Collaboration of AMPK and PKC to induce phosphorylation of Ser413 on PIP5K1B resulting in decreased kinase activity and reduced PtdIns(4,5)P 2 synthesis in response to oxidative stress and energy restriction Iman VAN DEN BOUT*, David R. JONES*, Zahid H. SHAH*, Jonathan R. HALSTEAD†, Willem-Jan KEUNE*, Shabaz MOHAMMED‡, Clive S. D’SANTOS§ and Nullin DIVECHA*1 *Inositide laboratory, The Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, U.K., †Syngenta Cereals, Syngenta, 4006 Hawthorne Circle, Longmont, CO, U.S.A., ‡Biomolecular Mass Spectrometry and Proteomics Group, Padualaan 8, Utrecht, 3584 CH, The Netherlands, and §Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, U.K.

The spatial and temporal regulation of the second messenger PtdIns(4,5)P2 has been shown to be crucial for regulating numerous processes in the cytoplasm and in the nucleus. Three isoforms of PIP5K1 (phosphatidylinositol 4-phosphate 5-kinase), A, B and C, are responsible for the regulation of the major pools of cellular PtdIns(4,5)P2 . PIP5K1B is negatively regulated in response to oxidative stress although it remains unclear which pathways regulate its activity. In the present study, we have investigated the regulation of PIP5K1B by protein phosphorylation. Using MS analysis, we identified 12 phosphorylation sites on PIP5K1B. We developed a phospho-specific antibody against Ser413 and showed that its phosphorylation was increased in response to treatment of cells with phorbol ester, H2 O2 or energy restriction.

Using inhibitors, we define a stress-dependent pathway that requires the activity of the cellular energy sensor AMPK (AMPactivated protein kinase) and PKC (protein kinase C) to regulate Ser413 phosphorylation. Furthermore, we demonstrate that PKC can directly phosphorylate Ser413 in vitro. Mutation of Ser413 to aspartate to mimic serine phosphorylation decreased both PIP5K1B activity in vitro and PtdIns(4,5)P2 synthesis in vivo. Our studies show that collaboration between AMPK and PKC dictates the extent of Ser413 phosphorylation on PIP5K1B and regulates PtdIns(4,5)P2 synthesis.

INTRODUCTION

neurite formation [6,7] and has been shown to be regulated in response to oxidative stress [8,9]. The regulation of PIP5K1B is complex and still poorly understood. It can interact with numerous proteins including the small GTPases Rac, Rho and Arf6, and PLD (phospholipase D), all of which can regulate its activity and/or its localization [5]. PIP5K1B is also regulated by phosphorylation. For instance, Ser214 is phosphorylated by PKA (protein kinase A) which results in decreased lipid kinase activity [10]. Oxidative stress induced by exposure to H2 O2 causes a decrease in cellular PtdIns(4,5)P2 levels as a result of reduced PIP5K1B kinase activity and its delocalization away from the plasma membrane where its substrate resides [8,9]. The decrease in PtdIns(4,5)P2 appears to be important in initiating cell death in response to oxidative stress as the overexpression of PIP5K1B can protect cells from apoptosis [9]. How PIP5K1B is regulated in response to oxidative stress is not clear although enhanced tyrosine phosphorylation by the protein tyrosine kinase Syk has been implicated in regulating both PIP5K1B activity and localization [8]. Cellular stressors also activate a host of stress-responsive kinases including AMPK (AMP-activated protein kinase) [11,12]. AMPK activation in response to cellular stress is dependent on the activation of upstream kinases such as LKB1 (liver kinase B1) and CaMKK1β (calcium/calmodulin-dependent protein kinase kinase 1β) and on its allosteric activation by AMP [11].

Cells are constantly under attack by damaging agents present in the extracellular matrix and by toxic reactive oxygen species generated as by-products of metabolic processes within the cell. These cellular stressors can cause DNA damage, lipid peroxidation and protein modification, which if not dealt with eventually lead to cell death. Cells also experience an ever-changing environment in terms of their accessibility to nutrients and depend on numerous pathways to detect cellular damage or changes in metabolic status to initiate response pathways to combat these insults. The phosphoinositide PtdIns(4,5)P2 is a lipid second messenger present in the plasma membrane and in the nucleus that has emerged as a critical signalling molecule controlling responses to cellular stress. The PIP5K (phosphatidylinositol 4-phosphate 5-kinase) family is responsible for the synthesis of PtdIns(4,5)P2 and comprises three catalytically active isoforms designated PIP5K1A, PIP5K1B and PIP5K1C. The maintenance of PtdIns(4,5)P2 levels by PIP5K has been shown to be essential for viability as deletion of the gene for the sole PIP5K homologue in both Saccharomyces cerevisiae and Caenorhabditis elegans is lethal [1,2]. In mammalian cells, PIP5K isoforms have been shown to regulate vesicle trafficking, actin cytoskeleton remodelling, ion balance and calcium homoeostasis, and their deregulation has been implicated in human diseases [3– 5]. PIP5K1B has been implicated in focal adhesion turnover and

www.biochemj.org

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doi:10.1042/BJ20130259

Key words: energy restriction, oxidative stress, phosphatidylinositol 4-phosphate 5-kinase, phosphoinositide.

Abbreviations used: ACC, acetyl-CoA carboxylase; AICAR, 5-amino-4-imidazolecarboxamide riboside; AMPK, AMP-activated protein kinase; DMEM, Dulbecco’s modified Eagle’s medium; FTICR, Fourier-transform ion cyclotron resonance; HEK, human embryonic kidney; HRP, horseradish peroxidase; PH, pleckstrin homology; PIP5K, phosphatidylinositol 4-phosphate 5-kinase; PKB, protein kinase B; PKC, protein kinase C; PLC, phospholipase C; PS, phosphatidylserine; TUP, theoretical upper phase; WT, wild-type. 1 To whom correspondence should be addressed (email [email protected]).  c The Authors Journal compilation  c 2013 Biochemical Society

Biochemical Journal

Biochem. J. (2013) 455, 347–358 (Printed in Great Britain)

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I. van den Bout and others

Essentially AMPK acts as a sensor for the energy status of the cell and, by rewiring the activity of metabolic enzymes and transcription programmes, AMPK activation reduces ATP consumption and induces ATP generation [13,14]. PKC (protein kinase C) is another kinase family intimately involved in signalling downstream of oxidative stress. PKCs are a family of serine/threonine kinases, which are involved in the regulation of diverse functions such as proliferation, differentiation and cell death [15]. Notably, PKCδ has been implicated in regulating processes downstream of cellular stress-induced pathways [16–19]. Using MS, we identified a number of potential phosphorylation sites on PIP5K1B. The sites include tyrosine and serine/threonine sites of which the serine/threonine sites mostly cluster in a small region in the C-terminus of the protein. Most of these sites are not conserved between PIP5K isoforms, suggesting that they play a role in isoform-specific regulation. We focused our investigation on Ser413 and show that Ser413 phosphorylation regulates PIP5K1B activity in response to stress activation. MATERIALS AND METHODS Chemicals

The AMPK inhibitor compound C was obtained from Merck, AICAR (5-amino-4-imidazolecarboxamide riboside) was from Tocris, and the PKC inhibitor G¨o6983, PMA, glucose-free DMEM (Dulbecco’s modified Eagle’s medium) and phosphatefree DMEM were from Sigma–Aldrich. Cell lines

