PI3K drives the de novo synthesis of coenzyme A from vitamin B5

Abstract

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis¹. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling^2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells^4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)^6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT⁸. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK4⁹ is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K-PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

Fig. 1. PI3K–AKT signalling stimulates de novo CoA synthesis.

a, The CoA de novo synthesis pathway. b, Insulin (100 nM) and PI3K inhibitor (GDC-0941 or GDC-0032; 2 μM) treatments with concurrent ¹³C₃¹⁵N₁-VB5 labelling (3 h), preceded by serum/growth factor deprivation (18 h) and inhibitor pretreatment (15 min) in MCF10A cells. c, PIK3CA^+/+ and PIK3CA^p.H1047R/+-knockin MCF10A cells with PI3K inhibitor (GDC-0941; 2 μM) treatment. Labelling and conditions were otherwise as described in b. d, Acid-extracted CoA and short-chain acyl-CoAs with the cells and conditions as described in b, except with 4 h treatments and labelling. e, Radioactive ¹⁴C-VB5 labelling (3 h) with the cells and conditions otherwise as described in b, followed by a chase (1 h) in medium without VB5. Disintegrations per minute (DPM) normalized to protein. f, AKT inhibitor (GDC-0068, 2 μΜ) and mTORC1 inhibitor (rapamycin, 100 nM) treatments with concurrent ¹³C₃¹⁵N₁-VB5 labelling (3 h) of MCF10A cells expressing doxycycline (Dox)-inducible HA-tagged wild-type (WT) or constitutively active (E17K) AKT. Treatments and labelling were preceded by doxycycline incubation (48 h), serum and growth factor deprivation (18 h) and inhibitor pretreatment (15 min). g, AKT inhibitor (GDC-0068, 2 μΜ) and ACLY inhibitor (NDI-091143, 15 μM) treatments with the cells, labelling and conditions otherwise as described in f. For b–d, f and g, metabolites were measured using LC–MS/MS and normalized to protein; labelled metabolites (mass + 4 [M + 4]); fractional abundance is [M + 4]/total. For the percentage change graphs, the left-most treatment group mean was set to 0%. For b–g, n = 3 biological replicates (circles). Data are mean ± s.e.m. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey test; asterisks (*) indicate significant differences compared with the treatment groups marked with daggers (†) or between treatments indicated with brackets (P < 0.05). Immunoblotting analysis probed for total and phosphorylated (p) proteins.

Fig. 2. PANK2 and PANK4 are direct AKT substrates.

a, CoA synthesis pathway enzymes. The full names and accession numbers are provided in Supplementary Table 1. b, Enzymes of the CoA synthesis pathway that are candidate AKT substrates. Amino acid residues that fall within low- to high-quality AKT substrate motifs according to the kinase–substrate prediction program Scansite, and that are reported to be phosphorylated in the phosphoproteomic database Phosphosite are listed. Numbering is based on the human sequence. c, Endogenous PANK1, PANK2 and PANK4 immunoprecipitation (IP) from MCF10A cells with insulin (100 nM) and PI3K inhibitor (GDC-0941, 2 μM) treatments (30 min), preceded by serum and growth factor deprivation (18 h) and inhibitor pretreatment (15 min). d, Endogenous PANK4 immunoprecipitation from orthotopic mammary allograft tumours in C57BL/6J treated with vehicle or PI3K inhibitor (BYL-719, 45 mg kg⁻¹) daily for 10 days. e, Endogenous PANK4 immunoprecipitation from skeletal muscle (gastrocnemius) of C57BL/6J mice treated with PI3K inhibitor (BYL-719, 50 mg kg⁻¹) for 1 h. f, Endogenous PANK2 and PANK4 immunoprecipitation from MCF10A cells treated with insulin (100 nM), AKT inhibitor (MK-2206, 2 μM), and mTORC1 inhibitor (rapamycin, 20 nM) with conditions otherwise as in c. g,h, In vitro AKT kinase assays. Untagged PANK2 (g) or PANK4 (h) (WT or alanine point mutants) were immunopurified from respective reconstituted knockout cells treated with PI3K and AKT inhibitors. PANK immunopurifications were incubated with purified GST–AKT (30 min). i, Diagram of the human PANK2 and PANK4 domains and AKT-targeted phosphorylation sites. The asterisks indicate the location of evolutionary mutations inactivating PANK4 kinase domain. For c–h, immunoblotting analysis probed for total and phosphorylated proteins including AKT phospho-substrate motifs; representative of two independent experiments. IgG, control IgG immunoprecipitation.

