The nuclear phosphoinositide response to stress

ABSTRACT

Accumulating evidence reveals that nuclear phosphoinositides (PIs) serve as central signaling hubs that control a multitude of nuclear processes by regulating the activity of nuclear proteins. In response to cellular stressors, PIs accumulate in the nucleus and multiple PI isomers are synthesized by the actions of PI-metabolizing enzymes, kinases, phosphatases and phospholipases. By directly interacting with effector proteins, phosphoinositide signals transduce changes in cellular functions. Here we describe nuclear phosphoinositide signaling in multiple sub-nuclear compartments and summarize the literature that demonstrates roles for specific kinases, phosphatases, and phospholipases in the orchestration of nuclear phosphoinositide signaling in response to cellular stress. Additionally, we discuss the specific PI-protein complexes through which these lipids execute their functions by regulating the configuration, stability, and transcription activity of their effector proteins. Overall, our review provides a detailed landscape of the current understanding of the nuclear PI-protein interactome and its role in shaping the coordinated response to cellular stress.

KEYWORDS:

• Nuclear localization
• phosphoinositides signaling
• stress response
• phosphoinositide effectors

Introduction

Phosphoinositides (PIs) are lipid messengers that play essential roles in the regulation of a wide array of cellular processes. The myo-inositol ring in phosphatidylinositol (PtdIns) can be phosphorylated on the 3, 4, and 5 hydroxyls in all possible combinations to generate 7 distinct PIs (Figure 1). The monophosphorylated PtdIns phosphates (PtdIns3P, PtdIns4P, and PtdIns5P) are the simplest inositol phospholipids. They can be further phosphorylated forming PtdIns(3,4)P₂, PtdIns(3,5)P₂, PtdIns(4,5)P₂, and PtdIns(3,4,5)P₃. The PI signaling cycle was discovered by Lowell and Mabel Hokin in the 1950s [1]. As the minor constituents of membranes, PIs are present in the plasma membrane, Golgi, endoplasmic reticulum, endosomes, lysosomes, as well as in the nucleus [2]. Initially, research efforts focused on the biochemical and functional mechanisms of the cytosolic membrane-associated PIs.

Figure 1. Nuclear PI metabolism.

Phosphatidylinositol (PtdIns) structure and nuclear PI cycle. Sites of phosphorylation on the hydroxyl groups of the inositol head are indicated with yellow stars and carbon atom numbers are shown in red. The nuclear PIs, kinases, phosphatases, and phospholipases are shown as in Tables 1 and 2. IP₃: Ins(1,4,5)P₃. PtdIns(4,5)P₂ 4-Ptase I: Type I PtdIns(4,5)P₂ 4-phosphatase.

In 1965 and 1977, researchers demonstrated the existence of phospholipids in the nucleus [3,4]. Subsequently in 1983, Smith and Wells detected the activity of phosphatidylinositol kinase and phosphatidylinositol phosphate kinase in the nucleus [5], which inspired the research work of Cocco et al. in 1987 to discover the generation of PIs in the nucleus [6]. We demonstrated that multiple PIs and the kinases that synthesize them are localized to nuclear speckles containing pre-mRNA processing factors that are separate from membranes [7]. These studies expanded the horizon of canonical cytoplasmic PI signaling into the nucleus. In the past 30 years, substantial evidence supports that multiple PIs are present in diverse sub-nuclear compartments along with kinases and phosphatases that generate different nuclear PI pools [8–10]. Additionally, it has become evident that nuclear PI signaling regulates proteins involved in diverse nuclear functions, particularly stress responses. This review will focus on nuclear PIs, their sub-nuclear localization, and their signaling in response to cell stress.

The nuclear PIs and their responses to cellular stress

As the precursor of all PIs (Figure 2), PtdIns is the most abundant inositol lipid in mammalian cells, accounting for approximately 80% of total cellular PIs [11]. PtdIns4P and PtdIns(4,5)P₂ are the most abundant mono- and bi-phosphorylated PIs, which are present at roughly equal abundance in cells (2 ~ 5% of total cellular PIs), constituting approximately 90% of the phosphorylated PtdIns [11–14]. PtdIns3P makes up about 0.2 ~ 0.5% of the total cellular PIs, while PtdIns5P represents approximately 0.01 ~ 0.2% of the total cellular PIs [11,15]. The other 3-phosphorylated PIs, PtdIns(3,4)P₂ (<0.1% of total cellular PIs), PtdIns(3,5)P₂ (<2% of total cellular PIs), and PtdIns(3,4,5)P₃ (<0.05% of total cellular PIs) are typically present at low level or remain undetectable in non-stimulated cells [11,12,14].

Figure 2. Abundance of PIs in eukaryotic cells.

Numbers showing here are the estimated percentages of each PI of the total PIs in eukaryotic cells.

In contrast to the cytosolic PIs which are mainly associated with membranes, the nuclear PIs localize to both membrane and non-membrane structures [16–19] (Figure 3). To date, all PIs except PtdIns(3,5)P₂ have been detected in the nucleus [8,20,21] (Table 1). The different compartmentalization of nuclear PIs suggests their broad involvement in multiple signaling pathways.

Table 1. Nuclear targeted PIs: types, locations, and response to cell stress

Inositol molecules	Nuclear Location	Response to cell stress
PtdIns3P	Nuclear Matrix [24], Nucleoli [22,23]	Nuclear level increased upon genotoxic stress [25], Induced generation in the nuclear matrix upon partial hepatectomy [24]
PtdIns4P	Nuclear envelope [27], Nuclear Matrix [24], Nucleoplasm [23], Nuclear Speckles [23], Nucleoli [23], Chromatin [28]	Accumulated at DNA damage sites and involved in the repair machinery [28] Induced upon hypertrophic stress in the perinuclear region to serve as the substrate for PLCε at the nuclear envelope [27]
PtdIns5P	Nuclear Matrix [29], Chromatin [29,133]	Nuclear level increased upon genotoxic and oxidative stress, reduced upon oxidative attenuation [30,31]
PtdIns(3,4)P2	Nuclear envelope [37], Nuclear speckles [35], Nucleoli [36]	Nuclear level increased upon oxidative stress [37]
PtdIns(4,5)P2	Nuclear envelope [40,44], Nuclear Matrix [23,24,95], Nucleoplasm [23], Nuclear Speckles [23,38,98], Nucleoli [23,38,40,44], Chromatin [40], Nucleoplasmic Ca^2+ store vesicles [39], Nuclear lipid islets [41]	Nuclear level increased upon genotoxic stress [25,28]; Local enrichment at DNA damage sites [25,28]; Induced generation in the nuclear speckles upon genotoxic and oxidative stress [42,43]; Induced generation in the nuclear envelope and nucleoli upon heat stress [44];
PtdIns(3,4,5)P3	Nuclear Matrix [35,45], Nucleoplasm [46], Nuclear speckles [35,98], Nucleoli [46], Chromatin [28,79]	Nuclear level increased upon genotoxic stress [25] and proliferative stimuli [45]; Induced generation in the nucleus upon oxidative stress [47] and proliferative stimuli [47]; Accumulation at DNA damage sites upon UV irradiation [28]
DAG	Nuclear envelope [27], Inner membrane [50], Nuclear lipid droplets [50], Nuclear Matrix [51]	Nuclear level increased upon hypertrophic stress [57], proliferative stimuli [56] and differentiation stimuli [56]; Synthesized by PLCε at the nuclear envelope upon hypertrophic stress [27]
Ins(1,4,5)P3	Nuclear envelope [52], Outer membrane [52], Inner membrane [52,53], Nucleoplasmic reticulum [54], Nucleoplasmic Ca^2+ store vesicles [39]	Mediating chemical-induced hypertrophy for pathological cardiomyocyte growth [58]

Figure 3. Sub-nuclear localizations of PIs, kinases, phosphatases, phospholipases and PI-binding proteins.