HeLa and HEK (human embryonic kidney)-293 cells were obtained from A.T.C.C. (Manassas, VA, U.S.A.) and cultured in DMEM containing 10 % fetal bovine serum, glutamine, 100 unit/ml penicillin and 0.1 mg/ml streptomycin. Doxycycline-inducible HeLa cell lines were established by stably expressing the pRetroX-Tet-On-Advanced (Clontech) and the pRetroX-Tight-Pur (Clontech) plasmid containing the WT (wild-type) or mutant forms of murine PIP5K1B. Plasmids

GFP and Myc-tagged versions of murine PIP5K1B have been described previously [11]. The S413A and S413D mutants were made by site-directed mutagenesis using PHUSION polymerase (Finnzyme). WT, S413A and S413D PIP5K1B constructs were cloned into pRetrox-Tight using a partial EcoRI digest. Mass spectrometry Sample preparation

HeLa cells were transfected with GFP-tagged murine PIP5K1B. After 20 h, cells were lysed with 1 % Nonidet P40, 50 mM Tris, pH 8, 10 mM EDTA, 50 mM KCl, 20 mM orthovanadate and 50 mM NaF. Cell lysates were centrifuged (20 000 g for 5 min) to remove nuclear debris and the supernatant was immunoprecipitated using the anti-GFP polyclonal antibody for 16 h at 4 ◦ C. Protein G–Sepharose beads were added for 1 h and subsequently washed three times with lysis buffer. Nupage LDS gel loading buffer was added to the beads and samples were boiled and separated by SDS/PAGE (4–12 % gradient gel). Gel digestions were performed as described previously [10] with some modifications. Briefly, after colloidal Coomassie Blue staining the protein bands were cut into pieces and after several washes the gel pieces were submitted to a reduction step using  c The Authors Journal compilation  c 2013 Biochemical Society

10 mM DTT in 100 mM NH4 HCO3 buffer at 56 ◦ C for 45 min. Alkylation was performed with a solution of 55 mM iodacetamide in 100 mM NH4 HCO3 for 30 min at room temperature (20 ◦ C) and the first digestion was performed with 7 μg/ml trypsin in 50 mM NH4 HCO3 at 37 ◦ C overnight. Subsequently, V8 protease was added and the resultant mixture was incubated for 4 h at room temperature. Finally, the mixture was acidified by adding 1 volume of 5 % formic acid. LC-MS/MS analysis

Nano-HPLC-MS/MS (nanoscale HPLC-MS/MS) experiments were performed on an Agilent 1100 nanoflow system connected to a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron) equipped with a nanoelectrospray ion source. Loading was accomplished by using a flow rate of 5 μl/min on to a homemade 2-cm-fused silica pre-column [100 μm I.D. (inner diameter), 375 μm O.D. (outer diameter), Reprosil C18 -AQ, 3 μm using autosampler]. Sequential elution of peptides was accomplished using a linear gradient from solution A (0.6 % acetic acid) to 50 % of solution B (80 % acetonitrile and 0.5 % acetic acid) in 40 min over the pre-column in line with a homemade 20–25-cm resolving column (50 μm I.D., 375 μm O.D., Reprosil C18 -AQ, 3 μm). The mass spectrometer was operated in the data-dependent mode or automatically switched between MS and MS/MS acquisition. Survey full-scan MS spectra (from m/z 300–1500) were acquired using FTICR (Fourier-transform ion cyclotron resonance) with resolution R = 25 000 at m/z 400 (after accumulation to a target value of 5×106 in the linear ion trap). The three most intense ions were sequentially isolated for accurate mass measurements by a FTICR ‘SIM scan’ which consisted of 15 Da mass range, R = 50 000 and target accumulation value of 8×104 . These were then simultaneously fragmented in the linear ion trap using collision-induced dissociation at a target value of 1×104 . All MS/MS spectra files from each LC run were centroided and merged to a single file using bioworks 3.2 (Thermo Electron), which was searched using the Mascot Search Engine (version 2.3, Matrix Science) against the Human SwissProt database (June 2012) containing additionally the PIP5K1B mouse sequence (20 238 non-redundant proteins including an equivalent number of decoy sequences) with a carbamidomethyl cysteine residue as a fixed modification. Oxidized methionine and phosphorylation (serine, threonine and tyrosine residues) were searched as variable modifications. Searches were performed with tryptic/V8 specificity allowing nine miscleavages and an initial tolerance on mass measurement of 100 p.p.m. in MS mode and 0.9 Da for MS/MS ions. A mascot score of 40 corresponds to P < 0.05. The subsequently acquired .dat file has been converted into a scaffold file which is available from N.D. on request. Antibodies Generation of phospho-specific antibodies

Peptides containing the phosphorylated epitopes for Ser413 (P8: CSKKRCNpSIAALKAT) were coupled with keyhole-limpet haemocyanin and used to immunize New Zealand white rabbits (carried out by Sanquin Blood Supply). The rabbit antiserum obtained was negatively selected using non-phosphorylated peptides and tested by ELISA. Indicated peptides were coated overnight on a 96-well ELISA plate (2 μg/50 μl per well), blocked with 1 % BSA in PBS and incubated with antiserum at the indicated dilutions. Wells were incubated with HRP (horseradish peroxidase)-conjugated secondary swine anti-rabbit antibody (Dako) for 1 h followed by 15 min incubation with TMB (3,3 5,5 -tetramethylbenzidine) substrate. Reactions were stopped

AMPK and PKC regulate PIP5K1B phosphorylation and PtdIns(4,5)P 2 synthesis

by adding 2 M H2 SO4 and the attenuance was read at 450 nm using a plate reader. Other antibodies

An antibody recognizing PIP5K1B was generated by immunizing rabbits as described above with a coupled peptide (LEEGTIYLTAEPNTLD) and obtaining the rabbit antiserum. Phospho-ACC (acetyl-CoA carboxylase) and total ACC antibodies were obtained from Cell Signaling Technology. Western blotting

Cells were washed in PBS before being lysed in Nonidet P40 buffer containing 50 mM Tris, pH 8, 50 mM KCl, 10 mM EDTA, 1 % Nonidet P40, phosphatase inhibitors sodium orthovanadate and sodium fluoride, and protease inhibitor cocktail (CompleteTM EDTA free, Roche). After centrifugation (20 000 g for 5 min), lysates were denatured using DTT and separated by SDS/PAGE. Proteins were blotted on to nitrocellulose membrane and blocked with 5 % BSA before being incubated with primary antibody overnight followed by three washes in PBS-Tween 20 (0.1 %). Secondary HRP-linked antibody was incubated for 30 min after which blots were washed and developed using ECL. Transfection

HeLa cells were transfected using FuGENE® 6 (Roche). HEK293 cells were transfected using the calcium phosphate method. In vitro protein phosphorylation assay

HeLa cells stably expressing GFP–PIP5K1B were lysed in the absence of phosphatase inhibitors and GFP–PIP5K1B was isolated using the GFP–Trap® beads (ChromoTek) (beads were split for the different treatments in the assay). The beads were incubated in kinase buffer (20 mM Hepes, pH 7.4, 1.67 mM CaCl2 , 10 mM MgCl2 and 1 mM DTT). A 1 μl aliquot of 5 mM ATP (final concentration 50 μM) was added to the samples and the relevant purified PKC proteins (Sigma), or the recombinant purified PIP5K1B was added. Samples were incubated at 30 ◦ C for 30 min. Loading buffer containing DTT was added and the samples were boiled and separated by SDS/PAGE. Purification of soluble PIP5K1B for in vitro lipid kinase assays

Myc–PIP5K1B expressed transiently in HEK-293 cells or GFP– PIP5K1B from doxycyclin-induced cells was affinity-purified using chromatography on heparin–Sepharose. Cells were lysed in buffer containing 20 mM Tris/HCl, pH 7.5, 0.5 mM EDTA, 0.1 mM EGTA, 2 mM MgCl2 , 1 % Nonidet P40 and 0.3 M NaCl, 10 % glycerol, 12 mM 2-mercaptoethanol and protease inhibitors (CompleteTM EDTA free, Roche). After centrifugation, lysates were incubated with heparin–Sepharose beads (Roche) for 1 h at 4 ◦ C. Beads were washed three times in wash buffer (lysis buffer containing only 0.01 % Nonidet P40 and 0.3 M NaCl). Proteins were eluted from the beads by incubation in elution buffer (lysis buffer without Nonidet P40 and containing 1 M NaCl). Elution was performed twice and eluates were pooled. In vitro lipid kinase assay

PtdIns4P and PS (phosphatidylserine) were obtained from Echelon Biosciences. The substrate was prepared by mixing 0.5 nmol of PtdIns4P with 10 nmol of PS. PIP5K1B isolates were diluted in 60 μl of 10 mM Tris/HCl, pH 7.4. A mastermix

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was prepared containing 10 μl of resuspended lipids and 50 μl of 2× PIPK buffer (100 mM Tris/HCl, pH 7.4, 20 mM MgCl2, 2 mM EGTA, 140 mM KCl, 50 μM ATP and 5 μCi [32 P]ATP). The 2× mastermix was added to the PIP5K solutions and incubated at 30 ◦ C for 10 min. The reactions were stopped by the addition of 500 μl of chloroform/methanol (1:1 containing Folsch-extracted brain lipids) and 125 μl of 2.4 M HCl. After centrifugation, the top phase was removed and the lower phase was washed using the TUP [theoretical upper phase; methanol/1 M HCl/chloroform in a mixture of (v/v) 235:245:15]. The lower phase containing the phosphorylated lipids was removed and dried by vacuum centrifugation. The lipid samples were resuspended in chloroform and separated by TLC [developed in chloroform/methanol/water/25 % concentrated ammonia in a mixture of (v/v) 45:35:8:2 respectively]. Plates were dried and exposed to film or phosphorimager screens and band intensity was quantified. Knockdown of PIP5K1B

Lentiviral constructs targeting PIP5K1B were from the TRC1/TRC2 library (Sigma) and viral particles were generated in HEK-293FT cells. HT115 cells (200 000) were plated on to six-well plates and transduced overnight with either control or PIP5K1B viral particles in the presence of polybrene. The following day the virus was removed, the cells were washed and left overnight before being selected with 2 μg/ml puromycin. Endogenous PIP5K1B immunoprecipitation and Ser413 phosphorylation analysis

HT115 cells (1×106 ) were lysed in Nonidet P40 lysis buffer and immunoprecipitated using a goat anti-PIP5K1B antibody (D-19) (sc-11778, Santa Cruz Biotechnology) overnight. The following day Protein G–agarose was added for 1 h to collect the immunoprecipitates. Immunoprecipitated PIP5K was eluted with SDS loading buffer and Ser413 phosphorylation was assessed by Western blotting with the anti-Ser413 -P antibody. Total immunoprecipitated PIP5K1B was assessed using Western blotting with the above anti-PIP5K1B antibody. ECL signals were captured using a digital camera and values were integrated. The quantitative data are presented as integrated Ser413 -P signal divided by the total PIP5K1B signal. In vivo phosphoinositide labelling

The cells in six-well plates were washed with phosphate-free DMEM, followed by incubation for 30 min with phosphatefree DMEM containing 125 μCi of [32 P]Pi . The cells were washed with phosphate-free DMEM and the labelling was stopped by the addition of 0.45 ml of 1.2 M HCl. The cells were scraped, collected and 0.6 ml of methanol and 0.5 ml of chloroform were added. After centrifugation the lower phase was washed with TUP, and the lower phase was removed and dried by vacuum centrifugation. The dried lipids were resuspended in chloroform. Of that, 70 % was used for total lipid phosphate measurement and 30 % was separated by TLC and analysed by phosphorimaging. RESULTS Identification of phosphorylation sites on PIP5K1B

Since PtdIns(4,5)P2 levels need to be regulated spatially and temporally, we hypothesized that PIP5K should be strictly regulated and that phosphorylation of the protein would play  c The Authors Journal compilation  c 2013 Biochemical Society

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Figure 1

I. van den Bout and others

Identification of potential phosphorylation sites within PIP5K1B

(A) Graphic representation of phosphorylation sites identified by MS. The letters in bold indicate identified phosphorylation sites with corresponding bold letters in other isoform sequences indicating conserved amino acids. The enlarged area covers the C-terminal tail of PIP5K1B where many identified phosphorylation sites were clustered. (B) The Ab P8 (anti-Ser413 -P antibody) shows specificity for the phosphorylated peptide after negative selection with the non-phosphorylated peptide. After negative selection using the non-phosphorylated peptide coupled with Sepharose, the anti-P8 serum strongly recognizes its phosphorylated peptide epitope (䊏), but shows little reactivity towards the non-phosphorylated epitope peptide (䉬). No reactivity was observed towards a control peptide (䉱). Antibody was added in serial dilution starting at 1/100 of serum down to 1/51 200 of serum (n = 3). (C) Specificity of the anti-Ser413 -P antibody in Western blotting. Lysates from cells overexpressing Myc–PIP5K1A , Myc–PIP5K1B and Myc–PIP5K1C were separated by SDS/PAGE transferred on to nitrocellulose and probed with either the anti-Ser413 -P antibody (upper panel) to ascertain isoform specificity or with an anti-Myc antibody (lower panel) to check their level of expression (n = 3). (D) Anti-Ser413 -P antibody does not recognize PIP5K1B after incubation of lysates at 37 ◦ C. Cells overexpressing PIP5K1B were lysed in the presence of protease inhibitors, but in the absence of phosphatase inhibitors. Lysates were incubated at 37 ◦ C for the indicated time points (minutes) after which the lysates were immunoblotted with the indicated antibodies. The recognition of PIP5K1B by the anti-Ser413 -P antibody decreased over the time of incubation, although the total levels of PIP5K1B remained the same (n = 3). (E) Recognition of PIP5K1B by the anti-Ser413 -P antibody requires Ser413 . Cells were transfected with plasmids expressing WT PIP5K1B or S413A or S413D mutants. Cell lysates were separated and analysed by Western blotting with the anti-Ser413 -P antibody to identify phosphorylated PIP5K1B. Total PIP5K1B protein levels were assessed using an anti-PIP5K1B antibody (n = 3).

an important part in this regulation. Indeed, we showed previously that oxidative stress results in an increase in tyrosine residue phosphorylation of PIP5K1B [9]. We endeavoured to identify potential phosphorylation sites that could regulate the activity or localization of PIP5K1B. Overexpressed murine PIP5K1B (using the human nomenclature) was isolated from HeLa cells and analysed using MS. The analysis revealed that PIP5K1B was phosphorylated at 12 different sites, including two tyrosine residues, nine serine residues and one threonine residue (Figure 1A, and Supplementary Figure S1 at http://www. biochemj.org/bj/455/bj4550347add.htm). The two tyrosine phosphorylation sites and one of the serine sites were located within the kinase domain, whereas the other nine phosphorylation sites were clustered in a 60-amino-acid region within the C-terminal domain of PIP5K1B (Figure 1A). There is a high degree of conservation between the PIP5K isoforms within the catalytic domain, but there is much less conservation in the C-terminal region and therefore most of the sites that we identified in this  c The Authors Journal compilation  c 2013 Biochemical Society

