Author response: PASK links cellular energy metabolism with a mitotic self-renewal network to establish differentiation competence

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Quiescent stem cells are activated in response to a mechanical or chemical injury to their tissue niche. Activated cells rapidly generate a heterogeneous progenitor population that regenerates the damaged tissues. While the transcriptional cadence that generates heterogeneity is known, the metabolic pathways influencing the transcriptional machinery to establish a heterogeneous progenitor population remains unclear. Here, we describe a novel pathway downstream of mitochondrial glutamine metabolism that confers stem cell heterogeneity and establishes differentiation competence by countering post-mitotic self-renewal machinery. We discovered that mitochondrial glutamine metabolism induces CBP/EP300-dependent acetylation of stem cell-specific kinase, PAS domain-containing kinase (PASK), resulting in its release from cytoplasmic granules and subsequent nuclear migration. In the nucleus, PASK catalytically outcompetes mitotic WDR5-anaphase-promoting complex/cyclosome (APC/C) interaction resulting in the loss of post-mitotic Pax7 expression and exit from self-renewal. In concordance with these findings, genetic or pharmacological inhibition of PASK or glutamine metabolism upregulated Pax7 expression, reduced stem cell heterogeneity, and blocked myogenesis in vitro and muscle regeneration in mice. These results explain a mechanism whereby stem cells co-opt the proliferative functions of glutamine metabolism to generate transcriptional heterogeneity and establish differentiation competence by countering the mitotic self-renewal network via nuclear PASK. Editor's evaluation The study by Xiao et al. presents an important finding in the area of metabolic regulation underpinning cell fate decisions in murine muscle stem cells. Combining multiple approaches, the study provides convincing evidence of glutamine-dependent control of the sub-cellular localisation of the kinase PASK and the consequent activation of myogenic programs. The study will be of interest to researchers in the areas of stem cells, regeneration, and metabolic signalling. https://doi.org/10.7554/eLife.81717.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Cell identity is a dynamic feature that must be continually reestablished in proliferating stem cells. To preserve stem cell identity, self-renewing stem cells rapidly reactivate the expression of genes linked with cell identity following cell division. Several mechanisms have been proposed for the post-mitotic reactivation of lineage-defining genes, including the mitotic recruitment of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) by WDR5 to transcriptional start sites of genes regulating pluripotency and H3K27ac mitotic bookmarking (Pelham-Webb et al., 2021; Liu et al., 2017; Oh et al., 2020). During mitosis, the WDR5-APC/C interaction is enhanced, and transient disruption of this interaction causes loss of pluripotency in embryonic stem cells (ESCs) (Oh et al., 2020). However, how differentiation signals counter mitotic self-renewal machinery remains poorly understood. Expression of PAS domain-containing kinase (PASK), a kinase involved in cellular energy balance and metabolic control, positively correlates with the undifferentiated, proliferative state of ESCs and adult stem cells. Functionally, PASK is required for the onset of ESC and adult stem cell differentiation programs downstream of nutrient signaling (Kikani et al., 2019; Kikani et al., 2016). In adult muscles, PASK is required to induce an early regenerative myogenesis program (Kikani et al., 2016). During muscle regeneration, normally quiescent Pax7+ muscle stem cells (MuSCs) enter the cell cycle and undergo proliferative bursts, resulting in the generation of a heterogeneous, activated, self-renewing myoblast population (Pax7+/-, MyoD+/-, Myf5+/-). Signaling cues stimulate the generation of a committed progenitor population (MyoD+, MyoG+), which orchestrates the myogenesis program. We and others have shown that PASK is required to generate the MyoD+, MyoG+ committed progenitor population during