Phosphoinositides and intracellular calcium signaling: novel insights into phosphoinositides and calcium coupling as negative regulators of cellular signaling

Abstract

Intracellular calcium (Ca²⁺) and phosphoinositides (PIPs) are crucial for regulating cellular activities such as metabolism and cell survival. Cells maintain precise intracellular Ca²⁺ and PIP levels via the actions of a complex system of Ca²⁺ channels, transporters, Ca²⁺ ATPases, and signaling effectors, including specific lipid kinases, phosphatases, and phospholipases. Recent research has shed light on the complex interplay between Ca²⁺ and PIP signaling, suggesting that elevated intracellular Ca²⁺ levels negatively regulate PIP signaling by inhibiting the membrane localization of PIP-binding proteins carrying specific domains, such as the pleckstrin homology (PH) and Ca²⁺-independent C2 domains. This dysregulation is often associated with cancer and metabolic diseases. PIPs recruit various proteins with PH domains to the plasma membrane in response to growth hormones, which activate signaling pathways regulating metabolism, cell survival, and growth. However, abnormal PIP signaling in cancer cells triggers consistent membrane localization and activation of PIP-binding proteins. In the context of obesity, an excessive intracellular Ca²⁺ level prevents the membrane localization of the PIP-binding proteins AKT, IRS1, and PLCδ via Ca²⁺-PIPs, contributing to insulin resistance and other metabolic diseases. Furthermore, an excessive intracellular Ca²⁺ level can cause functional defects in subcellular organelles such as the endoplasmic reticulum (ER), lysosomes, and mitochondria, causing metabolic diseases. This review explores how intracellular Ca²⁺ overload negatively regulates the membrane localization of PIP-binding proteins.

Fig. 1. Structures of phosphoinositol and seven interconvertible phosphoinositides found in mammalian cells.

Specific lipid kinases and phosphatases are phosphorylated or dephosphorylated at hydroxyl group positions 3, 4, and 5 within the inositol ring of phosphoinositol. PI phosphatidyl inositol, DAG diacylglycerol, PTEN phosphatase and tensin homolog on chromosome 10, SHIP SH2-containing inositol 5′-phosphatase, PI3K phosphatidylinositol 3-kinase, PI4K phosphatidylinositol 4-kinase, PI5K phosphatidylinositol 5-kinase, INPP4B inositol polyphosphate-4-phosphatase, type II.

Fig. 2. PIPs recruit PH domain–carrying proteins to the plasma membrane and regulate diverse cellular functions.

Upon activation, PI3K phosphorylates PI(4,5)P₂ to generate PI(3,4,5)P₃. Activation of PI3K by either a receptor tyrosine kinase, B-cell receptor (BCR)/T-cell receptor (TCR), or cytokine receptor enables it to phosphorylate PI(4,5)P₂, resulting in PI(3,4,5)P₃, which recruits PH domain–carrying signaling proteins, such as PDK, AKT, Rho guanine nucleotide exchange factors (GEFs), ADP-ribosylation factor (ARF) GAPs, ARF GEFs, ITK, and BTK, to the plasma membrane. These PH domain–carrying proteins are then activated at the plasma membrane and regulate various cellular functions, including cell survival, proliferation, cytoskeleton rearrangement, intracellular vesicle trafficking, cell metabolism, and immune and inflammatory responses.

Fig. 3. The PI3K-AKT pathway and major upstream effectors in the signaling network.

In the presence of insulin, the insulin receptor (IR) autophosphorylates its cytosolic tyrosine kinase, which then phosphorylates IRS proteins and creates binding sites that recruit p85 and the catalytic subunit of PI3K to the plasma membrane. PI3K phosphorylates PI(4,5)P₂ to produce PI(3,4,5)P₃, which recruits PDK1 and AKT to the plasma membrane, and at the plasma membrane, AKT is phosphorylated at T308 and S473 by PDK1/2 and mammalian target of rapamycin complex 2 (mTORC2), respectively. AKT signaling promotes glucose uptake and lipid, glycogen, and protein synthesis via the phosphorylation of the GTPase-activating AS160, GSK3β, and tuberous sclerosis complex (TSC1/2) complex. In contrast, AKT signaling inhibits apoptosis and gluconeogenesis via the phosphorylation of proapoptotic Bcl-2 protein (BAD) and forkhead box O family transcription factor (FOXOs). Alternatively, negative regulators of PI3K/AKT include PTEN, SHIP, inositol polyphosphate-4-phosphatase (INPP4B), phosphatase 2A (PP2A), and PH-domain leucine-rich-repeat protein phosphatase (PHLPP). The lipid phosphatase PTEN dephosphorylates the 3-phosphate of PI(3,4)P₂ and PI(3,4,5)P₃ to generate PI(4)P and PI(4,5)P_2. SHIP2 dephosphorylates the 5-phosphate of PI(3,4,5)P₃ to yield PI(3,4)P₂, and INPP4B dephosphorylates the 4-phosphate of PI(3,4)P₂ and PI(3,4,5)P₃ to generate PI(3)P and PI(3,5)P₂. Ultimately, a lipid phosphatase decreases the PI(3,4,5)P3 level and inhibits the membrane localization of PDK1 and AKT, inhibiting insulin signaling. Moreover, the protein phosphatases PP2A and PHLPP dephosphorylate and inhibit AKT activity. GLUT4, glucose transporter-4; RHEB, the small GTPase Ras homolog enriched in the brain; Raptor, regulatory associated protein of mTOR; Rictor, the rapamycin-insensitive companion of mTOR; 4EBthe P1, eukaryotic translation initiation factor 4E-binding protein 1; P70S6k, p70 ribosomal protein S6 kinase.

Fig. 4. Regulation of Ca²⁺ homeostasis.