C-terminal region were specific for PIP5K1B (Figure 1A). All identified sites are conserved between human and mouse, whereas six are also present on PIP5K in Drosophila melanogaster, but only Ser445 is present on the yeast homologue (Table 1). Analysis using NetPhosK to predict kinases which might phosphorylate these sites revealed that PIP5K1B might be subject to regulation in response to the activation of many different signalling pathways, including calcium signalling, DNA damage, MAPK (mitogenactivated protein kinase) and the WNK (‘with no K’) pathway (Table 2), implicating a potential role for PtdIns(4,5)P2 synthesis in all of these pathways. Phosphorylation of Ser413 has not been identified previously and therefore we further investigated the regulation of this phosphorylation site. Ser413 can potentially be phosphorylated by a large number of kinases as analysed using NetPhosK. In order to further study Ser413 phosphorylation, antibodies were raised against a peptide containing phosphorylated Ser413 and the antiserum was negatively selected using the

AMPK and PKC regulate PIP5K1B phosphorylation and PtdIns(4,5)P 2 synthesis Table 1

Conservation of identified phosphorylation sites

Table 3

Amino acid sequences of murine and human PIP5K1B, D. melanogaster SKTL, C. elegans PPK1 and S. cerevisiae MSS4 were aligned using ClustalW. Conserved phosphorylation sites are indicated. The numbering is representative of the murine amino acid sequence. Residue Tyr171 /Tyr174 Ser317 Ser405 Ser413 Thr420 Ser421 Ser429 Ser445 Ser447 Ser448 Ser465

Table 2 sites

Human

Murine

√ √ √ √ √ √ √ √ √ √ √

√ √ √ √ √ √ √ √ √ √ √

D. melanogaster −/

C. elegans

S. cerevisiae

√

√

√

√

√

√ √ √

√

√

Prediction of kinases responsible for phosphorylation of identified

The murine amino acid sequence was analysed using NetPhosK to identify which kinase could potentially phosphorylate the identified sites within PIP5K1B. Residue

Kinase(s)

Tyr171 /Tyr174 Ser317 Ser405 Ser413 Thr420 Ser421 Ser429 Ser445 Ser447 Ser448 Ser465

Axl Wnk CDC2, CDK5, DYRK, GSK3B, MAPK1/3 Akt2, PKA, RSK, CAMK2, CHK1, AMPK, CAMKL, MAPKAPK, RAD53, STE PKR GRK, MLCK, CK1, STE20, ATM, ATR, DNAPK PHK, ATM, DNAPK CAMK1 CK1a, CK2, PLK1 RAF, IKKa GRK1/2

non-phosphorylated peptide. The resulting serum (anti-Ser413 P serum) showed clear specificity for the phosphorylated peptide over the non-phosphorylated peptide in ELISA assays (Figure 1B). Anti-Ser413 -P antibody also specifically recognized murine Myc–PIP5K1B but, as expected, did not recognize Myc– PIP5K1A or Myc–PIP5K1C, although they were all expressed to similar levels (Figure 1C). To ascertain whether recognition of PIP5K1B by anti-Ser413 -P is phospho-specific, lysates derived from cells overexpressing human PIP5K1B were incubated at 37 ◦ C in the absence of phosphatase inhibitors allowing endogenous phosphatases to function. Although there was no reduction in the total amount of PIP5K1B, incubation for 10 or 30 min reduced recognition of PIP5K1B by anti-Ser413 -P (Figure 1D). Finally, mutation of Ser413 to an alanine or aspartate residue completely abrogated recognition of PIP5K1B by antiSer413 -P (Figure 1E). Thus we have identified a number of potential regulatory phosphorylation sites on PIP5K1B and have generated a phospho-specific antibody that specifically recognizes phosphorylated Ser413 .

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Stimulation of cells to induce Ser413 phosphorylation

HeLa cells expressing the WT PIP5K1B were stimulated with the indicated compounds. Cell lysates were prepared and immunoblotted to determine PIP5K1B Ser413 phosphorylation. Only stimulation with PMA was found to increase Ser413 phosphorylation. LPA, lysophosphatidic acid; HGF, human growth factor; EGF, epidermal growth factor; m-3M3FBS, 2,4,6-trimethyl-N -[3-(trifluoromethyl)phenyl]benzenesulfonamide; PDGF, platelet-derived growth factor; IGF, insulin-like growth factor. Compound

Concentration

Time (min)

LPA Insulin HGF EGF m-3M3FBS (PLC activator) PDGF IGF PMA

1 μM 1 μg/ml 100 ng/ml 1 ng/ml 100 μM 10 ng/ml 50 ng/ml 100 ng/ml

2–10 5–20 10–30 10–30 10–30 10–30 10–30 10–30

with a panel of agonists for the times indicated (Table 3) after which the phosphorylation status of Ser413 was analysed by Western blotting using the anti-Ser413 -P antibody. Of these various treatments, only PMA treatment resulted in the phosphorylation of Ser413 (Figure 2A). PMA stimulates the activity of a number of targets including PKC. In order to determine whether PKC was required for the PMA-induced phosphorylation of Ser413 , cells overexpressing PIP5K1B were pre-treated with the PKC inhibitor G¨o6983, which abrogated the increase in Ser413 phosphorylation in response to PMA (Figure 2B). To determine if PKC can directly phosphorylate Ser413 we performed in vitro protein kinase assays with immunoprecipitated PIP5K1B. GFP–PIP5K1B was affinity-purified, incubated with purified recombinant PKC isoforms and Ser413 phosphorylation was analysed by Western blotting. Ser413 phosphorylation of PIP5K1B increased markedly after incubation with recombinant PKCα, PKCδ or PKCθ , demonstrating that PKC can directly phosphorylate PIP5K1B on Ser413 . Incubation of purified PIP5K1B with PMA led to a slight increase in Ser413 phosphorylation. This increase could be due to co-immunopurified PKC (Figure 2C). Previous studies have suggested that a number of lipid kinases also possess protein kinase activity and are able to auto-phosphorylate. We tested whether the addition of recombinant purified GST–PIP5K1B isolated from insect cells could phosphorylate GFP–PIP5K1B. Although the recombinant PIP5K1B was phosphorylated, probably as a consequence of its expression in insect cells, it did not increase phosphorylation of GFP–PIP5K1B. To demonstrate that increased recognition of PIP5K1B by the anti-Ser413 -P antibody after incubation with purified PKC was dependent only on Ser413 phosphorylation, we assessed PKC-mediated phosphorylation of the S413A mutant. As expected, no increased recognition was observed with PKC incubation in the mutant, whereas an increase was observed when PKC was added to WT PIP5K1B (Figure 2D). We conclude that PMA induces the phosphorylation of PIP5K1B by stimulating PKC and that PKC directly phosphorylates Ser413 in vitro.

