MEL cells were preincubated and treated as shown. The mass of PtdIns5P in the CEF was determined. Data shown are from duplicate samples from one experiment that was performed twice.
(B) MEL cells expressing EGFP or EGFP-PIP4Kbeta were irradiated as indicated. Expressed proteins were detected in intact nuclei using an antibody against EGFP (indicated by arrows). In a parallel experiment, the CEF was isolated and the mass of PtdIns5P (expressed as percent of nonirradiated cells) was determined. The results are calculated from three independent experiments.
(C) MEL cells were transfected with either an empty (control) RNAi vector or an RNAi vector containing sequences targeting PIP4Kbeta (RNAi-PIP4Kbeta). After 40 hr, intact nuclei were isolated from the cells. The level of endogenous PIP4Kbeta was determined by immunoblotting using a specific antibody (left). The mass level of PtdIns5P in the CEF was determined (right, expressed as percent of control RNAi vector-transfected cells). Identical results were obtained in three identical experiments. In all cases, the graphical data show the mean plus the standard deviation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

7. Figure 6
ING2 Is an Endogenous Sensor for Changes in the Level of Nuclear PtdIns5P
(A) Exponentially growing MEL cells and differentiated MEL cells were treated as indicated. Endogenous ING2 and histone 4 were detected in the CEF by immunoblotting (left and middle). Right, exponentially growing MEL cells were treated as indicated. Endogenous ING2 and actin were detected in total cell lysates by immunoblotting.
(B) MEL cells were treated as indicated. ING2 and histone 4 were detected in the isolated CEFs by immunoblotting.
(C) (Left) MEL cells expressing EGFP or EGFP-PIP4Kbeta were treated as indicated. Endogenous ING2 and histone 4 were detected in the isolated CEFs by immunoblotting. (Right) MEL cells were transfected with either an empty (control) RNAi vector or an RNAi vector containing sequences targeting PIP4Kbeta (RNAi-PIP4Kbeta). After 40 hr, the CEF was isolated. ING2 and histone 4 were detected in the isolated CEFs by immunoblotting.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

8. Figure 7
A Proposed Model of How Cellular Stressors Modulate the Level of PtdIns5P
Stressors cause the activation of the p38 pathway (via upstream activators such as MKK6). This leads to the phosphorylation of PIP4Kbeta at Ser326, causing inhibition of its lipid kinase activity (represented by the size of PIP4Kbeta). As a consequence, the level of nuclear PtdIns5P increases (as its constitutive conversion [dotted line] to PtdIns(4,5)P2 is attenuated). PHD finger-containing proteins (including ING2) “sense” the increased level of nuclear PtdIns5P, which may act as a cofactor to regulate their function.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions