Signaling through non-membrane nuclear phosphoinositide binding proteins in human health and disease

Abstract

Phosphoinositide membrane signaling is critical for normal physiology, playing well-known roles in diverse human pathologies. The basic mechanisms governing phosphoinositide signaling within the nucleus, however, have remained deeply enigmatic owing to their presence outside the nuclear membranes. Over 40% of nuclear phosphoinositides can exist in this non-membrane state, held soluble in the nucleoplasm by nuclear proteins that remain largely unidentified. Recently, two nuclear proteins responsible for solubilizing phosphoinositides were identified, steroidogenic factor-1 (SF-1; NR5A1) and liver receptor homolog-1 (LRH-1; NR5A2), along with two enzymes that directly remodel these phosphoinositide/protein complexes, phosphatase and tensin homolog (PTEN; MMAC) and inositol polyphosphate multikinase (IPMK; ipk2). These new footholds now permit the assignment of physiological functions for nuclear phosphoinositides in human diseases, such as endometriosis, nonalcoholic fatty liver disease/steatohepatitis, glioblastoma, and hepatocellular carcinoma. The unique nature of nuclear phosphoinositide signaling affords extraordinary clinical opportunities for new biomarkers, diagnostics, and therapeutics. Thus, phosphoinositide biology within the nucleus may represent the next generation of low-hanging fruit for new drugs, not unlike what has occurred for membrane phosphatidylinositol 3-kinase drug development. This review connects recent basic science discoveries in nuclear phosphoinositide signaling to clinical pathologies, with the hope of inspiring development of new therapies.

Keywords: diabetes; endometriosis; glioblastoma; hepatocellular carcinoma; inositol phosphate multikinase; nuclear lipid signaling; phosphatidylinositol (3,4,5) triphosphate.

Fig. 1.

Nuclear receptor SF-1 binds PI(3,4,5)P3 (PIP3) with acyl chains buried in a deep hydrophobic pocket, with the PIP3 headgroup on the surface of the SF-1 protein. A: Surface representation of the 2.4 Å crystal structure of the ligand binding domain of SF-1 (colored pink) bound by dipalmitoyl (di-C16) PIP3 (represented as atom colored sticks) (13). The lower right contains the integrated surface representation of the cocrystallized nuclear receptor coactivator LXXLL peptide, PGC1α (colored gold), which functions to regulate SF-1 transcriptional activity in chromatin. This helical bundle architecture is conserved throughout the ligand binding domains of the nuclear receptor superfamily of transcription factors, most of which bind hydrophobic cholesterol-, fatty acid- or phospholipid-based ligands (54, 195). Note the absence of the DNA-binding domain from this structure; the full-length structures of SF-1 and LRH-1 are currently unknown. B: Cartoon representation of A, with identical coloring scheme, α helices represented as cylinders, highlighting the positioning of the dipalmitoyl acyl chains deep in the hydrophobic core of the SF-1 helical bundle. These structures exemplify how non-membrane nuclear phosphoinositides can exist outside membrane systems (8).

Fig. 2.

Solvent accessibility of the SF-1 bound PI(3,4,5)P3 headgroup is high and participates in an extensive water-mediated hydrogen bonding network. A: Cartoon representation of the crystal structure of SF-1 bound to dipalmitoyl PI(3,4,5)P3 (13) colored as a spectrum to solvent accessibility, where the least solvent accessible is blue

Fig. 3.

Model of PTEN- and IPMK-mediated nuclear phosphoinositide signaling through SF-1. A: IPMK is a nuclear protein conserved in all eukaryotes (144) that was first characterized functionally as a transcriptional coregulator (196, 197) before the inositol kinase enzyme activity was discovered (92). B: Shortly thereafter, Adam Resnick in Solomon Synder’s laboratory discovered that IPMK also has a PI3-kinase enzyme activity, with the ability to phosphorylate PI(4,5)P2 in membrane systems (90), confirmed to occur in vivo in mouse models (103). IPMK is a close structural homolog to the IP3-kinase superfamily and protein kinase A (99), but is structurally unrelated to the class 1 PI3-kinases. C: IPMK was then shown to bind and directly phosphorylate PI(4,5,)P2 bound to the nuclear receptor SF-1, generating PI(3,4,5)P3 bound to SF-1 resulting in robust activation of SF-1 transcriptional programs in human cell lines (1). Nuclear PTEN opposes this function of IPMK, dephosphorylating PIP3 bound to SF-1 and downregulating SF-1 transcriptional activity (1). This nuclear phosphoinositide signaling pathway is the first described in which the structural biology and mechanistic enzymology fully describe the mechanism of the signal transduction, which is extremely important in drug design efforts that could impact the clinic.

Fig. 4.

Superposition of two recently solved crystal structures of IPMK, a nuclear PI(4,5)P2 kinase. Cartoon views of recently published human IPMK catalytic core PDB 6C8A (apo) and PDB 5W2I (bound to ADP and di-C4 PI(4,5,)P2) (99, C. D. Seacrist and R. D. Blind, unpublished observations). These extensive crystallographic and kinetic studies revealed novel kinetic properties of IPMK and how IPMK interacts with ligands, ATP and PI(4,5)P2. However, because crystallization requires removal of the disordered domains of IPMK, the structure of these domains (one disordered domain on the N terminus and one which interrupts the core kinase domain) remains undescribed. Note that the PI(4,5)P2 glycerol backbone and the di-C4 acyl chains were not ordered (99).

Fig. 5.

GRP30 could be a good drug target in nuclear phosphoinositide signaling and endometriosis. Endometriotic uterine endometrial cells growing in the pelvic peritoneum or on the ovaries require estradiol to maintain growth in these ectopic locations. Ectopic endometriotic tissue itself is often steroidogenic, overexpressing SF-1. Estradiol or synthetic agonists can activate the G protein-coupled receptor, GPR30 (GPER1), present on the plasma membrane and nuclear envelope of endometriotic cells, resulting in accumulation of global cell levels of PIP3 (112). The mechanism GPR30 uses to increase PIP3 is unknown and it is further unknown whether IPMK might play a role in endometriosis. GPR30-mediated increases in PIP3 correlate with activation of SF-1, leading to increased gene expression of steroidogenic genes involved in biosynthesis of estradiol, such as aromatase (CYP19A1). Increased expression of these genes leads to increased production of steroids, which can then reactivate membrane-bound GPR30, resulting in a feed-forward activation loop that perpetuates ectopic growth of the endometrial tissue (112). For these reasons, GPR30 could be an excellent candidate for drug development efforts targeting endometriosis.

Fig. 6.

Human LRH-1 and SF-1 both bind nuclear PIP3 in very similar ways. A: Superposition of the 1.8 Å crystal structure of human LRH-1 ligand binding domain (14) (represented as purple cartoon) and the crystal structure of human SF-1 ligand binding domain (13) (represented as pink cartoon), each bound individually to one dipalmitoyl PI(3,4,5)P3 molecule. The two superposed structures have a root mean square deviation of less than 1 Å (0.898 Å) that, while totally identical, shows the high degree of homology between SF-1 and LRH-1. B: Close-up of A showing similar solvent accessibility of PI(3,4,5)P3 headgroups when the phosphoinositide is bound to either human SF-1 or human LRH-1. This structural comparison suggests that the same structural format of PI(3,4,5)P3 that grants special signaling capacity to SF-1 is conserved within LRH-1. It remains to be determined whether IPMK or PTEN can directly remodel phosphoinositides bound to LRH-1.