Ser413 is phosphorylated in response to oxidative stress and glucose deprivation Ser413 is phosphorylated directly by PKC

Having identified Ser413 as a potential regulatory phosphorylation site, we investigated which pathways might stimulate its phosphorylation. HeLa cells expressing PIP5K1B were stimulated

We have shown that PKC directly phosphorylates Ser413 in vitro and that PKC is essential for Ser413 phosphorylation in cells after PMA stimulation. From our initial studies (Table 1), we were unable to demonstrate changes in Ser413 phosphorylation  c The Authors Journal compilation  c 2013 Biochemical Society

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Figure 2

I. van den Bout and others

PKC phosphorylates Ser413 in vivo and in vitro

(A) Cells were treated with compounds as indicated and cell lysates were analysed by Western blotting with the anti-Ser413 -P antibody and the anti-(total) PIP5K1B antibody (n = 2). (B) Ser413 is phosphorylated in response to PKC activation. HeLa cells overexpressing GFP–PIP5K1B were treated with 100 ng/ml PMA for the indicated times (minutes) in the absence and presence of 1 μM of the PKC inhibitor G¨o6983. Cells were lysed and Ser413 phosphorylation status was determined using the anti-Ser413 -P antibody, whereas the total levels of PIP5K1B were determined using an anti-PIP5K antibody (n = 3). (C) PKC directly phosphorylates Ser413 in vitro . GFP–PIP5K1B was isolated using GFP– Trap® beads and incubated with 100 ng/ml PMA, recombinant PIP5K1B or recombinant isoforms of PKC as indicated. Ser413 phosphorylation was determined using anti-Ser413 -P, whereas the total level of GFP–PIP5K1B in the assay was determined using an anti-GFP antibody (n = 2). (D) WT or S413A PIP5K1B protein was mixed with PKCα in the presence of ATP. The reactions were quenched, proteins were separated by SDS/PAGE and analysed by immunoblotting with an anti-GFP antibody to monitor total PIP5K1B protein and with the anti-Ser413 -P antibody to monitor Ser413 phosphorylation (n = 2).

in response to G-protein-coupled receptor stimulation or in response to tyrosine kinase stimulation, suggesting that Ser413 phosphorylation of PIP5K1B does not occur in all situations when PKC is activated. We and others have demonstrated previously that phosphorylation of PIP5K1B regulates both the activity and the localization of PIP5K1B in response to H2 O2 treatment [8,9] and specific isoforms of PKC, such as the PKCδ isoform, are known to be activated in response to H2 O2 [16,17]. We therefore assessed whether cellular stresses such as oxidative stress or energy restriction could affect Ser413 phosphorylation in a PKCdependent manner. PIP5K1B is normally expressed at very low levels in many cell lines, and in order to study its phosphorylation we generated a HeLa cell line that expressed PIP5K1B under the control of an inducible promoter (doxycycline). This also avoided potential cell adaptation to its continuous overexpression. PIP5K1B expression was induced in cells overnight before they were exposed to H2 O2 for 20 min, after which cells were washed and left for different times. Western blot analysis shows that Ser413 phosphorylation was increased after H2 O2 exposure and remained elevated over the next 2 h (Figure 3A). As PKC is activated by H2 O2 treatment [16,17] and PKC can phosphorylate Ser413 , we assessed if H2 O2 -induced Ser413 phosphorylation required PKC activation. Treatment with the PKC inhibitor did not significantly reduce the basal levels of Ser413 phosphorylation, but abrogated the increase in Ser413 phosphorylation observed after H2 O2 treatment (Figure 3A). Energy restriction also activates stress response pathways and therefore we tested whether a reduction in the energy status of the cells, induced by depleting glucose and pyruvate, would induce Ser413 phosphorylation. Removal of serum caused an upward shift in PIP5K1B mobility, although it was difficult to discern any increase in Ser413 phosphorylation. Removal of glucose led to an increase in Ser413 phosphorylation as well as the expected increase in phosphorylation of ACC. Depletion of both pyruvate and glucose led to a further increase in Ser413 phosphorylation (Figure 3B). Although variable, the increase in Ser413 phosphorylation in response to glucose deprivation was robust (3.01 + − 1.4-fold increase compared with the presence of  c The Authors Journal compilation  c 2013 Biochemical Society

glucose from three separate experiments). To test whether the phosphorylation of Ser413 is dynamic and responds to changes in glucose concentration, cells were first incubated in glucosefree medium for 4 h before glucose (25 mM) was added back and Ser413 phosphorylation was analysed at different time points. Relieving glucose deprivation led to a rapid reduction in the phosphorylation of Ser413 , which was mirrored by a reduction in ACC phosphorylation (Figure 3C). Treatment of cells with the PKC inhibitor reduced Ser413 phosphorylation after energy restriction, although it had little effect on the phosphorylation of ACC (Figure 3D). Thus Ser413 phosphorylation is dynamically responsive to oxidative stress and energy restriction and in both cases Ser413 phosphorylation is dependent on PKC activity. Stress-induced AMPK activity regulates Ser413 phosphorylation

AMPK is a critical sensor of the energy status of cells. Its activation in response to energy restriction leads to a reduction in activity of pathways that utilize ATP and an increase in pathways that replenish ATP [4]. As AMPK is often activated downstream of cellular stress, we considered that AMPK activation may also play a role in Ser413 phosphorylation in response to cell stressors. Cells were treated with H2 O2 in the presence or absence of the AMPK inhibitor compound C (Figure 4A). Ser413 phosphorylation was elevated for several hours after H2 O2 exposure in non-treated cells, but this elevation was abrogated by pre-treatment with compound C. ACC phosphorylation also increased after H2 O2 exposure and, as expected, this was abrogated in cells treated with compound C. Energy restriction-induced Ser413 phosphorylation was also reduced by pre-treatment with compound C (Figure 4B). To determine whether AMPK activation is sufficient to induce Ser413 phosphorylation, we treated cells with the AMPK activator AICAR [20] in the presence of glucose (Figure 4B). AICAR led to an increase in Ser413 phosphorylation which was reduced by the AMPK inhibitor compound C. As PKC activity is required for phosphorylation of Ser413 in response to glucose deprivation we determined if PKC acted upstream or downstream of AMPK activation. Pre-treatment of cells with the PKC inhibitor reduced Ser413 phosphorylation in response to AICAR treatment,

AMPK and PKC regulate PIP5K1B phosphorylation and PtdIns(4,5)P 2 synthesis

Figure 3

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H2 O2 and energy restriction enhance PKC-dependent Ser413 phosphorylation

(A) Cells expressing PIP5K1B were maintained as controls ( − ) in the presence or absence of 1 μM of the PKC inhibitor G¨o6983 for 30 min and were then treated with 1 mM H2 O2 for 20 min ( + ). After H2 O2 stimulation, the cells were washed (wash out) and incubated for the indicated time points under normal serum conditions. Cells lysates were separated by SDS/PAGE and immunoblotted and Ser413 phosphorylation was determined using the anti-Ser413 -P antibody. The total level of PIP5K1B was determined by blotting with the anti-PIP5K antibody (n = 3). (B) Cells were maintained in the presence or absence of fetal bovine serum, glucose or both glucose and pyruvate. Cell lysates were separated by SDS/PAGE and immunoblotted with the indicated antibodies (n = 2). (C) Cells were starved of glucose for 4 h ( − ) before glucose was added back to a final concentration of 25 mM for the times indicated. Cell lysates were immunoblotted with the indicated antibodies (n = 3). (D) Glucose starvation triggers PKC-dependent Ser413 phosphorylation. Cells were deprived of glucose for 4 h ( − ) or kept in control medium containing 25 mM glucose ( + ) either in the absence or presence of the PKC inhibitor G¨o6983 as indicated. Cell lysates were immunoblotted with the indicated antibodies (n = 2).