Fig. 3. PANK4 suppresses CoA synthesis and phosphorylation of PANK4 Thr406 reduces this suppression.

a, PANK kinase inhibitor (0–10 μM; inhib.) treatments with concurrent ¹³C₃¹⁵N₁-VB5 labelling (3 h) of MCF10A cells expressing doxycycline-inducible HA-tagged wild-type or constitutively active (E17K) AKT. Treatment and labelling were preceded by doxycycline incubation (48 h), serum and growth factor deprivation (18 h) and inhibitor pretreatment (15 min). b, Individual siRNA-mediated knockdowns of PANK1, PANK2 and PANK4 (siP1, siP2 and siP4, respectively) or non-targeting siRNA (siC) in MCF10A AKT^p.E17K/+ cells with ¹³C₃¹⁵N₁-VB5 labelling (24 h) preceded by serum and growth factor deprivation (18 h). c, Wild-type PANK4 and PANK4-KO AKT^p.E17K/+ MCF10A cells with ¹³C₃¹⁵N₁-VB5 labelling (3 h) preceded by serum and growth factor deprivation (18 h). Divided blots are from same SDS–PAGE gel and image. d, PANK4-KO cells stably expressing vector (Vec) or untagged human PANK4 (WT or T406A) with conditions and labelling otherwise as described in c. Divided blots are from same SDS–PAGE gel and image. e, ACLY inhibitor (NDI-091143, 20 μM) treatment with concurrent ¹³C₃¹⁵N₁-VB5 labelling (3 h) of PANK4-KO cells stably expressing vector or untagged human PANK4 (T406A or T406E). Treatments and labelling were preceded by serum and growth factor deprivation (18 h) and inhibitor pretreatment (2 h). f, Two-dimensional (2D) proliferation of cell lines from d and e with growth factors. Statistical comparison was performed on day 4 data. For a–e, metabolites were measured using LC–MS/MS and normalized to protein; labelled metabolites (M + 4). For the graphs of percentage change, the mean value of the left-most treatment group was set to 0%. Immunoblotting analysis probed for total or phosphorylated proteins. For a–f, n = 3 biological replicates (circles). Data are mean ± s.e.m. Statistical analysis was performed using two-tailed Student’s t-tests (c), one-way ANOVA with Tukey test (a and d–f) and two-way ANOVA with Sidak test (b); asterisks (*) indicate significant differences compared with the treatment groups marked with daggers (†) or between treatments indicated with brackets (P < 0.05).

Fig. 4. PANK4 functions as a metabolite phosphatase.