Cytoplasmic, ER, and mitochondrial Ca²⁺ homeostasis is maintained by the actions of transporters and pumps, including PMCAs, SERCAs, and NCLX. Other plasma membrane channels, such as TRP channels and P2RXs, also mediate Ca²⁺ signaling during GPRC activation. Stimulation of G protein-coupled receptors (GPCRs) by glucagon and catecholamines or the TCR by specific antigens leads to the activation of PLC, resulting in I(1,4,5)P₃ (IP3) and DAG production. IP3 binds to IP3Rs, stimulating Ca²⁺ release from ER Ca²⁺ stores. A decrease in ER Ca²⁺ level is sensed by the low-affinity EF-hands of STIM1/2, which activates ORAI1 proteins at the plasma membrane, inducing SOCE. IP3R mediates the release of Ca²⁺, which is transferred into mitochondria through the MCU at highly specialized membrane contact sites, mitochondrion-associated membranes that effectively enhance bioenergetics and ATP production. Alternatively, GPCR activation leads to cAMP production, activating PKA, which phosphorylates IP3R and thus activates its activity. In the cytosol, increased intracellular Ca²⁺ levels activate two main Ca²⁺ sensors, Ca²⁺/calmodulin-dependent protein kinase (CaMK) and calcineurin (CnA). CaMKs directly phosphorylate many targets, including the inflammatory proteins JNK and p38 and transcription factors such as FOXO and cAMP response element-binding protein (CREB). High intracellular Ca²⁺ levels lead to the activation of CaN, which regulates the activity of at least two crucial transcription pathways involving the nuclear factor of activated T cells (NFAT) and CREB-regulated transcription coactivator 2 (CRTC2). TRP nonselective transient receptor potential, P2RX purinergic ionotropic receptor, MCU mitochondrial Ca²⁺ uniporter, PMCA plasma membrane Ca²⁺ ATPase, SERCA sarcoplasmic/ER Ca2+ ATPase, NCLX mitochondrial Na⁺/Ca²⁺/Li⁺ exchanger.

Fig. 5. Dysregulation of Ca²⁺ channels and pumps in metabolic diseases.

Under normal physiological conditions, insulin-stimulated PI3K phosphorylates PI(4,5)P₂ to produce PI(3,4,5)P₃. PI(3,4,5)P₃ recruits PH domain-carrying AKT to the plasma membrane, where activated AKT phosphorylates IP3Rs, reducing intracellular Ca²⁺ release from the ER. Alternatively, AKT phosphorylates PLN, a critical negative regulator of SERCAs in the ER, thereby promoting SERCA activity and decreasing intracellular Ca²⁺ levels by transporting Ca²⁺ from the cytosol to the ER lumen. Under pathological conditions, such as obesity and hyperlipidemia, however, metabolic stress may increase ORAI and IP3R expression, leading to intracellular Ca²⁺ overload. In the mitochondria, Ca²⁺ overload correlates with increased ROS levels and decreased ATP production. Reduced ATP production may decrease SERCA and PMCA expression and activity, contributing to the depletion of ER Ca²⁺ and increasing intracellular Ca²⁺ and mitochondrial Ca²⁺ overload. In addition, ER Ca²⁺ depletion may increase the UPR and ER stress response. Thus, ER Ca²⁺ depletion and mitochondrial Ca²⁺ overload may exacerbate intracellular Ca²⁺ overload. Increased intracellular Ca²⁺ also decreases autophagosome biogenesis and decreases the autophagy rate. Moreover, increased intracellular Ca²⁺ activates CnA to dephosphorylate CRTC2, promoting its nuclear translocation, and activates CaMK to phosphorylate FOXO, leading to increased gluconeogenesis in the liver. In metabolic diseases, Ca²⁺ is released from lysosomes through TRPML (transient receptor potential cation channel, mucolipin subfamily, member), leading to local CnA activation and TFEB dephosphorylation. Dephosphorylated TFEB transcriptionally activates the lysosomal/autophagic pathway. The resulting ER Ca²⁺ depletion and mitochondrial Ca²⁺ overload may trigger a vicious cycle of intracellular Ca²⁺ overload, which contributes to the development of insulin resistance and other metabolic disorders by activating Ca²⁺-dependent cellular pathways.

Fig. 6. Ca²⁺-mediated inhibition of membrane localization of PIP-binding proteins.

Under pathological conditions such as obesity and hyperlipidemia, metabolic stress can increase the expression of Ca²⁺ channel proteins such as ORAI and IP3R, thereby increasing the intracellular degree of Ca²⁺ overload. At the same time, the expression and activity of SERCAs and PMCAs may decrease, causing depletion of Ca²⁺ stores in the ER and increased intracellular and mitochondrial Ca²⁺ overload. In addition, dysregulation of intracellular Ca²⁺ homeostasis results in ER stress, activation of the UPR, increased ROS production, and oxidative stress, all of which may further impair the function of ATP-driven Ca²⁺ pumps such as SERCAs and PMCAs. The resulting ER and mitochondrial dysfunction can trigger a vicious cycle of intracellular Ca²⁺ overload and contribute to the development of insulin resistance and other metabolic disorders. In addition to affecting several Ca²⁺-dependent cellular pathways, such as the CaMK and CaN pathways, increased intracellular Ca²⁺ levels cause Ca²⁺ to bind PIPs and thus form Ca²⁺-PIPs, which disrupt the electrostatic interactions between PIPs and PH domains or C2C domains in proteins, inhibiting the membrane localization of PH domain- or C2C domain-carrying proteins, and disrupting PIP signaling. Thus, Ca²⁺-PIP mediates the inhibition of the membrane localization of PH domain- or C2C domain-carrying signaling proteins, which leads to modulated signaling pathway activation in response to changes in the intracellular Ca²⁺ level.