suggesting that AMPK does not directly phosphorylate Ser413 and that PKC lies downstream of AMPK activation (Figure 4B). Finally, to determine whether AMPK activation is required for Ser413 phosphorylation by PKC, cells overexpressing PIP5K1B were pre-treated with the AMPK inhibitor compound C before they were stimulated with PMA (Figure 4C). Western blot analysis shows that Ser413 phosphorylation induced by PMA was not reduced by pre-treatment with compound C (Figure 4C). These data also demonstrated that compound C does not directly inhibit PKC mediated phosphorylation of Ser413 . Taken together, these data suggest that, in response to oxidative stress or energy restriction, AMPK and PKC collaborate to regulate Ser413 phosphorylation and that direct phosphorylation of Ser413 is carried out by PKC.

observed when PIP5K1B was overexpressed. To demonstrate that PMA-induced phosphorylation required enhanced PKC activity, we pre-treated cells with the PKC inhibitor G¨o6983 (Figure 5C). PMA induced robust Ser413 phosphorylation which was blocked by pre-treatment with G¨o6983. As observed with the overexpressed PIP5K1B, energy restriction-induced Ser413 phosphorylation was dependent on both PKC and AMPK (Figure 5D). We confirmed our endogenous phosphorylation data in U2OS cells that express lower levels of PIP5K1B. In these cells PMA induced robust Ser413 phosphorylation of PIP5K1B (Figure 5E). These data show that endogenous PIP5K1B is phosphorylated at Ser413 in response to PMA, oxidative stress and energy restriction, and that energy restrictioninduced phosphorylation requires both PKC and AMPK activity.

Endogenous Ser413 is phosphorylated in response to PMA and energy restriction

Mimicking Ser413 phosphorylation reduces PIP5K1B kinase activity

PIP5K1B is expressed at very low levels in HeLa cells and therefore it is difficult to assess whether endogenous PIP5K1B is phosphorylated similarly to when it is heterologously overexpressed. HT115 colorectal cells express higher levels of PIP5K1B and therefore we used these cells to assess PIP5K1B Ser413 phosphorylation. As a control, PIP5K1B expression was supressed in HT115 cells using shRNA and total PIP5K1B, and Ser413 phosphorylation of PIP5K1B was assessed by Western blotting (Figure 5A). A 66-kDa protein was immune-precipitated that was phosphorylated on Ser413 and importantly this protein was absent after knockdown of PIP5K1B. We next assessed PIP5K1B phosphorylation after treatment with various stimuli (Figure 5B). Ser413 phosphorylation increased in response to energy restriction (no glucose and no pyruvate), PMA stimulation or H2 O2 addition. The largest increase was observed with PMA stimulation as also

Phosphorylation of Ser413 could influence the function of PIP5K1B either by altering its intracellular localization or by affecting its kinase activity. In order to investigate the functional consequences of Ser413 phosphorylation, we generated mutants of Ser413 in PIP5K1B. Ser413 was replaced with either an alanine residue to prevent its phosphorylation or to aspartate to mimic phosphorylation. HeLa cells were transiently transfected with the WT or mutant versions of GFP–PIP5K1B and their localization was monitored by confocal microscopy. WT PIP5K1B localized to the plasma membrane where the PH (pleckstrin homology) domain of PLCδ1 (phospholipase C δ1) as a probe for PtdIns(4,5)P2 was present (Figure 6A). Neither the localization of PIP5K1B nor the PH domain were significantly altered in cells expressing either the S413A or S413D mutant, suggesting that the presence of a negative charge at position 413 does not alter the localization of PIP5K1B at the membrane.  c The Authors Journal compilation  c 2013 Biochemical Society

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Figure 4

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H2 O2 - and energy restriction-, but not PMA, induced Ser413 phosphorylation requires AMPK activation

(A) Oxidative stress induces an AMPK-dependent increase in Ser413 phosphorylation. Cells expressing PIP5K1B were maintained as controls or were pre-treated with the AMPK inhibitor compound C (10 μM) for 30 min before the addition of 1 mM H2 O2 for 20 min (as indicated). Cells were then washed and maintained in serum containing medium for the indicated time (hours). If compound C was added then it was also present after the washout. Cell lysates were immunoblotted with the indicated antibodies (n = 2). (B) Cells expressing PIP5K1B were maintained as controls or treated with compound C as indicated. Left-hand panel: cells were starved of glucose for 4 h in the presence or absence of compound C. Cell lysates were immunoblotted with the indicated antibodies. Right-hand panel: direct activation of AMPK causes Ser413 phosphorylation. Cells expressing PIP5K1B were pre-treated for 1 h with 1 μM G¨o6983 or with 10 μM compound C in the presence of glucose and then stimulated with 2 mM AICAR for 1 h. Cell lysates were then immunoblotted with the indicated antibodies (n = 2). (C) AMPK activity is not required for PMA-induced Ser413 phosphorylation. Cells expressing PIP5K1B were maintained as controls or pre-treated with the AMPK inhibitor compound C. The cells were then treated with PMA (100 ng/ml) for the indicated times (hours). Cell lysates were immunoblotted with the indicated antibodies (n = 2).

Figure 5

Endogenous Ser413 phosphorylation in response to energy restriction requires both PKC and AMPK activity

(A) HT115 cells were transduced with either control (SHX) or lentivirus targeting PIP5K1B (SH-PIP5K1B). The cells were selected with puromycin and then lysed and analysed by Western blotting with the antibodies indicated. (B) HT115 cells were serum-starved and stimulated with PMA (15 min), H2 O2 (15 min) or were deprived of glucose (2 h). Cell lysates were immunoprecipitated with an anti-PIP5K1B antibody and analysed by Western blotting with the indicated antibodies. The histogram shows the ratio of phosphorylated PIP5K1B to total PIP5K1B + − S.D. from at least three different experiments. (C) HT115 cells were serum-starved and treated as indicated. Cell lysates were immunoprecipitated using an anti-PIP5K1B antibody. Proteins present in the immunoprecipitates were analysed with the indicated antibodies (n = 2). (D) HT115 cells were serum-starved, pre-treated with the indicated inhibitors and deprived of glucose as indicated. Cell lysates were immunoprecipitated using an anti-PIP5K1B antibody. Proteins present in the immunoprecipitates were analysed with the indicated antibodies (n = 2). (E) U2OS cells were serum-starved and stimulated with PMA (15 min). Cell lysates were immunoprecipitated using an anti-PIP5K1B antibody. Proteins present in the immunoprecipitates were analysed with the indicated antibodies (n = 3).

 c The Authors Journal compilation  c 2013 Biochemical Society

AMPK and PKC regulate PIP5K1B phosphorylation and PtdIns(4,5)P 2 synthesis

Figure 6

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Ser413 phosphorylation affects PIP5K activity

(A) HeLa cells were transiently transfected with GFP-tagged constructs of WT PIP5K1B, S413A PIP5K1B or S413D PIP5K1B together with RFP–PLCδ1 PH domain to monitor PtdIns(4,5)P 2 levels. The following day, the cells expressing both constructs were imaged using a spinning disk confocal microscope. All images were taken at 60× magnification and with the same exposure times. (B) WT, S413A and S413D mutants of PIP5K1B were expressed in HEK-293 cells and purified by heparin–Sepharose chromatography. Soluble purified protein (10 or 20 μl) was analysed by immunoblotting with the anti-PIP5K antibody to determine their concentration. Equal amounts of PIP5K1B protein as indicated were used in in vitro lipid kinase assays to determine PIP5K activity. The PIP5K activity, represented as arbitrary phosphoimager units incorporated into PtdIns(4,5)P 2 , is presented in the histogram. The histogram represents the mean + − S.E.M. from three experiments. The activity of WT PIP5K1B was found to be significantly different to that of the S413D mutant (*P < 0.05, one-way ANOVA with a post-hoc Tukey test).