a, Amino acid alignment of PANK4 with conserved catalytic aspartates (blue, bold) and other conserved residues (grey) of previously characterized DUF89 domains (non-PANK4 orthologues). At, Arabidopsis thaliana; Hs, Homo sapiens; Ph, Pyrococcus horikoshii; Sc, Saccharomyces cerevisiae. b, PANK4 phosphatase assay. Flag-tag immunopurifications from cells expressing vector or Flag–PANK4 (WT, D623A or D659A). Substrates: para-nitrophenyl phosphate (PNPP) and 4′-phosphopantetheine (p-PaSH). The wild-type mean was set to 1. c, PANK4 phosphatase assay. Flag–PANK4 as in b. Substrates: 4′-phosphopantothenate (p-Pa) and 4′-phosphopantetheine. The 4′-phosphopantetheine mean was set to 1. d, PANK4-KO AKT^p.E17K/+ MCF10A cells stably expressing vector or untagged PANK4 (WT, D623A or D659A). ¹³C₃¹⁵N₁-VB5 labelling (3 h) was performed with serum replacement and growth factors. Metabolites were measured using LC–MS/MS and normalized to protein. Labelled metabolites (mass + 4). Vector mean set to 0%. e, Unlabelled polar metabolomics using cells and conditions in d.Two independent experiments, ‘a’ and ‘b, were analysed (Methods). f, Unlabelled lipidomics using the cells and conditions as described in d. The analysis incorporates three independent experiments (Methods). g, Seahorse oxygen-consumption assay using the cells in d without serum or growth factors. OCR, oxygen consumption rate. h, 2D proliferation with the cells and conditions as described in d. Representative of three independent experiments. i, Three-dimensional (3D) soft agar colony formation using the cells in d with serum and growth factors. j, Orthotopic mammary xenograft tumours using SUM159 PANK4-KO cells with stable expression of vector or untagged PANK4 (WT or D623A) in nude mice. Individual tumour growth curves (left). Kaplan–Meier survival curves using tumour volume (750 mm³) or ulceration (X) end points (right). k, Model of PI3K-dependent CoA synthesis regulation. For b, d and j, immunoblotting analysis probed for total and phosphorylated proteins. For b–d and g–i, n = 3 (b–d, g and h), n = 4 (j) or n = 6 (i) biological replicates (circles). Data are mean ± s.e.m. For b–d and g–j, statistical analysis was performed using one-way ANOVA with Tukey test; asterisks (*) indicate significant differences compared with the treatment groups marked with daggers (†) or between treatments indicated with brackets (P < 0.05). Source data

Extended Data Fig. 1. Data supporting Fig. 1.

a, PI3K-regulated metabolite screen. Serum/growth factor-deprived (18 h) MCF10A cells treated (1 h) with insulin (100 nM) and PI3K inhibitor (GDC-0941, 2 μM). Graphed metabolites were significantly (one-way ANOVA, p<alpha 0.05) different by ≥ 50% between any two treatments in both independent experiments (A and B). b, Diagram of heavy isotope (¹³C₃¹⁵N₁)-labelled Vitamin B5 (VB5) tracing into CoA and acyl-CoAs. Labelled carbon (blue), nitrogen (red). Labelled VB5, CoA, and acyl-CoAs are all native mass + 4 [M+4]. c, Steady state ¹³C₃¹⁵N₁-VB5 labelling of MCF10A cells (4–6 days) in media containing only ¹³C₃¹⁵N₁-VB5, supplemented with either defined serum replacement or horse serum. Metabolites measured by LC-MS/MS and displayed as fractional abundance of labelled metabolites ((M+4)/total). Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. d, Growth of MCF10A cells over 4 days in serum-free media containing growth factors and defined serum replacement, in the presence or absence of 1 µM VB5. Cells were grown in the absence of VB5 for 48 h prior to plating. Mean (points on line); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-sided student’s t-test, (p<alpha 0.05). e, Time course of MCF10A cells serum/growth factor-deprived (18 h), pre-incubated (1 h) with ¹³C₃¹⁵N₁-VB5, and treated (1, 3, or 5 h) with insulin (100 nM) and ¹³C₃¹⁵N₁-VB5 labelling. Protein immunoblots probed with antibodies to total and phosphorylated (p) AKT (left); molecular weight markers in kD (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-way ANOVA with Sidak’s test, (p<alpha 0.05). f, Validation of MS/MS methods for ¹³C₃¹⁵N₁-VB5 tracing into VB5, CoA, and acetyl-CoA. Serum/growth factor-deprived (18 h) MCF10A cells were treated (3 h) with insulin (100 nM) and labelled with ¹³C₃¹⁵N₁-VB5. Unlabelled fructose-1,6-bisphosphate (F-1,6-BP) shown as control. Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). g, Time course showing intracellular levels of ¹³C₃¹⁵N₁-VB5 in serum/growth factor deprived (18 h) MCF10A cells upon incubation in media exclusively containing ¹³C₃¹⁵N₁-VB5 for 10–60 min. Graphed metabolites measured my LC-MS/MS. Mean (points on line); SEM (error bars); n = 3 biological replicates.

Extended Data Fig. 2. Data supporting Fig. 1.

a, Diagram of heavy isotope (¹³C₃¹⁵N₁)-labelled cysteine tracing into CoA and acyl-CoAs. Labelled carbon (blue) and nitrogen (red). Note that labelled cysteine is M+4 but that labelled CoA and acyl-CoAs are M+3 due to the decarboxylation step of CoA synthesis. b, Validation of MS/MS methods for ¹³C₃¹⁵N₁-cysteine tracing. Serum/growth factor-deprived (18 h) MCF10A cells were treated (3 h) with insulin (100 nM) and concurrently labelled with ¹³C₃¹⁵N₁-cysteine. Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-way ANOVA with Sidak’s test, (p<alpha 0.05). c, ¹³C₃¹⁵N₁-cysteine tracing (3 h) with concurrent insulin (100 nM) and PI3K inhibitor (GDC-0941, 2 μM) treatments in MCF10A cells. Cells were serum/growth factor-deprived (18 h) and pretreated with inhibitor prior to tracing. Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). d, Insulin (100 nM), Epidermal Growth Factor (EGF, 50 ng ml⁻¹), Insulin-like growth factor 1 (IGF-1, 50 ng ml⁻¹) treatments with concurrent ¹³C₃¹⁵N₁-VB5 labelling (3 h) of serum/growth factor-deprived (18 h) MCF10A cells. Protein immunoblots probed with indicated antibodies (left); molecular weight markers in kD (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars). *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). e, Insulin (100 nM), Epidermal Growth Factor (EGF, 50 ng ml⁻¹), PI3K inhibitor (GDC-0941, 2 µM) treatments with concurrent ¹³C₃¹⁵N₁-VB5 labelling (3 h) of serum/growth factor-deprived (18 h) MCF10A cells, pretreated with inhibitor prior to tracing. Protein immunoblots probed with indicated antibodies (left); molecular weight markers in kD (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05).

Extended Data Fig. 3. Data supporting Fig. 1.

a, Pulse-chase labelling of insulin-stimulated MCF10A cells. Cells were serum/growth factor-deprived (18 h), pulse-labelled (4 h) with ¹³C₃¹⁵N₁-VB5, incubated in media without VB5 (1 h), and then chased (3 h) in media with or without ¹³C₃¹⁵N₁-VB5 and insulin (100 nM). Protein immunoblots probed with indicated antibodies (left); molecular size makers in kD (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). b, Validation of LC-MS/MS methods for ¹³C₃¹⁵N₁-VB5 tracing into short-chain acyl-CoAs. Serum/growth factor-deprived (18 h) MCF10A cells were treated (4 h) with insulin (100 nM) and ¹³C₃¹⁵N₁-VB5. Metabolites were extracted under acidic conditions. Unlabelled (M+0) CoA and acetyl-CoA levels are shown for reference. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). c, Intracellular radioactivity counts during chase period after a pulse with radioactive ¹⁴C-VB5. Serum/growth factor-deprived (18 h) MCF10A cells were pulsed (3 h) with radioactive ¹⁴C-VB5 and insulin (100 nM) and then chased in media lacking ¹⁴C-VB5 for 15 min, 1 h, and 3 h to wash out unincorporated ¹⁴C-VB5. Intracellular levels of radioactivity at each timepoint were measured using scintillation counting of disintegrations per minute (DPM). d, PI3K inhibitor (GDC-0941, 2 μΜ) treatment of breast cancer cell lines SUM159, MDA-MB-468, and T47D grown in serum-containing media with concurrent ¹³C₃¹⁵N₁-VB5 labelling (3 h). Protein immunoblots probed with indicated antibodies (left); molecular weight markers (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-way ANOVA with Sidak’s test, (p<alpha 0.05). e, Mouse NIH-3T3 fibroblasts deprived of growth factors and treated with IGF-1 (50 ng ml⁻¹) and PI3K inhibitor (GDC-0941, 2 μM) with concurrent ¹³C₃¹⁵N₁-VB5 labelling (3 h). Cells were incubated with 1% serum but without growth factors (18 h) and pretreated with inhibitor (15 min) prior to treatments. Protein immunoblots probed with indicated antibodies (left); molecular weight markers in kD (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05).

Extended Data Fig. 4. Data supporting Fig. 1.

a, Insulin (100 nM), PI3K inhibitor (GDC-0941, 2 μΜ), and AKT inhibitor (MK-2206 or GDC-0068, 4 μΜ) treatments with ¹³C₃¹⁵N₁-VB5 labelling (3 h). Prior to treatments and labelling cells were serum/growth factor depleted (18 h) and pretreated with inhibitor (15 min). Protein immunoblots from one representative experiment probed with antibodies for total and phosphorylated (p) proteins (left); molecular weight markers in kD (right). Graphed metabolites measured by LC-MS/MS. Mean metabolite levels (bars) from four independent experiments (∇); SEM (error bars). Average for left-most treatment group set to 0% for each metabolite in % change graph. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). b, Total metabolite levels in MCF10A cells expressing doxycycline-inducible HA-tagged wild-type (WT) or constitutively active (E17K) AKT following doxycycline incubation (48 h) and serum/growth factor-deprivation (18 h). Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. Average for left-most treatment group set to 0% for each metabolite. *significant difference from treatment marked (†), two-sided student’s t-test, (p<alpha 0.05). c, Total metabolite levels for experiment shown in main Fig. 1g. AKT inhibitor (GDC-0068, 2 μΜ) and ACLY inhibitor (NDI-091143, 15 μM) treatments with concurrent ¹³C₃¹⁵N₁-VB5 labelling (3 h) of MCF10A expressing doxycycline-inducible HA-tagged wild type (WT) or constitutively active (E17K) AKT. Cells were treated with doxycycline (200 ng ml⁻¹; 48 h), serum/growth factor-deprived (18 h) and pre-treated (15 min) with inhibitor prior to labelling. Graphed metabolites measured by LC-MS/MS. Replicate samples (○); mean (bars); SEM (error bars); n = 3 biological replicates. Average for left-most treatment group set to 0% for each metabolite. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05).

Extended Data Fig. 5. Data supporting Fig. 2.

a, Validation of specificity for commercially available siRNAs and antibodies against endogenous PANK1/2/4. MCF10A cells were treated with a non-targeting control siRNA (siC) or siRNAs targeting individual PANK paralogs, PANK1–4 (siP1–4). Protein immunoblots of whole cell lysates were probed with the indicated PANK antibodies (manufacturer and catalogue numbers listed, left); molecular weight makers in kD (right); non-specific band (∅). Representative of two independent experiments. b, Immunopurifications (IPs) of endogenous PANK4 from growth factor-deprived NIH-3T3 mouse fibroblasts treated (30 min) with IGF-1 (50 ng ml⁻¹), PI3K inhibitor (GDC-0941, 2 μM), and AKT inhibitor (MK-2206, 4 μM). Cells were incubated without growth factors but with 1% serum (18 h) and pretreated with inhibitors (15 min) prior to treatments. IPs and whole cell lysates were probed with a phospho-RXRXXS/T motif antibody and other indicated antibodies (left); molecular weight markers in kD (right). Representative of two independent experiments. c, IPs of endogenous PANK4 from SUM159 breast cancer cells grown in media with 10% fetal bovine serum (FBS) and treated (30 min) with two PI3K inhibitors (GDC-0941, 2 μM and BYL-719, 2 μM). IPs and whole cell lysates were probed with a phospho-RXRXXS/T motif antibody and other indicated antibodies (left); molecular weight markers in kD (right). Representative of two independent experiments. d, Body weight, tumour weight, and tumour volume corresponding to treatments of the mouse mammary allograft from main Fig. 2d. Replicate samples (○). Mean (horizontal line). SEM (error bars); n = 3 biological replicates. e, Endogenous PANK2 and PANK4 IPs from MCF10A cells expressing doxycycline-inducible HA-tagged wild-type (WT) or constitutively active (E17K) AKT, with AKT inhibitor (GDC-0068, 2 μM) or mTORC1 inhibitor (rapamycin, 20 nM) treatments (30 min) preceded by doxycycline incubation (48 h) and serum/growth factor-deprivation (18 h). IPs and whole cell lysates were probed with a phospho-RXRXXS/T motif antibody and other indicated antibodies (left); molecular weight markers in kD (right). Representative of two independent experiments.

Extended Data Fig. 6. Data supporting Fig. 2.

a, Protein sequence alignments of PANK2 orthologues from indicated species based on amino acids surrounding S169 and S189 of the human protein. b, Protein sequence alignments of PANK4 orthologues from indicated species based on amino acids surrounding T406 of the human protein.

Extended Data Fig. 7. Data supporting Fig. 3.

a, Total VB5, CoA, acetyl-CoA levels, and VB5 labelling data from main Fig. 3a. Graphed metabolites measured using LC-MS/MS. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). b, Fractional abundance (M+4/total) of labelled CoA species in main Fig. 3b. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. c, siRNA-mediated knockdown of PANK4 in SUM159 cells with ¹³C₃¹⁵N₁-VB5 labelling (3 hours) in the presence of 10% fetal bovine serum. Protein immunoblots probed with indicated antibodies (left); size markers in kD (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-tailed student’s t-test, (p<alpha 0.05). d, siRNA-mediated knockdown of PANK4 in MDA-MB-468 cells with ¹³C₃¹⁵N₁-VB5 labelling (3 h). Cells were grown in media with dialysed fetal bovine serum (FBS). Protein immunoblots probed with indicated antibodies (left); size markers in kD (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-tailed student’s t-test, (p<alpha 0.05). e, Steady state ¹³C₃¹⁵N₁-VB5 labelling (5 μM, 3–5 days) in SUM159 cells grown in RPMI and 10% FBS which contain ~5 μM unlabelled VB5. Graphed metabolites measured by LC-MS/MS and expressed as fractional abundance of labelled metabolites (M+4/total). Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates.

Extended Data Fig. 8. Data supporting Fig. 3.

a, WT or PANK2 knockout AKT p.E17K/+ MCF10A cells labelled with ¹³C₃¹⁵N₁-VB5 (3 h) in serum-free media with or without defined serum replacement and growth factors. Protein immunoblots probed with indicated antibodies (left); molecular weight markers in kD (right). Graphed metabolites measured using LC-MS/MS. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. b, PANK2 knockout AKT p.E17K/+ MCF10A cells stably expressing empty vector or WT PANK2 in serum-free media, media with defined serum replacement and growth factors, or standard MCF10A media. Protein immunoblots probed with indicated antibodies (left); molecular weight markers in kD (right). Graphed metabolites measured using LC-MS/MS. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates.

Extended Data Fig. 9. Data supporting Fig. 3.

a, Fractional abundance (M+4/total) of labelled metabolites in main Fig. 3c. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-tailed student’s t-test, (p<alpha 0.05). b, Fractional abundance (M+4/total) of labelled metabolites in main Fig. 3d. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), two-tailed student’s t-test, (p<alpha 0.05). c, Two-dimensional proliferation assay with PANK4 KO AKT+/+ MCF10A cells stably expressing empty vector or PANK4 (WT, T406A, or T406E) in serum-free media with serum replacement and growth factors over 4 days. Replicate samples (○). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). Significance based on day 4 values. Representative of two independent experiments.

Extended Data Fig. 10. Data supporting Fig. 4.