myogenesis. Mechanistically, PASK phosphorylates WDR5, a member of the histone H3 lysine 4 methyltransferase (H3K4me3) complexes, in response to differentiation signaling. This phosphorylation sets in motion chromatin remodeling at the Myog promoter and its transcriptional activation resulting in the onset of myogenesis (Kikani et al., 2019; Kikani et al., 2016; Karakkat et al., 2019). This entire pathway is downstream of nutrient-activated mTOR-dependent phosphorylation of PASK, which stimulates the PASK-WDR5 interaction (Kikani et al., 2019). Intriguingly, the PASK function is specifically required to establish the initial committed myoblast progenitor population but is dispensable to sustain myogenesis (Kikani et al., 2019). Since PASK functions at the critical decision point between self-renewal and differentiation, understanding signals that regulate PASK activity and subcellular distribution could provide mechanistic insight into how the exit from self-renewal is regulated in stem cells to generate the committed progenitor population. Signaling cues from the early regenerating niche play critical regulatory roles in facilitating the transition from quiescent, non-proliferative MuSCs to activated, hyper-proliferative myoblasts during the early stages of tissue regeneration (Ryall et al., 2015). Mitochondrial uptake of glutamine is thought to play an essential role in sustaining stem cell proliferation by generating ATP and maintaining redox balance (Yu et al., 2019). During muscle regeneration, glutamine secreted by macrophages sustains myoblast proliferation and promotes differentiation (Shang et al., 2020); however, the precise mechanistic function of glutamine metabolism in driving myoblast differentiation is unclear. Our results discovered a novel mitochondrial-nuclear signaling axis that connects glutamine metabolism in the mitochondria with the mitotic self-renewal network in the nucleus. In this pathway, glutamine metabolic signaling is required to generate a differentiation-primed progenitor population by disrupting the cell cycle-linked WDR5-APC/C interaction via PASK acetylation and nuclear localization. This axis provides new insights into the regulation of stem cell self-renewal and differentiation and identifies key signaling pathways that could be targeted to enhance tissue regeneration. Results PASK inhibition preserves self-renewal and sustains the proliferation of adult stem cells and ESCs PASK is highly expressed in proliferating pluripotent, embryonic, and adult stem cells from mice and humans (Kikani et al., 2016). It is rapidly downregulated following the onset of the differentiation program in all systems resulting in the near absence of PASK expression in most adult tissues under normal physiology (Kikani et al., 2016). Functionally, PASK is required for the onset of terminal differentiation program in embryonic and adult mouse stem cells; however, the role of PASK in self-renewal and stemness properties of stem cells remains unclear. To answer these questions, we cultured mouse embryonic stem cells (mESCs) in the 2i+LIF (2i) condition designed to maintain pluripotency and subsequently replaced the 2i media with PASKi (PASK inhibitor, BioE-1197, Kikani et al., 2016; Wu et al., 2014)+LIF (PASKi) to assess if PASKi can sustain pluripotency after the withdrawal of 2i. Strikingly, replacing 2i media with PASKi in mESCs resulted in a further increase in the expression of genes associated with self-renewal and stemness (Pou5f1, Sox2, and Prex1 mRNA) when compared with mESCs cultured in the 2i conditions (Figure 1A, Figure 1—figure supplement 1A). Additionally, using a Rex1-GFP reporter mESC line (Wray et al., 2011), we observed that cells cultured in PASKi maintained GFP reporter expression at levels comparable to those cultured in 2i (Figure 1B, Figure 1—figure supplement 1B). To compare the differentiation competence of 2i vs. PASKi cultured mESCs, we performed an embryoid body (EB) formation assay of cells grown in 2i or PASKi. Cells grown in the 2i culture condition differentiate well, as seen from the emergence of several fluid-filled cavitated structures after the withdrawal of 2i. Remarkably, PASKi cultured cells showed a substantial increase in the numbers and size of fluid-filled cavitated structures compared with 2i pretreated cells (~52% for PASKi versus ~12% for 2i-treated cells, Figure 1—figure supplement 1C) after PASKi withdrawal. Consistent with our previous study, the presence of PASKi during EB formation attenuated differentiation as assessed morphologically (Figure 1—figure supplement 1C; Kikani et al., 2016). Thus, PASK inhibition in ESCs preserved self-renewal and enhanced differentiation potential upon PASK inhibitor withdrawal. Figure 1 with 4 supplements see all Download asset Open asset Inhibition of PAS domain-containing kinase (PASK) preserves pluripotency, decreases muscle stem cell (MuSC) heterogeneity, and inhibits precocious differentiation. (A) RT-qPCR analysis of indicated transcript levels from 2i+LIF cultured mouse embryonic stem cells (mESCs) after transitioning into 2i+LIF or PASKi+LIF conditions and cultured for 4 days. *p<0.05, error bars ± SD. (B) Rex1-GFP intensity levels from mESCs cultured in 2i vs. in PASKi as in (A). Rex1-GFP reporter expression was quantified by flow cytometry against a non-fluorescent control (control). (C) Isolated primary myoblasts were treated with DMSO or 50 µM PASKi for 4 days during the normal growth phase. Microscopy images were taken at 24 hr or 48 hr post-isolation and treatment. Scale bar = 40 µm. (D) Myogenic transcription factor progression of MuSCs after initial activation from quiescent (Pax7+) state. Activated MuSCs (ASC) are a highly heterogeneous population marked by varying levels and coexpression patterns of Pax7, MyoD, and Myf5. MyoD+ASC further differentiates into MyoG+ committed stem cells (CSC). CSC progenitors initiate a differentiation program to generate myotubes. (E) Expression pattern of myogenic regulatory factors in fluorescence-activated cell sorting (FACS)-sorted PaskWT and PaskKO myoblasts 48 hr after isolation. (F) Quantification of percent Pax7+, MyoD+, and MyoG+ cells from PaskWT and PaskKO animals. Error bars ± SD. *p<0.05, **p<0.005, ***p<0.0005 (KO vs. WT). (G) Fusion index (% nuclei in myotubes/total nuclei) was calculated from MHC-stained cells isolated from PaskWT or PaskKO animals. Error bars ± SD. ***p<0.0005 (KO vs. WT). (H) Experimental setup designed to compare the in vivo regeneration capabilities between PaskWT and PaskKO animals. (I) Embryonic myosin heavy chain (eMHC) staining of fresh-frozen muscle sections from mice of indicated genotype 5 days after muscle injury. DAPI marked nuclei, which are centrally localized in muscle sections from both animals, indicate of muscle regeneration in progress. (J) Quantification of eMHC+ myofiber numbers from experiment in Figure (I). Error bars ± SD. ***p<0.0005 (KO vs. WT). (K) Muscle sections from PaskWT or PaskKO animals were stained with anti-Pax7 (green) antibodies 5 days post-injury and centrally located nuclei were visualized using DAPI. (L) Quantification of Pax7+ cell numbers in PaskWT vs. PaskKO muscle sections 5 days post-injury. Error bars ± SD, ***p<0.0005 (KO vs. WT). (M) Heatmap of differentially expressed genes associated with stemness and myogenesis in C2C12 myoblasts treated with DMSO (control) or PASKi for 2 days. Figure 1—source data 1 Source data used to generate Figure 1. https://cdn.elifesciences.org/articles/81717/elife-81717-fig1-data1-v2.zip Download elife-81717-fig1-data1-v2.zip Figure 1—source data 2 Source data used to generate Figure 1. https://cdn.elifesciences.org/articles/81717/elife-81717-fig1-data2-v2.xlsx Download elife-81717-fig1-data2-v2.xlsx PASK is highly expressed in adult proliferating stem cells (Kikani et al., 2016). Adult MuSCs undergo successive transitions through quiescence, activation, commitment, and differentiation during regeneration in vivo and in culture upon their isolation. While PASK is expressed at negligible levels in adult quiescent MuSCs, its expression increases rapidly as MuSCs are activated and begin to proliferate (Figure 1—figure supplement 2A; Kikani et al., 2016; Liu et al., 2013). To test if PASK inhibition during in vitro activation of MuSCs affects self-renewal, stemness, and differentiation dynamics of adult stem cells, we isolated primary myoblasts from hindlimbs of uninjured mice using flow cytometry-based sorting of Sca-1-, CD31-, CD45-, VCAM1+, α7-Integrin+ cells and cultured them in the presence or absence of PASKi. PASK inhibition was well tolerated by isolated primary myoblasts and caused no apparent proliferation defect. On the other hand, PASKi robustly blocked the precocious myogenesis observed in the control myoblasts for as long as 4 days post-isolation (Figure 1C, Figure 1—figure supplement 2B–D). Taken together, these results suggest that PASK inhibition preserves the self-renewal property of cultured ESCs and adult MuSCs and prevents their precocious differentiation. To mechanistically understand how PASK functions during the onset of myoblast differentiation, we isolated primary myoblasts from PaskWT and PaskKO animals using fluorescence-activated cell sorting (FACS)-based purification (Figure 1—figure supplement 3A). MuSCs isolated from PaskWT or PaskKO were indistinguishable in size 12 hr post-isolation and did not show overt proliferation defects, which is consistent with our previous results and the notion that PASK is not expressed or required for the maintenance or release from a quiescent state. Finally, while PaskWT cells began to form nascent myotubes by 48 hr post-isolation, PaskKO cells continued to proliferate and showed little signs of myotube formation (Figure 1—figure supplement 3B). Thus, genetic or pharmacological loss of PASK results in continued self-renewal of adult myoblasts and impaired differentiation. Quiescent MuSCs express Pax7 but lack MyoD or Myf5 protein expression (Figure 1—figure supplement 2A). Upon their isolation, Pax7+ MuSCs rapidly activate MyoD mRNA translation, and by 48 hr, Pax7+/MyoD+ SCs diverge into a heterogeneous population expressing a combination of Pax7, MyoD, and/or Myf5 (Figure 1D–E; Rocheteau et al., 2012). Interestingly, PaskKO MuSCs showed an increased percentage of Pax7+ myoblasts numbers compared with control (~83% for PaskKO vs. 30% for PaskWT) during 48 hr of culture. Similarly, loss of PASK resulted in reduced levels of MyoD+ myoblasts (Figure 1E–F). Furthermore, the increased proportion of Pax7+ myoblasts observed in PaskKO is independent of the method chosen for stem cell isolation (FACS, magnetic-activated cell sorting [MACS], and pronase-based method) (Figure 1—figure supplement 3C–D). Finally, PaskKO myoblasts showed a marked reduction in committed MyoG-expressing cells and myogenesis, as measured by the fusion index (Figure 1E–G). Thus, genetic loss of PASK increases self-renewing Pax7+ myoblast numbers and decreases the generation of committed (MyoD+/MyoG+) myoblasts in vitro. During adult muscle regeneration, Pax7+ myoblasts begin to proliferate and generate MyoD+/MyoG+ committed progenitor population by 3 days post-injury. These cells begin the regenerative myogenesis program, marked by the emergence of embryonic myosin (eMHC) expression. Loss of PASK severely affected the progression through the regenerative myogenesis program, as seen from decreased eMHC+ myofiber numbers in PaskKO animals compared with littermate controls 5 days post-injury (Figure 1H–I). Furthermore, loss of PASK resulted in a significant expansion of Pax7+ MuSC numbers in PaskKO muscles compared to the littermate control (Figure 1K–L). Combined with decreased MyoD+ myoblasts numbers and loss of MyoG expression in cells isolated from these animals in vitro (Figure 1E–G), our results indicate delayed generation of committed progenitor population in PaskKO animals. Thus, the loss of PASK impairs the transition from Pax7+ stem cells to the MyoD+/MyoG+ committed progenitor population required for the onset of the differentiation program. The increase in Pax7+ cell numbers seen in isolated myoblasts in PaskKO or PASKi-treated cells could be attributed to the suppression of differentiation by the loss of active PASK. Thus, we turned to non-transformed, cultured myoblasts such as C2C12, which can be maintained in a proliferative state for an extended duration under appropriate culture conditions. Furthermore, C2C12 cells express low levels of Pax7 compared with isolated myoblasts and show increased heterogeneity in Pax7 expression (Olguin and Olwin, 2004). Thus, the C2C12 system has been employed to discover cell-intrinsic pathways regulating Pax7 expression and function (Olguin and Olwin, 2004; McKinnell et al., 2008; Sincennes et al., 2021). Using this system, we asked if PASKi treatment during the proliferative phase affects the expression of genes associated with stemness and self-renewal in cultured myoblasts. We performed RNAseq analysis of C2C12 myoblasts treated with PASKi during proliferative, early differentiating, and late differentiating conditions. Our global transcriptomic analysis revealed a strong enrichment of genes associated with stemness and self-renewal at all time-points when PASK is inhibited, starting with the proliferative condition (Figure 1M, Figure 1—figure supplement 4). Furthermore, PASK inhibition preserved self-renewal and stemness despite culture conditions that otherwise stimulate differentiation (Figure 1M, Figure 1—figure supplement 4). Our results in ESCs and MuSCs show that the PASK inhibition is a viable strategy to preserve in vitro self-renewal and pluripotency of ESCs and adult stem cells and is indicative of its functional role in balancing stemness and pluripotency with differentiation. PASK is dynamically redistributed from cytoplasmic granules to the nucleus in a cellular heterogeneity-dependent manner To mechanistically understand how PASK inhibition sustains stem cell proliferation, we examined PASK subcellular distribution in proliferating versus early differentiating myoblasts. In proliferating C2C12 myoblasts (day 0), most of the PASK is localized in the cytosol and excluded from the nucleus (Figure 2A). Curiously, PASK appears to be localized into cytoplasmic granules in cultured myoblasts (Figure 2A, inset). Upon induction of the differentiation program, a significant loss of the cytoplasmic granular staining pattern occurs, and a large fraction of PASK was redistributed into the nucleus, as seen by overlapping intensities of the PASK signal with the nuclear marker DAPI and by biochemical fractionation (Figure 2A–B, Figure 2—figure supplement 1). We next asked if the subcellular distribution of PASK affects Pax7 expression in isolated primary myoblasts. Similar to cultured myoblasts, isolated primary myoblasts showed a strong granular staining pattern of PASK in proliferating myoblasts (Figure 2C). Furthermore, nearly all cells in which PASK was localized into the cytoplasmic granules were strongly positive for Pax7 expression (Figure 2D). In contrast, cells with any extent of nuclear-localized PASK lacked Pax7 expression, even under proliferating conditions (Figure 2D). In addition, in nascent myotubes (Figure 2E, marked by arrow), the PASK localization pattern is switched from within cytoplasmic granules to diffused nuclear, which correlated with the loss of Pax7 positivity (Figure 2F). These results suggest that nuclear translocation of PASK might be associated with heterogeneity in Pax7+ cell numbers seen in proliferating myoblasts (Figure 2D). Consistent with this, under proliferating conditions, Pax7hi (Figure 2G, yellow arrow indicates cells with stronger nuclear levels of Pax7) cells are more frequently associated with cytoplasmic PASK presence, and Pax7L0 (Figure 2G, white arrows indicate cells with relatively weaker nuclear levels of Pax7) or Pax7ab (cells lacking nuclear Pax7) are more frequently seen in cells with noticeable nuclear PASK presence (Figure 2G–H). Interestingly, we noticed asymmetric nuclear distribution of PASK in rapidly dividing myoblasts wherein a cell that asymmetrically retained nuclear PASK exhibited the loss of Pax7 expression post-mitosis, thereby creating heterogeneity in Pax7 expression in culture conditions (Figure 2I–J). Combined with data from Figure 1, our results indicated the mechanistic connection between PASK nuclear translocation and heterogeneity in Pax7 expression in proliferating myoblasts. Figure 2 with 1 supplement see all Download asset Open asset PAS domain-containing kinase (PASK) is localized in the cytoplasmic granules in self-renewing stem cells, which is redistributed to nucleus in Pax7-deficient cells. (A) Proliferating or differentiating C2C12 myoblasts were stained with anti-PASK (green), anti-MHC (MF20, red), or nuclei marker, DAPI antibody. Scale bar = 20 µm. The inset picture shows an enlarged view of the relative distribution of PASK in proliferating or differentiating myoblasts. Notice a granular or punctate staining pattern of endogenous PASK in C2C12 myoblasts in proliferative conditions. (B) Quantification of PASK subcellular distribution as a function of myoblasts state. Error bars ± SD, ***p<0.0005 (Nuclear PASK in Differentiating vs. proliferating). (C) Fluorescence-activated cell sorting (FACS)-sorted muscle stem cells (MuSCs) from uninjured WT mice were stained for PASK and Pax7 24 hr after isolation. Notice a strong, granular staining pattern of PASK in the top panel, which is correlated with Pax7 positivity. The bottom panel shows uniform distribution of PASK, including in the nucleus. The inset picture shows the cytoplasmic granular staining pattern of PASK. (D) Violin plot showing a relationship between the subcellular localization of PASK and Pax7 expression. (E) FACS-sorted myoblasts were fixed 36 hr post-isolation and stained with PASK (green) and Pax7 (red). Arrow indicates nuclear PASK in early mononucleated myotubes. (F) Quantification of images from the experiment in (F). Error bars ± SD, ***p<0.0005 (Pax7+ in nuclear vs. cytosolic PASK). (G) Distribution of Pax7 expression (red) (high, low, or absent, as indicated by Pax7hi, Pax7lo, Pax7ab) in primary myoblasts with more cytosolic (C>N) vs. more nuclear (C<N) PASK (green). The yellow arrow indicates an example of a Pax7hi cell in which the PASK is cytoplasmic. The white arrow indicates Pax7L0 cells in which a large proportion of PASK is diffused nuclear. (H) Quantification of % Pax7+ myoblasts numbers as a function of relative PASK subcellular distribution. (I) Asynchronously proliferating primary myoblasts were stained with PASK and Pax7 antibodies. Notice the exclusion of Pax7 from daughter mitotic cells with asymmetric nuclear localization of PASK (white arrow). Scale bar = 40 µm. Error bars ± SD, ***p<0.0005 (Pax7+ in nuclear vs. cytosolic PASK). Signal-regulated nuclear import-export machinery regulates nucleo-cytoplasmic shuttling of PASK Since PASK inhibition or genetic loss resulted in increased Pax7+ myoblast numbers (Figure 1E–F, Figure 1—figure supplement 3), we hypothesize that nuclear translocation of PASK is the cause and not the effect of decreased Pax7 expression. To directly test this hypothesis, we asked whether forced nuclear retention of PASK could inhibit Pax7 expression and drive exit from self-renewal. To do that, we first fused a powerful SV40 Nuclear Localization Sequence (PKKKRKV, NLS) to GFP-tagged human PASK. To our surprise, NLS-tagging was insufficient to drive PASK into the nucleus (Figure 3A), indicating the possible presence of a powerful nuclear export sequence (NES) in PASK that ensures cytoplasmic localization of PASK in proliferating cells. Consistent with this, treatment of cells expressing NLS-hPASK but not WT-hPASK with nuclear exportin 1 (CRM1) inhibitor, leptomycin B (LMB), resulted in a modestly increased nuclear-localized PASK (Figure 3A). Figure 3 with 1 supplement see all Download asset Open asset Signal regulated nuclear import mechanism for PAS domain-containing kinase (PASK). (A) HEK-293T cells were transfected with GFP-tagged full-length human PASK (aa 1–1323, WT) or GFP-tagged full-length SV40 NLS-PASK (aa 1–1323, NLS-WT). Cells were treated with 25 nM leptomycin B (LMB) for 2 hr as indicated and analyzed by confocal microscopy. The percentage of cells containing any GFP signal in the nucleus in each condition was quantified (% cells showing nuclear [N± SD] GFP). All scale bars = 40 µm. (B) Domain illustration depicting GFP-tagged WT hPASK (aa 1–1323) and its truncated versions, GFP-tagged fragment 737 (aa 1–737), 668 (aa 1–668), and 400 (aa 1–400). The PAS domain is highlighted in red. (C) HEK-293T cells were transfected with GFP-tagged full-length PASK (aa 1–1323, WT) and various truncations. Cells were analyzed by confocal microscopy. The percentage of cells containing any GFP signal in the nucleus was included in the quantification (%N± SD). (D) Diagram showing the locations of two nuclear export sequences (NES1 and NES2) and their sequence homology relative to PGC1 (NES1) and PDPK1 (NES2), respectively. (E) HEK-293T cells were transfected with GFP-tagged full-length WT PASK (aa 1–1323, WT) or NES1 (L403AL405S) or NES2 (L666SL671A). Cells were analyzed by confocal microscopy. The percentage of cells containing any GFP signal in the nucleus in each condition was quantified (% N± SD). (F) HEK-293T cells were transfected with GFP-tagged SV40 NLS-PASK (NLS-hPASK-GFP) containing mutated nuclear export sequences (NES1, NES2, or combined NES 1+2). Cells were analyzed by microscopy. The percentage of cells containing strong GFP signal (GFPhi) in the nucleus in each condition was quantified (% N± SD). (G) HEK-293T cells were transfected with GFP-tagged full-length SV40 NLS-hPASK (aa 1–1323, NLS-PASK-GFP). Cells were serum-starved (0.1% serum) for 12 hr and then subsequently stimulated with either 0.1% serum (serum free) or 20% serum (serum) for 2 hr. Cells were treated with 25 nM leptomycin B (LMB), where indicated and analyzed by confocal microscopy. The percentage of cells containing any GFP signal in the nucleus in each condition was quantified (% N± SD). Figure 3—source data 1 Source data used to generate Figure 3. https://cdn.elifesciences.org/articles/81717/elife-81717-fig3-data1-v2.zip Download elife-81717-fig3-data1-v2.zip Figure 3—source data 2 Source data used to generate Figure 3. https://cdn.elifesciences.org/articles/81717/elife-81717-fig3-data2-v2.zip Download elife-81717-fig3-data2-v2.zip Figure 3—source data 3 Source data used to generate Figure 3. https://cdn.elifesciences.org/articles/81717/elife-81717-fig3-data3-v2.zip Download elife-81717-fig3-data3-v2.zip Previous high-throughput studies have shown the interaction between human PASK and CRM1 (Kırlı et al., 2015). Thus, we considered the possibility that PASK is a nucleo-cytoplasmic shuttling protein containing one or more NES and that regulated import and/or export may result in its nuclear localization at the onset of differentiation. Proteins smaller than 60 kDa could migrate to the nucleus by diffusion (Nigg, 1997). Considering this size limitation, we performed a series of C-terminal truncations in PASK to identify the region that mediates PASK nuclear export (Figure 3B). Scoring for cells showing at least some nuclear GFP presence, we found that the GFP-WT-PASK (MW=~200 kDa) and GFP-1–737 (MW=~115 kDa) fragment remained predominantly cytoplasmic (Figure 3C). A smaller fragment, GFP-1–660 (MW=~100 kDa) showed nuclear GFP expression in ~22% of cells (Figure 3C), while fragment GFP-1–400 (MW=~70 kDa) showed increased nuclear localization of PASK with ~67% of cells showing at least some nuclear GFP presence (Figure 3C). These results suggested the presence of NES between amino acids 400 and 660 and between amino acids 660 and 737. We used multiple bioinformatic tools to identify L401–L409 (NES1) and L666–L671 (NES2) as putative NES in PASK (Figure 3D, Figure 3—figure supplement 1; Figure 3—source data 1 and 2; Xu et al., 2015; Xu et al., 2021). The NES1 residues are similar to an experimentally verified NES in PGC1-α (Chang et al., 2010), and the NES2 residues are similar to the NES of PDK1 that we previously discovered (Figure 3D; Lim et al., 2003). We found that mutation of either NES1 or NES2 residues resulted in a modest but statistically significant increase in the number of cells with nuclear PASK localization compared to WT-PASK or NLS-PASK in the absence of LMB (Figure 3E). Combined mutation of NES1 and NES2 resulted in a significantly increased proportion of cells that contain nuclear PASK, with the extent of nuclear PASK in each cell similar to what we observed for PASK during myogenesis (Figure 3E). However, incomplete nuclear retention of NES1+NES2 mutated PASK prompted us to examine if the nuclear import of PASK might be rate-limiting, preventing stronger PASK nuclear accumulation despite NES1 and NES2 mutations (see Figure 3—source data 3 for a list of mutant nomenclature in Figure 3—figure supplement 1). Consistent with that, while mutations of individual NES1 or NES2 in NLS-PASK improved nuclear retention of NLS-PASK (Figure 3F), mutating both NES1 and NES2 together in NLS-PASK resulted in robust nuclear retention of NLS-PASK in nearly 100% of cells (Figure 3F). These results conclusively s