Demonstration of sub-nuclear localizations of PIs, kinases, phosphatases, phospholipases and PI-binding proteins as listed in Tables 1, 2, and 3. IP₃: Ins(1,4,5)P₃. PtdIns(4,5)P₂ 4-Ptase I: Type I PtdIns(4,5)P₂ 4-phosphatase.

PtdIns3P

PtdIns3P was detected in the dense fibrillar component within nucleoli by PtdIns3P-binding FYVE domains using electron/fluorescent microscopy [22,23]. Newly-synthesized PtdIns3P was found in membrane-depleted nuclei of liver cells post partial hepatectomy, which is proposed to trigger oxidative stress and liver injury [24]. These observations indicate PtdIns3P localization in the nuclear matrix, the structure remaining after harsh nuclear extraction, which includes but is not limited to the nucleoskeleton, and suggest the involvement of PtdIns3P in stress response. Our recent work shows that cisplatin increases the nuclear level of PtdIns3P identified by immunofluorescent staining [25], suggesting a potential role of PtdIns3P in response to genotoxic stress.

PtdIns4P

Newly-synthesized PtdIns4P was detected in the nucleus by incorporating radiolabeled phosphate in purified liver nuclei [24]. The incorporation of ³²P into PtdIns4P was also detected in membrane-depleted liver nuclei and after addition of exogenous PtdIns as the substrate [24], suggesting the de novo synthesis of PtdIns4P in the nuclear matrix and the nuclear activation of PtdIns 4-kinases. Using a specific antibody and immunofluorescence, PtdIns4P localized within nuclear speckles, small foci in the nucleoplasm, and nucleoli [23]. Moreover, PtdIns4P was detected at larger foci in the nucleoplasm and nuclear speckles using purified PtdIns4P-binding domain OSH1-PH in fluorescence microscopy [23]. Interestingly, the distribution pattern of PtdIns4P resembles that of nuclear PtdIns(4,5)P₂ [23]. Additionally, during the cell cycle, nuclear PtdIns4P levels peak at G₁ and then decreases through S phase [26]. The nuclear PtdIns(4,5)P₂ turnover also fluctuates through the cell cycle with a peak at the G₁/S transition [26]. These studies suggest nuclear PtdIns4P serves as a precursor for PtdIns(4,5)P₂.

PtdIns4P also functions at the nuclear envelope, where it serves as the substrate for the perinuclear phospholipase Cε (PLCε) to generate diacylglycerol (DAG), which further activates nuclear protein kinase D (PKD) [27]. This pathway is activated by hypertrophic stimuli and is responsible for the stress-induced pathological hypertrophy [27]. Upon UV irradiation, PtdIns4P detected by SidM-P4M domain rapidly accumulated at DNA damage sites, and the sequestration of PtdIns4P by the SidM-P4M domain partially suppressed Ataxia telangiectasia and Rad3-related protein (ATR)-dependent DNA damage signaling [28], suggesting an association of PtdIns4P with chromatin and a role in DNA damage repair.

PtdIns5P

Nuclear PtdIns5P was first detected in murine erythroleukaemia cells with a 20-fold increase at G₁ phase [26]. In 2003, ING2 (inhibitor of growth protein-2) was identified as a PtdIns5P-binding protein through the plant homeodomain (PHD) finger domain in membrane-stripped nuclei, suggesting an association of PtdIns5P-ING2 complex with the nuclear matrix and chromatin [29]. Strikingly, this localization was decreased upon overexpression of PIPKIIβ, which controls the PtdIns5P pool by catalyzing its conversion into PtdIns(4,5)P₂ [29]. This finding suggests the complex with PtdIns5P is required for the nuclear retention of ING2. Genotoxic stress (UV irradiation and etoposide) and oxidative stress (H₂O₂ and L-buthionine[S,R]-sulphoximine (BSO)) specifically induce the accumulation of PtdIns5P in the nucleus [30,31]. In contrast, attenuation of oxidative stress by the antioxidant N-acetyl-cystine (NAC) lowers the level of nuclear PtdIns5P [30]. There is also evidence that activation of the nuclear ubiquitin ligase Cul3-SPOP by the generation of PtdIns5P and its conversion to PtdIns(4,5)P₂ by PIPKIIβ [32]. The connection between cellular stressors and nuclear PtdIns5P levels points to multiple roles of nuclear PtdIns5P in stress signaling [33].

PtdIns(3,4)P₂

PtdIns(3,4)P₂ is actively involved in cellular stress responses. Proliferative stimuli, oxidative stress, and osmotic stress all induce PtdIns(3,4)P₂ generation in cells [34]. However, little is known about nuclear PtdIns(3,4)P₂ and its role in stress. Incubation of radiolabeled PtdIns(3,4,5)P₃ with nuclear extract resulted in the detection of radiolabeled PtdIns(3,4)P₂ [35], suggesting de novo synthesis of PtdIns(3,4)P₂ and activation of PtdIns 5-phosphatases in the nucleus. Indeed, Src homology 2 domain-containing inositol phosphatase isoform 2 (SHIP2) is the primary enzyme for metabolizing PtdIns(3,4,5)P₃ into PtdIns(3,4)P₂ in the nucleus [35]. The detection of SHIP2 in nuclear speckles [35] and another PtdIns(3,4,5)P₃ 5-phosphatase, SHIP1, in nucleoli [36] suggests the localization of PtdIns(3,4)P₂ in those sub-nuclear compartments. Using a specific monoclonal antibody, PtdIns(3,4)P₂ was detected at the nuclear envelope [37], while its potential intranuclear localization could not be assessed in this study because the nuclear membrane was not permeabilized. Interestingly, H₂O₂ induced the nuclear surface staining of PtdIns(3,4)P₂ [37], suggesting the involvement of PtdIns(3,4)P₂ in oxidative stress response.

PtdIns(4,5)P₂

Newly-synthesized PtdIns(4,5)P₂ was detected in the nucleus by incorporating radiolabeled phosphate in highly purified liver nuclei [24]. The incorporation of ³²P into PtdIns(4,5)P₂ was also detected in membrane-depleted liver nuclei and after addition of exogenous PtdIns4P as the substrate [24], suggesting the de novo synthesis of PtdIns(4,5)P₂ in the nuclear matrix and the nuclear activation of PtdIns4P 5-kinases. Using a specific antibody, PtdIns(4,5)P₂ was detected by immunofluorescence at nuclear speckles as well as weak staining in nucleoli [23,38]. Also, a monoclonal antibody detected PtdIns(4,5)P₂ at Ins(1,4,5)P₃-sensitive nucleoplasmic vesicles, serving as the precursor for Ins(1,4,5)P₃ generation, which controls the nuclear Ca²⁺ pool [39]. Interestingly, while purified PtdIns(4,5)P₂-binding domain-Tubby detected PtdIns(4,5)P₂ predominantly in the nucleoli, the other purified PtdIns(4,5)P₂-binding domain PLCδPH recognized PtdIns(4,5)P₂ in both nucleoli and nuclear speckles by fluorescence microscopy [23]. Moreover, using purified PLCδPH followed by immunoelectron microscopy, PtdIns(4,5)P₂ labeling was concentrated at heterochromatin with weaker labeling at the nuclear envelope [40]. Additionally, a recent study shows PtdIns(4,5)P₂ decorates nuclear lipid islets, where it associates with Pol II transcription machinery, pointing to a potential role in transcriptional regulation [41].

Genotoxic stress, including UV irradiation, the radiomimetic alkylating reagent MMS, and cisplatin increase nuclear PtdIns(4,5)P₂ levels [25,28]. Furthermore, the DNA damage agent etoposide and oxidative stress inducer-tert-Butylhydroquinone (tBHQ) specifically promote the generation of PtdIns(4,5)P₂ in the nuclear speckles [42,43]. When exposed to UV irradiation, PtdIns(4,5)P₂ detected by PLCδPH domain rapidly accumulated at DNA damage sites, and the sequestration of nuclear PtdIns(4,5)P₂ by PLCδPH domain suppressed ATR-dependent DNA damage signaling [28]. This is in accord with our recent results that cisplatin treatment induced the local enrichment of PtdIns(4,5)P₂ at DNA damage sites [25], suggesting a role of PtdIns(4,5)P₂ upon DNA damage stress. In plants, heat shock rapidly induced the generation of PtdIns(4,5)P₂ with a specific accumulation at the nuclear envelope and nucleoli [44], suggesting a functional role of PtdIns(4,5)P₂ in the heat shock response, which maintains plant homeostasis and facilitates survival during periods of elevated temperatures.

PtdIns(3,4,5)P₃

Using the purified PtdIns(3,4,5)P₃-binding GST-Grp1-PH domain and electron microscopy, PtdIns(3,4,5)P₃ was detected in the nuclear matrix [45]. Consistently, dephosphorylation of [³²P]PtdIns(3,4,5)P₃ by its 5-phosphatase SHIP2 and 3-phosphatase PTEN (phosphatase and tensin homolog) was found in membrane-depleted nuclei [35], supporting PtdIns(3,4,5)P₃ localization in the nuclear matrix. Additionally, SHIP2 colocalized with the SC35 splicing factor [35], suggesting the presence of PtdIns(3,4,5)P₃ in nuclear speckles. Moreover, PtdIns(3,4,5)P₃ was detected in the nucleoplasm and nucleoli by immunofluorescence [46].

Newly-synthesized PtdIns(3,4,5)P₃ was detected in the nucleus by incorporating radiolabeled phosphate in highly purified liver nuclei [24]. Interestingly, when nuclei were depleted of their membrane, and after addition of exogenous PtdIns(4,5)P₂ as the substrate, no radiolabeling of PtdIns(3,4,5)P₃ was detected [24], suggesting either the nuclear PtdIns(4,5)P₂-3 kinases are membrane-associated or they function only on anchored substrates.

Genotoxic stress by cisplatin [25] and proliferative stimulation by platelet-derived growth factor (PDGF) [45] have been reported to increase nuclear PtdIns(3,4,5)P₃. Upon UV irradiation, PtdIns(3,4,5)P₃ detected by Btk-PH domain was rapidly accumulated at DNA damage sites, and the sequestration of nuclear PtdIns(3,4,5)P₃ by Btk-PH domain suppressed ATR-dependent DNA damage signaling [28], suggesting an association of PtdIns(3,4,5)P₃ with chromatin and a role in DNA damage repair. Other cellular stimuli such as oxidative stress (H₂O₂) and proliferative stimuli (nerve growth factor (NGF) and PDGF) have also been reported to induce the generation of PtdIns(3,4,5)P₃ in the nucleus [47].

DAG and Ins(1,4,5)P₃

Hydrolysis of PtdIns(4,5)P₂ by PI-PLC (PI-specific phospholipase C) leads to the generation of diacylglycerol (DAG) and Ins(1,4,5)P₃, and subsequent activation of Ins(1,4,5)P₃ receptors for Ca²⁺ release and the DAG-mediated protein kinase C (PKC) pathway [48,49]. DAG localizes with the nuclear envelope [27] (especially at the inner membrane [50]), nuclear lipid droplets [50], and the nuclear matrix [51], whereas Ins(1,4,5)P₃ receptors localize with the nuclear envelope [52], including both outer and inner membrane [52,53], nucleoplasmic reticulum [54], and nucleoplasmic Ca²⁺ store vesicles [39], mostly governing the intranuclear calcium signaling for cell survival. The presence of PLCβ1 at nuclear speckles implicates that its activity generates DAG and Ins(1,4,5)P₃ at these sites [55]. The nuclear DAG level increases in response to proliferative signals such as insulin-like growth factor-I (IGF-I) and differentiation stimuli such as dimethyl sulfoxide [56]. The nuclear DAG and Ins(1,4,5)P₃ are actively involved in Endothelin-1 (ET1)-induced hypertrophic stress as ET-1 elevates nuclear DAG levels [57] and induces the synthesis of DAG by PLCε-dependent hydrolysis of PtdIns4P at the nuclear envelope [27]. Moreover, the induction of nuclear Ins(1,4,5)P₃ mediates ET1-induced hypertrophy for pathological cardiomyocyte growth [58].

The nuclear PI kinases, phosphatases, and phospholipases

Nuclear PI metabolism is tightly regulated by multiple kinases, phosphatases, and phospholipases that allow dynamic lipid turnover that is temporally and spatially regulated. The different compartmentalized enzymes actively participate in nuclear signaling and stress response (Table 2 and Figure 3).

Table 2. Nuclear PI-metabolizing enzymes, their substrates, nuclear location and response to cell stress

Nuclear PI kinases
Enzyme	Substrate	Nuclear location	Response to cell stress
PI4KIIα	PtdIns [59]	Nucleus [61], Nucleoplasmic Ca^2+ store vesicles [39]	Unknown
PI4KIIβ	PtdIns [59]	Nucleoplasmic Ca^2+ store vesicles [39]	Unknown
PI4KIIIα	PtdIns [59]	Nucleoplasm [61], Nucleoli [61,157]	Unknown
PI4KIIIβ	PtdIns [59]	Nuclear lamina-pore complexes [62], Nuclear speckles [63]	Unknown
PIPKI⍺	PtdIns4P [25]	Nuclear matrix [8], Nucleoplasm [25], Nuclear speckles [7,25,43], Nucleoli [66], Chromatin [25]	Accumulated in the nuclear speckle under oxidative stress [43]; Induced localization to DNA damage sites [25]
PIPKIɣ_i4	PtdIns4P [65]	Nuclear matrix [8], Nuclear speckles [65]	Unknown
PIPKII⍺	PtdIns5P [7]	Nuclear matrix [8], Nuclear speckles [7]	Unknown
PIPKIIβ	PtdIns5P [69]	Nuclear matrix [8], Nuclear speckles [7,32], Chromatin [69,70]	Activated for PtdIns(4,5)P2 generation and E-cadherin upregulation upon anti-cancer drug [69]; Antagonizing the genotoxic stress-induced PtdIns5P response [70]
Class I PI3K, p85⍺	-	Nucleus [47]	Increased nuclear level with considerable tyrosine phosphorylation upon oxidative stress [47]
Class I PI3K, p85β	-	Nucleus [72]	Unknown
Class I PI3K, p110⍺	PtdIns(4,5)P2 [71]	Nucleus [74], Chromatin [74]	Unknown
Class I PI3K, p110β	PtdIns(4,5)P2 [71]	Nucleoli [46], Chromatin [74]	Increased association with chromatin upon proliferative stimuli [72]; Induced accumulated in DNA damage sites [73].
Class II⍺ PI3K	PtdIns [71], PtdIns4P [71]	Nuclear speckles[75]	Unknown
Class IIβ PI3K	PtdIns [71], PtdIns4P [71]	Nuclear envelope [158], Nuclear matrix [24,76]	Induced nuclear level and activity upon proliferative stimuli [76]; Activated for PtdIns3P generation in the nucleus upon partial hepatectomy [24]
IPMK	PtdIns(4,5)P2 [79]	Chromatin [80]	Recruited to the DNA damage sites for PtdIns(3,4,5)P3 production and ATR repair machinery [28]; Induced association with p53 for transcription regulation and cell death upon genotoxic stress [80]
Nuclear PI phosphatases
PTEN	PtdIns(3,4,5)P3 [35]	Nucleoli [62], Chromatin [83,84]	Induced nuclear import, chromatin association, and Rad52 interaction for DNA damage repair upon genotoxic and oxidative stress [84]
SHIP1	PtdIns(3,4,5)P3 [36]	Nucleoli [36]	Induced accumulation and association with p53 in nucleoli upon ER stress [36]
SHIP2	PtdIns(3,4,5)P3 [35], PtdIns(4,5)P2 [91]	Nuclear speckles [35,91]	Unknown
Type I PtdIns(4,5)P2 4-phosphatase	PtdIns(4,5)P2 [92]	Nucleus [92]	Translocated into the nucleus upon genotoxic stress [92]
Nuclear phospholipases
PLCβ1	PtdIns(4,5)P2 [56]	Nuclear matrix [93], Nuclear speckles [55]	Activated for the generation of DAG from PtdIns(4,5)P2 upon proliferative stimuli [56]; Protected cells from oxidative stress by inducing c-fos mRNA generation [94]
PLCδ1	PtdIns(4,5)P2 [95]	Nuclear matrix [95]	Translocated to the nucleus upon cell cycle arresting [95]
PLCδ4	PtdIns(4,5)P2 [96]	Nucleus [96]	Unknown

PtdIns 4-kinases

PtdIns4P is primarily produced by the action of type II (α and β) and III (α and β) PtdIns 4-kinases [59]. The missing type I was a historical artifact which turned out to be PtdIns 3-kinase [60]. Although PtdIns 4-kinase activity has been detected in the nucleus [24], very little is known about the nuclear PtdIns 4-kinases. Using immunofluorescence, PI4KIIIα was detected in the nucleoplasm and nucleoli, whereas PI4KIIα displayed moderate cytoplasmic localization and more intense nuclear staining [61]. PI4KIIα and PI4KIIβ have been detected on nucleoplasmic Ca²⁺ store vesicles, where they produce PtdIns4P as the precursor for PtdIns(4,5)P₂ and downstream Ins(1,4,5)P₃ generation for the control of nucleoplasmic Ca²⁺ concentration [39]. Through subcellular fractionation, PI4KIIIβ was detected at the nuclear lamina-pore complexes, and the inhibition of nuclear export by leptomycin B resulted in its accumulation in the nucleus [62]. Consistently, the association of specifically-phosphorylated forms of PI4KIIIβ (Ser-496/Thr-504) with nuclear speckles also supports the nuclear localization of PI4KIIIβ [63]. All PtdIns 4-kinases contain a nuclear localization signal (NLS) and nuclear export signal (NES) [64], suggesting their involvement in nucleo-cytoplasmic traffic. Although the functional role of nuclear PtdIns 4-kinases remain unclear, the involvement of nuclear PtdIns4P in stress responses suggests a role in nuclear signaling.

PIP kinases

Both type I and II PIP kinases contain α, β, and γ isoforms. While type I PIP kinases primarily catalyze the conversion of PtdIns4P into PtdIns(4,5)P₂, type II PIP kinases predominantly catalyze synthesis of PtdIns(4,5)P₂ from PtdIns5P. To date, PIPKIα, PIPKIγ_i4, PIPKIIα, and PIPKIIβ have all been found in the nucleus in association with nuclear matrix [7,8,25,43,65]. PIPKIα and PIPKIIα localize to nuclear speckles, where they colocalize with PtdIns(4,5)P₂ and mRNA-processing machinery [7]. Moreover, immunofluorescence shows the enrichment of PIPKIα in nucleoli at G₁/S phase of the cell cycle [66]. PIPKIα contains a nuclear speckle-targeting region (440–562) and accumulates in nuclear speckles upon oxidative stress where it associates with the poly (A) polymerase Star-PAP, controlling 3ʹ-end processing of select stress-induced mRNAs [43]. In response to genotoxic stress, PIPKIα localizes to sites of DNA damage, suggesting a role in DNA damage repair consistent with an accumulation of phosphoinositides at these sites [25,28]. PIPKIγ generates PtdIns(4,5)P₂ in focal adhesion sites and adherens junctions [65]. There are at least four PIPKIγ splicings in humans, i1, i2, i4, and i5 [65]. Among them, PIPKIγ_i4 is found in both the cytosol and the nucleus, and in nuclei colocalizes with speckle markers [65]. PIPKIIβ is enriched in nuclear speckles [7,32,67,68]. Three-dimensional structural analyses and point mutations indicate that α-helix 7 and insert region of PIPKIIβ is essential for its nuclear localization and its interaction with the speckle-type POZ domain protein (SPOP) a component of Cul3-based ubiquitin ligases [32,67]. Upon treatment with the anti-cancer vitamin D metabolite-1α,25(OH)2D3, nuclear PIPKIIβ is activated and generates PtdIns(4,5)P₂, leading to E-cadherin upregulation and inhibition of cell motility [69]. PIPKIIβ functions in the PtdIns5P pathway [32], PtdIns5P responds to the genotoxic agent etoposide and regulates the expression of myogenic genes during myoblast differentiation [70].

PtdIns 3-kinases

PtdIns 3-kinases (PI3Ks) are divided into three classes. Class I PI3Ks are the dominant kinases that synthesize PtdIns(3,4,5)P₃, and class III PI3Ks are responsible for synthesizing PtdIns3P. Class II PI3Ks synthesize both PtdIns3P and PtdIns(3,4)P₂ to a lesser extent [71].

Class I PI3Ks functions as heterodimers consisting of a regulatory subunit (p85⍺ or its two splicing variants (p55⍺ and p50⍺), p85β, p55γ, p101 or p84/p87) and a catalytic subunit (p110⍺, p110β, p110δ or p110γ). The class I PI3Ks are involved in nuclear signaling and stress response. Upon H₂O₂-induced oxidative stress, the nuclear fraction of p85⍺ is markedly increased with considerable tyrosine phosphorylation [47]. p85β contains an NES whereas p110β contains an NLS in its C2 domain [72]. Together these signals regulate the nuclear localization of p85β/p110β and control cell survival [72,73]. While p110β is mainly nuclear in starved cells, proliferative stimuli (NGF, serum, and PDGF) increase its association with chromatin [72]. Consistently, both p110⍺ and p110β are associated with Akt and proliferating cell nuclear antigen (PCNA) in the nucleus, with p110β being the dominant isoform that regulates PCNA loading onto chromatin for DNA replication and cell proliferation [74]. Moreover, p110β and its product-PtdIns(3,4,5)P₃ accumulate at sites of DNA damage following UV irradiation, where p110β senses double-strand breaks (DSB) and recruits the Nbs1 sensor protein to damaged DNA [73]. Inhibiting the kinase activity of p110β inhibits DNA damage repair [73], indicating a functional role of PtdIns(3,4,5)P₃ in genotoxic stress signaling.

The C2 domain of the class II PI3K-CIIα also contains an NLS, which is essential for its nuclear localization [75]. Indeed, PI3K-CIIα concentrates in nuclear speckles and colocalizes with snRNA [75]. Treating cells with transcription inhibitors α-amanitin and actinomycin D results in the phosphorylation of PI3K-CIIα [75]. Similarly, the C2 domain of PI3K-CIIβ also contains an NLS [76]. PI3K-CIIβ co-localizes with lamin A/C in the nuclear matrix and is activated in membrane-depleted nuclei [24,76]. PI3K-CIIβ is also found in the nuclear envelope where it exhibits robust activity [75]. Stimulation with epidermal growth factor augments PI3K-CIIβ expression and kinase activity in the nucleus [76]. Post partial hepatectomy, PI3K-CIIβ activation occurs in membrane-depleted nuclei, leading to PtdIns3P production during compensatory liver growth [24], suggesting a role of PI3K-CIIβ in stress responses.

IPMK

Inositol polyphosphate multikinase (IPMK) functions as PI 3-kinase in the nucleus, converting PtdIns(4,5)P₂ to PtdIns(3,4,5)P₃ [77,78]. Overexpressed IPMK colocalizes with and increases nuclear PtdIns(3,4,5)P₃ [77]. IPMK converts nuclear receptor Steroidogenic Factor-1 (SF-1)-bound PtdIns(4,5)P₂ into PtdIns(3,4,5)P₃, and knockdown of IPMK results in decreased abundance of SF-1 targets [79]. UV irradiation recruits IPMK to DNA damage sites, where it produces PtdIns(3,4,5)P₃ and contributes to the ATR DNA damage repair machinery [28]. The chemotherapy drug etoposide recruits IPMK to promoters of p53 target genes, PUMA (p53 up-regulated modulator of apoptosis), Bax (Bcl-2 associated X) and p21, where it interacts with p53 and regulates transcriptional control of select p53 targets [80].

PTEN

PTEN is a phosphatase and key tumor suppressor [81]. It preferentially dephosphorylates the D-3 position of PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, antagonizing PI3K signaling [35]. PTEN is present in the nucleus, where its phosphatase activity was detected [35]. Studies have demonstrated the presence of PTEN in nucleoli and chromatin by immunofluorescence, where depletion of PTEN induced nucleolar morphologic changes and spontaneous DSBs [82,83]. Both genotoxic and oxidative stress promote the chromatin association of PTEN in an inverse correlation with the amount of cytosolic PTEN [84], suggesting nuclear import of PTEN in response to cellular stress. Significantly, PTEN interacts with Rad52, a key regulator of DNA DSB repair, at DNA damage sites [84], suggesting its role in DNA damage repair. PTEN also interacts with and regulates p53 [85–90].

SHIP1 and SHIP2

SHIP1 and SHIP2 catalyze the dephosphorylation of PtdIns(3,4,5)P₃ at the D-5 position in the nucleus [35,36]. SHIP1 is present in nucleoli, and its expression is induced in response to the proteasome inhibitor MG132 [36]. Detailed studies show that 8 hours of MG132 treatment induced SHIP1-p53 colocalization in the nucleolar cavity; however, after 16 hours of treatment, SHIP1 and p53 start to localize to different regions in the nucleoli [36], suggesting spatial and temporal roles of SHIP1 during stress responses. Immunofluorescence reveals the localization of SHIP2 in nuclear speckles [35]. The transcriptional inhibitor ⍺-amanitin, causes SHIP2 to redistribute together with the speckle component SC35 into fewer and larger structures [35]. Moreover, phospho-SHIP2 (Ser¹³²) co-localizes with PtdIns(4,5)P₂ in nuclear speckles, which demonstrate PtdIns(4,5)P₂ phosphatase activity [91]. As PtdIns(4,5)P₂ responds to genotoxic and oxidative stresses in nuclear speckles [42,43], SHIP2 may play a role in nuclear stress signaling.

Type I PtdIns(4,5)P₂ 4-phosphatase

PtdIns(4,5)P₂ 4-phosphatases, including type I and II, dephosphorylate PtdIns(4,5)P₂ to produce PtdIns5P. In response to genotoxic stress induced by etoposide or doxorubicin, type I PtdIns(4,5)P₂ 4-phosphatase translocates to the nucleus and augments PtdIns5P levels [92]. The elevated nuclear PtdIns5P activates the ING2-p53 apoptotic pathway by facilitating ING2-dependent p53 acetylation and stabilization [92]. Consistently, the pro-apoptotic effect of p53 can be inhibited by PIPKIIβ, an enzyme that converts PtdIns5P into PtdIns(4,5)P₂ [92], indicating a functionally important role of PtdIns5P, type I PtdIns(4,5)P₂ 4-phosphatase and PIPKIIβ in p53-dependent stress responses.

PLC family

PI-PLC is a family of 13 isozymes classified into 6 isotypes (β, γ, δ, ε, ζ, η) that participate in PtdIns(4,5)P₂ catabolism, producing Ins(1,4,5)P₃ and DAG, mobilizing cellular calcium and activating PKC. All four PLCβ isozymes have been detected in the nucleus, especially in the nuclear matrix when membranes are depleted, with PLCβ1 the most abundant nuclear isoform [93]. IGF1 selectively activates PLCβ1 in the nucleus without affecting its localization [56]. PLCβ1 is also present in nuclear speckles [55]. Indeed, overexpression of PLCβ1 protects cells from oxidative stress by facilitating the mRNA generation of c-Fos for cell survival [94]. PLCδ1 is found in the nuclear matrix and partially colocalizes with PtdIns(4,5)P₂ in DAPI-low areas [95]. Upon cell cycle arrest by serum starvation or double thymidine blockade, PLCδ1 accumulates in the nucleus at the G₁/S transition by a PtdIns(4,5)P₂-dependent mechanism, as mutation of the PtdIns(4,5)P₂-binding site reduced the nuclear localization of PLCδ1 [95]. Nuclear-localized PLCδ4, when knocked down, is reported to cause cell cycle arrest, with an increased G₁ phase and decreased S and G₂/M phases [96].

The nuclear PI-binding proteins

The nuclear PIs are signaling messengers that function in precise locations with specific PI-binding proteins. This is achieved by specific PI-binding domains/motifs in proteins that anchor to or constantly sample the environmental phospholipids. These PI-protein complexes are controlled both at temporal and spatial levels by various mechanisms, and they are actively involved in nuclear signaling and stress responses (Table 3 and Figure 3).

Table 3. Nuclear PI-binding proteins, their interacting PIs, associated PI enzymes, nuclear location, response to cell stress and functions

PI-binding proteins	Binding PI	Associated PI enzymes	Nuclear location	Stress response	Function
Aly	PtdIns(4,5)P2 [98], PtdIns(3,4,5)P3 [98]	IPMK [98,99]	Nucleoplasm [98], Nuclear speckle [98]	Unknown	mRNA export [98]
BASP1	PtdIns(4,5)P2 [100]	Unknown	Chromatin [100]	Sequestration abrin toxin in the nucleus [102]	Transcriptional regulation [100] and toxin resistance [102]
BRG1	PtdIns(4,5)P2 [107]	Unknown	Chromatin [103], Nuclear Matrix [107]	Potential roles in DNA damage repair, oxidative and metabolic stress [104–106]	Chromatin modification and transcriptional regulation [107,108]
EBP1	PtdIns(3,4,5)P3 [46]	PI3KI-p110β [46]	Nucleoli [46,159]	Induced sumoylation and nucleolar distribution upon genotoxic stress [159]	Post-translational modification and transcriptional regulation [159]
Fibrillarin	PtdIns(4,5)P2 [110]	Unknown	Nucleoli [111]	Reduced nucleolar level upon bacterial infection, heat shock and chemical treatment [111,113].	Configuration change [102] and transcriptional regulation [110]
ING2	PtdIns5P [29,92]	PIPKIIβ [29], Type I PtdIns(4,5)P2 4-phosphatase [92]	Nuclear Matrix [29], Chromatin [29,31]	Increased nuclear level, PtdIns5P binding, chromatin localization, regulation of p53 acetylation and apoptotic pathway upon genotoxic/oxidative stress [31,92,114]	Chromatin modification, transcriptional regulation, and stabilizing p53 [29,31,92,114]
NPM1	PtdIns(3,4,5)P3 [119]	PTEN [119], SHIP [119]	Nucleoli [115] Nucleoplasm [115]	Translocated from nucleoli to nucleoplasm upon genotoxic and viral stress [117]	Anti-apoptotic function [119] and stabilizing p53 [117]
p53	PtdIns(4,5)P2 [25]	PIPKI⍺ [25], IPMK [80], PTEN [87], SHIP1 [36]	Nucleoplasm [25], Nuclear speckles [25], Chromatin [25,143]	Induced nuclear level upon genotoxic/oxidative stress, hypoxia and oncogene activation [25,142–144]; Induced association with PtdIns(4,5)P2/PIPKI⍺/HSP27 upon genotoxic/oxidative stress [25]; Recruited to the nucleoplasm, nuclear speckles and DNA damage sites upon genotoxic stress [25]	Transcriptional regulation [143], stabilizing p53, and DNA damage repair [25]
PHF8	PtdIns(4,5)P2 [122]	Unknown	Chromatin [122]	Induced nuclear level upon genotoxic stress [123]	Transcriptional regulation [122] and cell proliferation [123]
SAP30/ SAP30L	PtdIns3P [124], PtdIns4P [124], PtdIns5P [124]	Unknown	Nucleoli [125], Chromatin [124]	Relocating from chromatin to cytoplasm and reduced repression activity upon oxidative stress [124]	Protein translocation, chromatin modification, and transcriptional regulation [124]
SF-1	PtdIns(4,5)P2 [127], PtdIns(3,4,5)P3 [127]	IPMK [79], PTEN [79]	Chromatin [79]	Protective roles under restrain, food deprivation, inflammatory inducer [128]; Induced accumulation at DNA damage sites [28].	Transcriptional regulation and DNA damage repair [28,127]; Adrenal development and stress response [128]
Star-PAP	PtdIns(4,5)P2 [43]	PIPKI⍺ [43]	Nuclear speckles [43]	Induced activity, association with PIPKIα/PtdIns(4,5)P2 in nuclear speckles upon genotoxic and oxidative stress [42,43,87,141^]	3′-end processing and post-transcriptional regulation [42,43,87,141]
Syntenin-2	PtdIns(4,5)P2 [129]	Unknown	Nucleoplasm [160], Nuclear speckles [129], Nucleoli [129]	Unknown	Maintain nuclear PtdIns(4,5)P2 pattern and contributes to cell division and survival [129]
TAF3	PtdIns5P [70]	PIPKIIβ [70]	Chromatin [70]	Enhanced interaction with PtdIns5P and transcriptional regulation upon genotoxic stress [70]	Transcriptional regulation and cell differentiation [70]
UHRF1	PtdIns(5)P [133]	Unknown	Chromatin [133] (Pericentric heterochromatin [130], Euchromatin [131,132])	Recruited to DNA damage sites [134], Protective role upon oxidative stress [135]	Chromatin modification, DNA damage repair, and transcriptional regulation [134,135]

Aly

Aly, a nuclear export factor, is involved in the assembly and recognition of spliced messenger RNA with TREX [97]. The function of Aly on mRNA export depends on nuclear speckle residency, which is directly impacted by binding to PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ through its N-terminal RG motif, as mutants lacking PtdIns(4,5)P₂/PtdIns(3,4,5)P₃-binding accumulate in the nucleoplasm [98]. Conversion of Aly-bound PtdIns(4,5)P₂ into PtdIns(3,4,5)P₃ by IPMK, and phosphorylation on T219 by Akt1 are further required for specific mRNA motif recognition [98,99]. The Aly-PIs complex hasn’t been explored in the context of cell stress, but multiple stressors increase nuclear PtdIns(4,5)P₂/PtdIns(3,4,5)P₃ levels [25,28,45,47], with some specifically causing PtdIns(4,5)P₂ accumulation in nuclear speckles [42,43], suggesting stress roles at play for Aly-regulated transcripts.

BASP1

The brain acid-soluble protein 1 (BASP1) is a transcriptional repressor regulating Wilms’ tumor 1 protein (WT1) target genes. The myristoylated BASP1 binds to nuclear PtdIns(4,5)P₂ through its N-terminal polybasic region (PBR), which leads to the recruitment of PtdIns(4,5)P₂ to the promoter region of WT1 target genes, where they recruit Histone Deacetylase 1 (HDAC1) and elicit transcriptional repression [100]. As an oncogene, WT1 has been reported to enhance cisplatin-resistance of advanced non-small-cell lung cancer (NSCLC), suggesting a role of BASP1-PtdIns(4,5)P₂ complex in genotoxic stress response [101]. BASP1 also protects cells from cellular stress induced by abrin toxin, a type II ribosome-inactivating protein, by sequestering the abrin A chain to the nucleus to alleviate its inhibitory effect on protein synthesis [102].

BRG1

The Brahma Related Gene 1 (BRG1) protein is an ATP-dependent helicase subunit of the BAF chromatin remodeling complex that controls gene expression [103]. It has roles in DNA damage repair, oxidative stress, and metabolic stress [104–106]. BRG1 directly binds to PtdIns(4,5)P₂, and this binding facilitates its association with nuclear β-actin, which in turn enhances the ATPase activity of BRG1 [107]. The BRG1-PtdIns(4,5)P₂ complex also stabilizes nuclear β-actin. Interestingly, four regions of BRG1 bind to β-actin, and PtdIns(4,5)P₂ replaces two of them while leaving the others still available for binding [108], suggesting that PtdIns(4,5)P₂-binding exposes/uncaps β-actin so β-actin can interact with actin filaments or other proteins. BRG1 may utilize β-actin polymerization and interactions to assist in chromosomal remodeling, allowing for the repression or expression of target genes. As BRG1 regulates expression of stress response genes [106], PtdIns(4,5)P₂ may play a role in transcriptional regulation of these genes.

EBP1

ErbB3-binding protein (EBP1), a putative tumor suppressor, translocates to the nucleus and functions as a transcriptional corepressor. It directly binds to several PIs via two lysine-rich PBR domains at the N- and C-terminus of the protein [46]. The C-terminal PBR mediates the nucleolar localization of EBP1, where it associates with PtdIns(3,4,5)P₃ via both electrostatic and hydrophobic interactions and co-localizes with the PI3K catalytic subunit p110β [46]. Interestingly, genotoxic stress triggers SUMOylation of EBP1, facilitating its nucleolar localization, protein stability, and transcriptional repressive activity, suggesting a potential role of the EBP1-PtdIns(3,4,5)P₃ complex in stress response.

Fibrillarin

Fibrillarin is a small nucleolar ribonucleoprotein (snoRNP) involved in pre-ribosome assembly and rRNA modification [109,110]. It also functions in the innate immune response as it is downregulated upon bacterial infection, which causes enhanced cell survival, inflammation reduction, and bacteria clearance [111]. Fibrillarin directly binds to PtdIns(4,5)P₂, and together they form a complex that co-localizes with nascent rRNA dependent on rRNA transcription [110,112]. Inhibition of rRNA transcription during mitosis or via chemical inhibition (actinomycin D), disrupts the co-localization of fibrillarin with PtdIns(4,5)P2 and rRNA synthesis foci [110,112,113]. Fibrillarin also loses its localization to nucleoli in response to diverse cell stressors such as heat shock, bacterial infection, or various drugs (actinomycin D, 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), cycloheximide, and Roscovitine/Seliciclib) [111,113]. The exact role of fibrillarin-PtdIns(4,5)P₂ binding remains unclear, but data does suggest that PtdIns(4,5)P₂ binding induces a conformational change in fibrillarin [102], which could stabilize it as in the case of p53 [25], or aid in its localization as in the case of Aly [98].

ING2

ING2 is a tumor suppressor that forms a chromatin-modifying complex for modulating methylation and acetylation of lysine residues [31]. It contains a PHD finger domain that coordinates with zinc to bind PtdIns5P [29]. Upon DNA damage, ING2 localizes to chromatin, mediates p53 acetylation, and represses a subset of specific target genes during the DNA damage response [31,92]. ING2 localization with chromatin, p53 acetylation, and ING targeted gene repression are all dependent on Ptdins5P binding [29,31]. Loss of ING2 binding to Ptdins5P reduces p53-mediated apoptosis in response to etoposide or H₂O₂ [29]. This is consistent with studies that show ING2 upregulation and increased nuclear localization of Type I PtdIns(4,5)P₂ 4-phosphatase, the main PtdIns5P generating enzyme, upon DNA damage [31,92,114]. The generation of PtdIns5P likely occurs at specific target sites on chromatin to recruit and activate ING2 [29]. Taken together, these studies show that PtdIns5P plays a key role in localizing ING2 to chromatin and activating it in response to genotoxic and oxidative stress.

NPM1

NPM1 (also known as B23 or nucleophosmin) is a multifunctional chaperone implicated in ribosome biogenesis, chromatin remodeling, and DNA damage repair, and is frequently overexpressed in proliferating cells and interacts with both p53 and the retinoblastoma (Rb) tumor suppressor gene product [115–117]. NPM1 is predominantly located in nucleoli, but a smaller fraction also can be detected in the nucleoplasm [115]. With both NLS and NES, NPM1 shuttles between the nucleus and cytoplasm and facilitates the nucleolar targeting of proteins [115,118]. Genotoxic stress and viral stress (viral K cyclin) induce the re-localization of NPM1 from nucleoli to the nucleoplasm, where it binds HDM2 and inhibits p53 degradation by disrupting HDM2-p53 complex formation [117]. NPM1 was also identified in a complex with PtdIns(3,4,5)P₃ that prevents cells from undergoing apoptosis [119]. This effect was abrogated by hydrolyzing PtdIns(3,4,5)P₃ with PTEN or SHIP [119]. Interestingly, a mutant NPM1 lacking the p53-interacting domain fails to confer cellular resistance to stress-induced apoptosis [116], suggesting that NPM1 protects cells from apoptosis through a mechanism involving p53, potentially in a PtdIns(3,4,5)P₃-dependent manner. Recent studies show that NPM1 undergoes liquid-liquid phase separation (LLPS), a process important for defining the membrane-less compartmentalization in nucleoli [120,121]. In the future, it would be of interest to test the role of lipid messengers in LLPS, especially under stressed conditions.

PHF8

The histone lysine demethylase PHF8 (PHD finger protein 8) directly interacts with PtdIns(4,5)P₂ through the C-terminal lysine/arginine-rich motif [122]. This interaction represses demethylation of H3K9me2 at the rDNA promoter, which represses the transcription of rRNA genes [122]. Genotoxic stress induced by UV irradiation and the radiomimetic DNA damage agent neocarzinostatin (NCS) increases protein levels of PHF8 via USP7 deubiquitinase-dependent stabilization, leading to the upregulation of a group of genes, including cyclin A2, which is critical for cell growth and proliferation in breast cancer [123]. Interesting, the C-terminal region, where PtdIns(4,5)P₂ binds, is also required for USP7 binding and transcriptional regulation of the DNA damage response [123], suggesting a potential regulatory role of the PHF8-PtdIns(4,5)P₂ complex in the cellular stress response.

SAP30 and SAP30L

The Sin3A-associated proteins SAP30 and SAP30L are linker molecules between various components of the Sin3A corepressor complex that regulates histone deacetylation and transcriptional activity. SAP30 and SAP30L not only facilitate the nucleolar targeting of Sin3A, but also directly associate with core histones and naked DNA [124,125]. SAP30 and SAP30L interact specifically with monophosphoinositides, most tightly to PtdIns5P (followed by PtdIns3P and PtdIns4P) through a PBR and supported by a hydrophobic region and a zinc-coordination structure [124]. Interestingly, the same region also contributes to DNA binding. Mutation and deletion of this region blocked the chromatin association of SAP30L, and decreased the repressive activity of SAP30L. Oxidative stress, which induces nuclear PtdIns5P accumulation [30], led to relocalization of SAP30L from the nuclear chromatin to the cytoplasm and attenuated its repressive activity [124]. Potentially, the SAP30L-chromatin interaction is mediated by the PI-/DNA-binding region, and PI binding disrupts this, leading to decreased transcriptional repression by SAP30L.

SF-1

The nuclear receptor SF-1 is an orphan nuclear receptor that serves as an essential regulator of multiple hormone-induced genes in the vertebrate endocrine system. It binds PIs and other phospholipids in a large hydrophobic pocket [119,126]. This pocket sequesters the hydrophobic tails of PtdIns(4,5)P₂/PtdIns(3,4,5)P₃ with their head groups fully solvent-exposed, which may define a new interaction surface between SF-1 and coregulatory proteins [127]. Indeed, PtdIns(3,4,5)P₃ binding increases coactivator recruitment to SF-1 and increases SF-1 activity [127]. Nuclear IPMK and PTEN switch SF-1-bound PIs between PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃, regulating SF-1 transcriptional activity [79]. UV irradiation-induces SF-1 accumulation at DNA damage sites, where it forms a complex with IPMK-catalyzed PtdIns(3,4,5)P₃ and contributes to the ATR DNA damage repair machinery [28]. Analysis of a patient heterozygous for the SF-1 gene indicated that reduced expression of SF-1 leads to adrenal failure [128]. In SF-1 heterozygous mice, adrenal deficits were revealed upon stress induction, including food deprivation, and inflammation induced by injection of lipopolysaccharide [128], suggesting potential roles of SF-1 in stress responses, which is possibly PI-regulated.

Syntenin-2

Syntenin-2 binds with high affinity to PtdIns(4,5)P₂ through its two PDZ (Postsynaptic density protein/Disc large/Zona occludens) domains [129]. These domains target syntenin-2 to nuclear speckles and nucleoli. Although the response of syntenin-2 to cell stress is unknown, it likely plays a role in stress signaling as depletion of syntenin-2 disrupts PtdIns(4,5)P₂ localization at nuclear speckles [129], where the PtdIns(4,5)P₂ generation is induced by genotoxic and oxidative stress [42,43], suggesting that syntenin-2 may function as an organizer of nuclear PtdIns(4,5)P₂. Moreover, depletion of syntenin-2 decreased cell division and survival [129], indicating that the syntenin-2-PtdIns(4,5)P₂ complex may play important roles in cell viability and proliferation.