a, Divalent metal cation dependence of in vitro PANK4 phosphatase activity. Flag-tag immunopurifications from HEK-293T cells expressing empty vector or full-length wild-type flag-PANK4 were incubated (30 min, 30 °C) with no metal, the chelator EDTA, or indicated metals and the substrate para-nitrophenyl phosphate (PNPP). Chromogenic reaction products measured by spectrophotometry. Relative activity with Co²⁺ reaction set to 1. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). b, In vitro PANK4 phosphatase assay with PANK4 phospho-mutants. Flag-tag immunopurifications from HEK-293T cells expressing empty vector or full-length flag-PANK4 (WT, T406A, or T406E) were incubated (30 min, 30 °C) with Co²⁺ and the substrate PNPP. Chromogenic reaction products measured by spectrophotometry. Relative activity with WT reaction set to 1. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). c, Fractional abundance (M+4/total) of labelled metabolites in main Fig. 4d. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. d, SUM159 PANK4 KO cells stably expressing empty vector, PANK4-WT, or PANK4-D623A with ¹³C₃¹⁵N₁-VB5 labelling (3 h), in the presence of 10% fetal bovine serum. Protein immunoblots probed with indicated antibodies (left); molecular weight markers in kD (right). Graphed metabolites measured by LC-MS/MS. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). e, Protein immunoblot associated with main Fig. 4e. Probed with indicated antibodies (left); molecular weight markers in kD (right). f, Insulin (100 nM) and PI3K inhibitor (GDC-0941, 2 µM) treatment (3 h) in PANK4 KO AKT p.E17K/+ MCF10A cells stably expressing empty vector or PANK4-WT. Cells were pretreated with inhibitor (15 min) prior to insulin stimulation. Total VB5 levels measured by LC-MS/MS. Replicate samples (○). Mean (bars). SEM (error bars); n = 3 biological replicates. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). g, Protein immunoblot associated with main Fig. 4f. Probed with indicated antibodies (left); molecular weight markers in kD (right). h, Mass spectrometry-based lipidomics analysis of PANK4 KO AKT p.E17K/+ MCF10A cells stably expressing empty vector or PANK4 (WT, T406A, or T406E) grown in serum-free media with serum replacement and growth factors. Protein immunoblots probed with indicated antibodies (left); molecular weight markers in kD (right). Significantly altered lipids (n = 3 biological replicates, one-way ANOVA, false discovery rate < 0.1, fold change > 25%) that correlated with CoA levels between cell lines (Patternhunter, Metaboanalyst 4.0, false discovery rate < 0.1) are represented by heatmap as fold change relative to the row mean for each respective lipid species. Lipid classes altered by PANK4 T406 phosphorylation status are represented in pie charts. Relative levels of triacylglycerols in PANK4-expressing cell lines are graphed separately as fold change relative to the mean of the three PANK4-expressing samples for each given triacylglycerol. Replicate samples (○). Mean (bars). SD (error bars); n = 39 triacylglycerol species. *significant difference from treatment marked (†), one-way ANOVA with Tukey’s test, (p<alpha 0.05). i, ¹³C₆-glucose labelling (16 h) of PANK4 KO AKT p.E17K/+ MCF10A cells stably expressing empty vector or PANK4-WT, grown in serum-free media with serum replacement and growth factors, followed by mass spectrometry-based lipidomics analysis. Protein immunoblots probed with indicated antibodies (left); molecular weight markers in kD (right). Since M+3 and above lipid isotopologues were enriched in labelled over unlabelled samples, the lipids for which the total signal for M+3 and above isotopologues was significantly different between vector and PANK4-WT cell lines (n = 3 biological replicates, two-sided student’s t-test, false discovery rate < 0.1, fold change > 20%) were represented by heatmap as fold change relative to the average of vector control samples. Total levels and fractional abundance ((sum of ≥M+3)/total) of significantly altered labelled lipid species are also shown by heatmap. Lipid classes colour- and letter-coded (A-H). j, Diagram of labelled ¹³C₆-glucose tracing into metabolites that generate fatty acids, glycerol backbones, and phospholipid headgroups. Polar metabolites were measured by mass spectrometry from PANK4 KO AKT p.E17K/+ MCF10A cells expressing empty vector, PANK4-WT or PANK4-D623A, and grown in serum-free media with serum replacement and growth factors. Metabolites are colour-coded by significant increase (red), no change (blue), and decrease (green) in PANK4-WT-expressing cells relative to empty-vector-expressing cells. k, Immunoblots of acid-extracted histones from PANK4 KO AKT p.E17K/+ MCF10A cells stably expressing empty vector, PANK4-WT, or PANK4-D623A, and grown in serum-free media with serum replacement and growth factors. Probed with indicated antibodies (left); molecular weight markers (right). Band intensities were quantified using ImageJ and acetyl-histone abundance was normalized to total histone abundance. Relative normalized acetyl-histone quantities are shown below each acetyl-histone band with the vector group set to 1. Representative of two independent experiments. Source data