Modification of protein sub-nuclear localization by synthetic phosphoinositides: Evidence for nuclear phosphoinositide signaling mechanisms
Introduction
Phosphoinositides (PtdInsPs) regulate diverse cellular functions that influence survival, growth, and proliferation, and dysregulated PtdInsP homeostasis is implicated in many disease processes (Cantley, 2002; Maehama et al., 2001; Payrastre et al., 2001). For example, in tumor tissues, the PtdInsP phosphatase gene, PTEN (phosphatase and tensin homologue deleted on chromosome 10; EC 3.1.3.67) is frequently mutated, whereas expression of the PtdInsP kinase, phosphatidylinositol 3-kinase (PI3K; EC 2.7.1.153) is often upregulated (Cantley, 2002). Mouse models have also implicated PI3K in the pathogenesis of diabetes and immunodeficiencies, and mutations in the myotubularin family of PtdInsP phosphatases have been linked to myopathies and neuropathies (Fruman and Cantley, 2002; Mauvais-Jarvis et al., 2002; Taylor et al., 2000). These and many other PtdInsP kinases and phosphatases generate seven different PtdInsP species, which are thought to mediate signaling pathways by modulating the sub-cellular localization and/or activity of PtdInsP-binding proteins (DiNitto et al., 2003; Tolias and Cantley, 1999; Vanhaesebroeck and Alessi, 2000).
The majority of study on PtdInsP regulatory mechanisms has centered on cytoplasmic processes. Nevertheless, there is a great deal of evidence that the functions of PtdInsPs in the nucleus are as diverse as their roles in the cytoplasm (Cocco, et al., 2000; Irvine, 2003; Jones and Divecha, 2004; Martelli et al., 1992, Martelli et al., 2002, Martelli et al., 2003, Martelli et al., 2004a, Martelli et al., 2004b; Ye et al., 2000). For instance, a number of clinically important PtdInsP kinases and phosphatases, including PI3 K, PTEN and src homology 2 domain-containing inositol phosphatase 2 (SHIP2; EC number not identified), translocate into the nucleus, often upon activation (Deleris et al., 2003; Lachyankar et al., 2000; Neri et al., 2002). Specifically, PI3K translocates into the nucleus during its activation and the nuclear GTPase PIKE (EC number not identified) enhances its activity (Rong et al., 2003; Ye et al., 2002, Ye et al., 2000). In addition, SHIP2 and PTEN have been observed to partly reside in the nucleus and, for PTEN, this localization is specifically lost in some cancer tissue (Deleris et al., 2003; Ginn-Pease and Eng, 2003; Neri et al., 2002; Tachibana et al., 2002; Tanaka et al., 1999; Whiteman et al., 2002; Ye et al., 2000). Many other phosphoinositide kinases and phosphatases are reported to localize within the nucleus and moreover, a number of PtdInsP species, including PtdIns(3)P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, have been directly observed to be intranuclear (Boronenkov et al., 1998; Chen et al., 2002; Deleris et al., 2003; Garcia-Bustos et al., 1994; Gillooly et al., 2000; Neri et al., 2002; Tabellini et al., 2003; Watt et al., 2002; Yokogawa et al., 2000). The presence of these molecules and their modifying enzymes in the nucleus argues that many nuclear events are regulated by, at present, undiscovered PtdInsP-signaling mechanisms.
Indeed, functional evidence has emerged that PtdInsPs modulate a number of nuclear processes (Martelli et al., 2004a). For example, PtdIns(4,5)P2 induces association of the SWI/SNF-like BAF complex with chromatin in T cells during antigen stimulation and likely has a role in pre-mRNA splicing (Boronenkov et al., 1998; Osborne et al., 2001; Zhao et al., 1998). Finally, we have recently identified the plant homeodomain (PHD) finger, a protein domain commonly found on nuclear-localized, chromatin-regulatory proteins, as a nuclear PtdInsP receptor (Gozani et al., 2003). We have shown that interactions between the PHD finger of the candidate tumor suppressor ING2 and PtdIns(5)P regulate the ability of ING2 to associate with chromatin and induce p53-mediated apoptosis in response to genotoxic stimuli (Gozani et al., 2003). Thus, it is becoming abundantly clear that understanding the role of PtdInsPs in the nucleus is essential for understanding how altered PtdInsP homeostasis leads to disease.