TAF3

The highly conserved RNA polymerase II transcription factor (TFIID) comprises the TATA box-binding protein (TBP) and a set of TBP-associated factors (TAFs, including TAF3) that play key roles in promoter recognition. Through a targeted screen for PI interactors, TAF3 was found to strongly interact with PtdIns5P through its PHD finger [70]. This interaction modulates the association of TAF3 with chromatin and regulates TAF3-dependent gene expression during myoblast differentiation. Etoposide-induced genotoxic stress, which largely increases nuclear PtdIns5P [31], enhances the TAF3-PtdIns5P interaction and alters transcriptional output to promote cell differentiation [70]. Notably, PIPKIIβ, the kinase that converts PtdIns5P to PtdIns(4,5)P_2, also regulates specific gene expression dependent on the PI-binding site of TAF3 upon etoposide treatment [70], suggesting that TAF3-mediated nuclear alterations contribute to its transcriptional regulation.

UHRF1

Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) is a nuclear multi-domain protein, localized to chromatin that plays key roles in DNA methylation, histone H3 modification (H3K9me3), and gene regulation, including silencing tumor suppressors [130–132]. UHRF1 is allosterically regulated by PtdIns5P binding [133]. Without PtdIns5P, the PBR in the C terminus of UHRF1 occupies an essential peptide-binding groove in its tandem tudor domain (TTD) to block its interaction with H3K9me. In this state, UHRF preferentially interacts with unmodified H3. In contrast, binding of PtdIns5P to the PBR of UHRF1 leads to a conformational change that releases the TTD for H3K3me binding, suggesting that PtdIns5P or other nuclear PIs may directly impact gene expression by regulating the chromatin binding activity of interactors. Upon psoralen/cisplatin-induced DNA damage, UHRF1 was enriched at the DNA lesions, serving as a lesion recognition factor and nuclease scaffold for DNA damage repair [134]. Depletion of UHRF1 increases DNA damage sensitivity [134]. Also, UHRF1 protects cancer cells against diethyl maleate (DEM)-induced oxidative stress by activating the oncogenic Keap1-Nrf2 pathway [135]. Given that UHRF1 is over-expressed in many cancer cells [135–137], the UHRF1-PtdIns5P complex may contribute to the malignant behavior of cancer cells and mediate chemotherapy-resistance.

Star-PAP

Speckle targeted PIPKIα regulated-poly(A) polymerase (Star-PAP) [43] (also called TUT1) [138] is a noncanonical poly(A) polymerase localized in nuclear speckles [43]. It controls the expression of about 40% of human genes and is activated by DNA damage and oxidative stress signals [42,43,139]. Star-PAP processes mRNAs by directly binding to the 3ʹUTR upstream of the poly(A) signal, where it recruits the cleavage and polyadenylation specificity factor (CPSF) subunits [140]. In nuclear speckles, especially in response to oxidative stress (tBHQ), Star-PAP complexes with PIPKIα and directly binds PtdIns(4,5)P₂, which enhances the polyadenylation activity of Star-PAP to control the expression of selected genes [43]. Oxidative stress also stimulates CKIα/ε-mediated Star-PAP phosphorylation, which is critical for Star-PAP activity and its stimulation by PtdIns(4,5)P₂ [141]. Moreover, etoposide-induced genotoxic stress stimulates the nuclear localization of PKCδ, which is integrated into the Star-PAP complex and directly interacts with PIPKIα [42]. PIPKIα-produced PtdIns(4,5)P₂ further activates PKCδ, which in turn promotes the activity of Star-PAP for 3ʹ end processing of BIK mRNA and induction of apoptosis [42]. In contrast, activation of the Star-PAP pathway by oxidative stress activates a distinct subset of genes, suggesting specificity of Star-PAP in stress signaling. Other Star-PAP target genes are independent of PIPKIα and may be independent of PI signaling [139].

p53

The tumor suppressor p53 is activated by a wide range of cellular stressors, protecting the genome from genotoxic stress, oxidative stress, hypoxia, and oncogene activation [142–144]. Most cellular stressors induce accumulation of wild-type p53 in the nucleus for the transcriptional regulation of gene networks that lead to cell cycle arrest, DNA damage repair, senescence, apoptosis, and/or metabolic reprogramming [143]. Mutant p53 dramatically accumulates in the nucleus and stress further enhances this accumulation [25]. Significantly, a subset of mutant p53 proteins acquire oncogenic activities [145,146]. We have demonstrated that p53 is a novel PI-binding protein, and the stability of mutant p53 and stress-induced wild-type p53 is regulated by PtdIns(4,5)P₂ [25]. Under genotoxic or oxidative stress, nuclear PIPKIα binds to p53, leading to the production and association of PtdIns(4,5)P₂ with the C-terminal PBR of p53 [25], and this sequence is known to regulate many p53 functions [25,143,144,146]. PtdIns(4,5)P₂-binding recruits small heat shock proteins HSP27/HSPB1 and αB-Crystallin/HSPB5 to the p53 complex, which stabilizes p53 and displaces PIPKIα from the complex [25]. Using sub-nuclear makers and proximity ligation assay (PLA), an approach to detect in situ interaction of two epitopes within 40 nm [147–150], the PI-p53 complex was identified in the nucleoplasm, nuclear speckles, and DNA damage sites, indicating multiple roles for this complex. Mutant p53, PIPKIα, and sHSPs have independently been implicated in cancer progression [146,151–153]. The discovery of a nuclear complex containing all three proteins points to multiple drug targets to disrupt this complex therapeutically in cancer. In addition to PIPKIα [25], multiple nuclear PI enzymes, including IPMK [80], PTEN [87], and SHIP1 [36] are associated with p53, illuminating a central role for PI signaling in regulating p53 function.

The nuclear compartment for phosphoinositides

A fundamental issue in nuclear PI signaling is the question of how the PIs are compartmentalized in the nucleus [8,9,20,50,127,154]. Although there is evidence for vesicular compartments that contain PIs that major PI content appears to be separate from membranes, suggesting novel protein-lipid complexes that remain to be defined.

Conclusions

A flourishing body of literature from many groups provide unequivocal evidence that nuclear PIs serve as central signaling hubs for regulating many nuclear processes, especially in response to cellular stress (Figure 4). However, it is also clear that we are only starting to scratch the surface in understanding the diverse biological roles of nuclear PIs and their effectors. Many crucial questions remain unanswered. For example, how do these phospholipids exist outside of membrane structures within the nucleoplasm? Do they play roles in LLPS for membrane-less compartmentalization? Where is their lipid origin? How are they held soluble by nuclear proteins? Do they link to nuclear proteins covalently or non-covalently? What advantage would phosphorylating a nuclear protein-bound PI have over simply phosphorylating one amino acid in the nuclear protein itself? How do the nuclear PIs and effectors sense cellular stress? Are there nuclear PI-scaffolds similar to the cytosolic ones [155,156]? Continued research efforts are needed to address these questions, to discover novel interactors, to decipher the nature of the PI-protein interaction, and to clarify the role of nuclear PIs. With many new tools in place to accelerate discovery in this field, many new insights regarding nuclear PIs and their associated proteins are likely to emerge in the next decade.

Figure 4. Schematic illustration of nuclear PI signaling in response to cellular stress.

In response to cellular stress, the levels of nuclear PI are altered and conversions are induced due to activation of different PI-metabolizing enzymes. Through direct interaction with downstream PI-binding proteins, the PI signals are transduced into changes in cellular functions.

Acknowledgments

We wish to thank members of the Anderson and Cryns lab and apologize to the authors whose work was not cited due to space limitations. This work was supported in part by a National Institutes of Health grant GM114386 (R.A.A.), Department of Defense Breast Cancer Research Program grants W81XWH-17-1-0258 (R.A.A.) and W81XWH-17-1-0259 (V.L.C.), and a grant from the Breast Cancer Research Foundation (V.L.C.).

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Institutes of Health [GM114386];U.S. Department of Defense [W81XWH-17-1-0259];U.S. Department of Defense [W81XWH-17-1-0258].