To investigate whether mimicking the phosphorylation of Ser413 might affect the kinase activity of PIP5K1B, WT PIP5K1B and the serine mutants were expressed in the inducible system in HeLa cells, purified and their kinase activity was assessed. Heparin– Sepharose chromatography was used to purify the PIP5Ks as it is rapid, affords a high level of purification directly from cell lysates and, in contrast with most antibody purifications, allows simple elution of the PIP5K1B from the purification matrix. The eluted protein concentration was assessed by Western blotting (Figure 6B) and similar amounts of protein were used to assay PIP5K activity using the substrate PtdIns4P and radiolabelled ATP (Figure 6B). Using this protocol approximately five to ten times more PIP5K activity was eluted from the heparin beads when PIP5K1B was expressed compared with control transfected cells. We found that the WT and S413A mutant of PIP5K1B protein had similar kinase activity levels. However, PIP5K1B carrying the phosphomimetic mutation S413D showed a reduction in kinase activity of 40 %. These data are consistent with a role for Ser413 phosphorylation in decreasing the catalytic activity of PIP5K1B, but not in modulating its localization at the plasma membrane.

Effects of Ser413 mutation on PtdIns(4,5)P 2 synthesis in vivo

In order to examine whether mimicking Ser413 phosphorylation of PIP5K1B could regulate the levels of intracellular PtdIns(4,5)P2 , we measured cellular PtdIns(4,5)P2 synthesis in inducible cells expressing the WT or mutants of PIP5K1B (Figure 7A). Overexpression of WT PIP5K1B resulted in a 5-fold increase in cellular PtdIns(4,5)P2 labelling over that of non-overexpressing cells. A similar increase in PtdIns(4,5)P2 labelling was also observed in cells overexpressing the S413A mutant. However, overexpression of the S413D phosphomimetic mutant increased PtdIns(4,5)P2 labelling significantly less than the overexpression of the WT protein. Analysis of the expression levels of WT and mutant PIP5K1B protein showed that they were expressed to

similar levels (Figure 7B). The labelling with [32 P]orthophosphate was carried out for only 30 min and therefore the incorporation of 32 P more likely reflects both the rate of PtdIns(4,5)P2 labelling and the steady-state level. The reason for the decrease in labelling observed in the S413D mutant might reflect changes in its PIP5K1B activity, PtdIns(4,5)P2 phosphatase activity, or basal PLC activity or a combination of these events. However, given that purified S413D PIP5K1B has approximately half the activity of the WT PIP5K1B (Figure 6B), it is reasonable to postulate that the decrease in PtdIns(4,5)P2 labelling is, in part, due to the decrease in PIP5K1B activity in the mutant. As H2 O2 also induces inhibitory phosphorylation of Ser413 , we analysed whether attenuating Ser413 phosphorylation might block the decrease in PtdIns(4,5)P2 levels. H2 O2 induced a similar reduction in PtdIns(4,5)P2 labelling in cell lines expressing either the WT or the alanine mutant of PIP5K1B. The PtdIns(4,5)P2 level in the S413D mutant was not significantly different compared with untreated cells, but the trend to a decrease was similar to the WT enzyme. The reduction in PtdIns(4,5)P2 labelling observed in the S413A mutant suggests that other mechanisms besides Ser413 phosphorylation also affects PtdIns(4,5)P2 levels in response to H2 O2 . H2 O2 leads to a delocalization of PIP5K1B from the membrane, as well as to a decrease in its activity [9], and therefore we assessed the localization of PIP5K1B and its mutants (Figure 7C). H2 O2 induced the translocation of the WT and both mutants of PIP5K1B from the membrane, where its substrate PtdIns4P resides, into the cytosol. The decrease in membranebound PIP5K1B is likely to decrease the rate of PtdIns(4,5)P2 labelling, perhaps explaining why the alanine mutant did not suppress the H2 O2 -mediated decrease in PtdIns(4,5)P2 labelling. A decrease in PtdIns(4,5)P2 levels in response to H2 O2 is also due to the activation of PLC [21]. That a single phosphomimetic point mutant leads to a change in PtdIns(4,5)P2 levels concordant with a change in its in vitro activity indicates the importance of Ser413 phosphorylation. Our data are consistent with a role for phosphorylation of Ser413 in regulating PIP5K1B activity which affects PtdIns(4,5)P2 synthesis in vivo.  c The Authors Journal compilation  c 2013 Biochemical Society

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Figure 7

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Effect of Ser413 mutation on cellular PtdIns(4,5)P 2 levels and protein localization during oxidative stress

(A) S413D PIP5K1B mutant synthesizes less PtdIns(4,5)P 2 than the WT or S413A mutant. HeLa cells expressing an inducible empty vector (EV) or WT, S413A or S413D PIP5K1B were treated with doxycycline overnight to induce the expression of the proteins. Cells were maintained as controls or stimulated with H2 O2 and then labelled with [32 P]Pi . Labelled lipids were extracted, separated by TLC and the incorporation of 32 P into PtdIns(4,5)P 2 was determined by phosphoimaging and is presented in the histogram. Error bars show S.E.M. for independent data points. Statistical significance was determined using a one-way ANOVA with a post-hoc Tukey test. The asterisks indicate P < 0.05. NS, not significant. (B) HeLa cells expressing an inducible empty vector as above WT, S413A or S413D PIP5K1B were treated overnight with doxycycline to induce the expression of the proteins. Cell lysates were then immunoblotted with the indicated antibodies. (C) HeLa cells were transfected with the indicated GFP–PIP5K1B constructs together with RFP–PLCd1 PH domain. The following day, the cells were stimulated with H2 O2 for the times indicated and GFP and RFP were imaged by confocal microscopy. (D) Diagram showing the pathway that regulates Ser413 phosphorylation leading to decreased kinase activity. The broken line represents the potential for AMPK to prime PIP5K1B for phosphorylation by PKC.

DISCUSSION

The cellular level of PtdIns(4,5)P2 is tightly regulated both spatially and temporally by the enzymes that synthesize it and those that remove it. PIP5K is a synthetic kinase that is responsible for the generation of the majority of PtdIns(4,5)P2 in cells. To understand how PIP5K1B is regulated, we undertook a mass spectrometric approach to define sites of post-translational modification. We found that PIP5K1B is phosphorylated at many sites and further investigated the phosphorylation of PIP5K1B at Ser413 . Our data are consistent with a model that cellular stress such as oxidative stress or glucose deprivation lead to the activation of AMPK, which enables the direct phosphorylation of Ser413 by PKC (Figure 6C). We provide a number of lines of evidence that support this conclusion: (i) PKC activity is required for H2 O2 and glucose deprivation-induced Ser413 phosphorylation; (ii) AMPK activity is required for H2 O2 and glucose deprivation-induced Ser413 phosphorylation; (iii) direct activation of AMPK increases Ser413 phosphorylation which is attenuated by PKC inhibition; (iv) direct activation of PKC increases Ser413 phosphorylation, but this does not require AMPK activity; and (v) PKC isoforms directly phosphorylate Ser413 in vitro. Functionally, mimicking Ser413 phosphorylation by its mutation to an aspartate residue, decreases the kinase activity of PIP5K1B, but has little effect on PIP5K1B localization. Stress-induced activation of AMPK functions to redirect cell metabolism to inhibit anabolic pathways and ATP utilization  c The Authors Journal compilation  c 2013 Biochemical Society

and to increase ATP generation through an increase in fatty acid metabolism [14]. As PtdIns(4,5)P2 synthesis is an energydependent process, AMPK-mediated inhibition of PIP5K could act to conserve ATP. A reduction in PtdIns(4,5)P2 synthesis would probably also lead to a reduction in the generation of PtdIns(3,4,5)P3 and a consequent reduction in PKB (protein kinase B)/mTOR (mammalian target of rapamycin) activation. The inhibition of both of these pathways would attenuate cell growth and proliferation and would also induce autophagy, providing substrates to fuel the cell’s metabolic requirements. Furthermore, the reduction in PKB signalling would also lead to the activation of the FOXO (forkhead box O) transcription factors [22] required for autophagy and cell maintenance under conditions of stress [23]. How AMPK and PKC pathways co-operate to regulate Ser413 phosphorylation is not clear. Whereas AMPK activity is required for Ser413 phosphorylation in response to stress, it is not clear exactly how AMPK functions in this process. Although NetPhosK predicts that Ser413 might be a direct site for AMPK (Table 2), we were unable to demonstrate that AMPK could directly phosphorylate PIP5K1B in vitro (results not shown). In response to stress signalling, AMPK could direct the activation of PKC which would then phosphorylate Ser413 . If and how AMPK regulates PKC activation in response to stress is not clear [23]. Alternatively, AMPK could phosphorylate PIP5K1B at other sites priming its phosphorylation at Ser413 by PKC. However, priming of PIP5K1B by AMPK is unlikely to be wholly responsible,

AMPK and PKC regulate PIP5K1B phosphorylation and PtdIns(4,5)P 2 synthesis

as PMA-induced Ser413 phosphorylation did not require AMPK activity and in vitro PKC isoforms were able to phosphorylate Ser413 directly. Although we show that the PKCα, PKCδ and PKCθ isoforms are able to directly phosphorylate PIP5K1B in vitro, it is possible that in vivo a specific isoform is utilized. H2 O2 is a strong inducer of Ser413 phosphorylation and is known to strongly induce the activation of PKCδ, suggesting that the δ isoform might be responsible for Ser413 phosphorylation [16,17]. Phosphorylation of PIP5Ks is a critical mechanism for negatively regulating their catalytic activity. For example, phosphorylation of PIP5Kγ on Ser645 by cdk5 (cyclindependent kinase 5) leads to its inactivation which can be reversed by calcineurin activation [24]. cAMP-dependent protein kinase phosphorylates Ser214 which decreases the activity of PIP5K1B, whereas lysophosphatidic acid treatment activates PIP5K1B through a PP1 (protein phospatase 1)-mediated dephosphorylation of Ser214 [10]. Oxidative stress decreases cellular PtdIns(4,5)P2 levels, which has been linked to the activation of caspases and an increase in apoptosis [7]. Interestingly, in control cells the decrease in PtdIns(4,5)P2 labelling was less than in cells overexpressing WT PIP5K1B (Figure 6A). HeLa cells express all three PIP5K isoforms, suggesting that different isoforms of PIP5K might be differentially regulated by H2 O2 signalling. This is in accordance with previous studies which showed that the activities of PIP5Kα and PIP5Kγ were not reduced in response to H2 O2 treatment [8]. In the present study we show that PMA, oxidative stress and energy restriction induce the phosphorylation of Ser413 and that mimicking this phosphorylation by replacing the serine residue with aspartate inhibits the catalytic activity of PIP5K1B in vitro and decreases its ability to synthesize PtdIns(4,5)P2 in vivo. The decrease in PtdIns(4,5)P2 levels induced by oxidative stress was not attenuated by mutating Ser413 to an alanine residue. This suggests that other mechanisms also play a role in regulating PtdIns(4,5)P2 levels in response to oxidative stress. Previous studies have shown that oxidative stress increased phosphorylation of Tyr105 on PIP5K1B which induced membrane delocalization and reduced PIP5K1B activity. Mutation of Tyr105 prevents its delocalization away from the membrane [8]. Also, the decrease in PtdIns(4,5)P2 levels in response to H2 O2 is also a consequence of PtdIns(4,5)P2 hydrolysis mediated by the activation of PLCγ by H2 O2 [21]. Furthermore, in the present study, we also identified a number of other phosphorylation sites on PIP5K1B. Interestingly, these sites, including Ser413 , cluster within the C-terminal domain of PIP5K. Although the membrane localization of PIP5K1B was not dependent on Ser413 phosphorylation, it is conceivable that multiple phosphorylation events at these sites might contribute to a negative local surface charge on the protein that influences PIPK1B localization to the negatively charged plasma membrane. It is noteworthy that these phosphorylation sites are not present in other PIP5K isoforms, suggesting that they will likely regulate specific non-redundant functions of the PIP5K1B isoform.

AUTHOR CONTRIBUTION All authors contributed to the acquisition of data for the paper. Nullin Divecha and Iman van den Bout designed the experiments and wrote the paper.

ACKNOWLEDGEMENTS We acknowledge Professor A. Heck for discussion and help with the paper. We thank all members of the Inositide Laboratory, The Paterson Institute for Cancer Research, U.K.

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FUNDING Preliminary studies were supported by a grant from the Dutch Cancer Society (KWF) (to N. D.) and after by funding from Cancer Research UK.

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Biochem. J. (2013) 455, 347–358 (Printed in Great Britain)

doi:10.1042/BJ20130259

SUPPLEMENTARY ONLINE DATA

Collaboration of AMPK and PKC to induce phosphorylation of Ser413 on PIP5K1B resulting in decreased kinase activity and reduced PtdIns(4,5)P 2 synthesis in response to oxidative stress and energy restriction Iman VAN DEN BOUT*, David R. JONES*, Zahid H. SHAH*, Jonathan R. HALSTEAD†, Willem-Jan KEUNE*, Shabaz MOHAMMED‡, Clive S. D’SANTOS§ and Nullin DIVECHA*1 *Inositide laboratory, The Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, U.K., †Syngenta Cereals, Syngenta, 4006 Hawthorne Circle, Longmont, CO, U.S.A., ‡Biomolecular Mass Spectrometry and Proteomics Group, Padualaan 8, Utrecht, 3584 CH, The Netherlands, and §Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, U.K.

Supplementary Figure S1 is on the following pages

1

To whom correspondence should be addressed (email [email protected]).  c The Authors Journal compilation  c 2013 Biochemical Society

I. van den Bout and others

Figure S1

Fragmentation spectra for the observed phosphorylation sites

 c The Authors Journal compilation  c 2013 Biochemical Society

AMPK and PKC regulate PIP5K1B phosphorylation and PtdIns(4,5)P 2 synthesis

Figure S1

(continued)

 c The Authors Journal compilation  c 2013 Biochemical Society

I. van den Bout and others

Figure S1

(continued)

 c The Authors Journal compilation  c 2013 Biochemical Society

AMPK and PKC regulate PIP5K1B phosphorylation and PtdIns(4,5)P 2 synthesis

Figure S1

(continued)

Received 19 February 2013/25 July 2013; accepted 5 August 2013 Published as BJ Immediate Publication 5 August 2013, doi:10.1042/BJ20130259  c The Authors Journal compilation  c 2013 Biochemical Society

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