One method of studying PtdInsP biology is to deliver synthetic PtdInsP analogs into living cells for chemical activation of functions normally mediated by endogenous PtdInsPs (Ozaki et al., 2000). This approach of “chemical rescue” has been used to discover and investigate diverse PtdnsP-biological activities; examples include activation of the proto-oncogene AKT/PKB, rescue of SHIP2 deficiency, trafficking of GLUT4, the establishment of cellular polarity, insulin-regulated F-actin stress fiber breakdown and golgi function (Derman et al., 1997; Franke et al., 1997; Maffucci et al., 2003; Padron et al., 2003; Sbrissa et al., 2004; Scheid et al., 2002; Wang et al., 2002, Wang et al., 2003; Weiner et al., 2002). Moreover, the repertoire of potential chemical applications has been greatly expanded by the development of carrier systems that allow for fluorescent, biotinylated, spin-labeled or photoactivatable PtdInsP analogs to be introduced into cells (Ozaki et al., 2000). Previously, treatment of cells with exogenous PtdInsPs has not been employed to probe nuclear PtdInsP functions in live cells, primarily because there has not been a well-defined PtdInsP-binding domain that resides and functions in the nucleus. We now use the PHD finger as a physiologically relevant nuclear PtdInsP receptor to demonstrate that exogenous PtdInsPs can access and function within the nucleus of living cells.
Section snippets
Materials, plasmids and cell culture
All PtdInsPs were from Echelon Biosciences, Inc. (Salt Lake City, UT). Vectors: pEGFP-C and pDsRed2 (Clontech). BalbC-3T3 or NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, penicillin, and streptomycin, at 5% CO2 at 37 °C. Transfections were carried out using LT1 (Mirus).
Confocal and time-lapse microscopy
Confocal microscopy was carried out as described (Gozani et al., 2003), using a BioRAD uRadience2000 system. For PtdInsP treatment, BalbC-3T3 cells grown on coverslips
Sub-nuclear targeting of the PtdInsP-binding PHD finger protein domain to nuclear foci
The majority of PHD-containing proteins are nuclear, and in many cases, mutations that disrupt the PHD finger have been shown to alter the sub-nuclear localization of these proteins (Sutherland et al., 2001). Consistently, we have previously reported that the fluorescent signal observed when the PHD finger of ING2 is fused to GFP (green fluorescent protein) is largely within the nucleus (Gozani et al., 2003). In addition, we have also previously found that endogenous ING2, which contains a
Summary
PtdInsPs are critical signaling molecules that regulate diverse cellular functions. One method to study PtdInsP biology involves using synthetic PtdInsP analogs to activate endogenous PtdInsP-mediated events in living cells. Such methodology has been successfully employed to explore the role of several PtdInsP-biological outcomes in the cytoplasm. However, this strategy has not previously been used to examine the function of PtdInsPs in the nucleus of live cells, primarily because there has not
Acknowledgments
We thank K. Chua for critical reading of the manuscript. This work was supported in part by NIH grants to JY (AG16674), GDP (GM57705 and NS29632), and LCC (GM36624). O.G. was supported by KO8AG19245 and is a recipient of a Burroughs Wellcome Career Development Award in Biomedical Sciences.
References (56)
- et al.
Inositides and the nucleus: phospholipase C beta family localization and signaling activity
Advan Enzyme Regul
(2000) - et al.
The lipid products of phosphoinositide 3-kinase increase cell motility through protein kinase C
J Biol Chem
(1997) - et al.
Phosphoinositide 3-kinase in immunological systems
Sem Immunol
(2002) - et al.
The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor
Cell
(2003) - et al.
Linking lipids to chromatin
Curr Opin Genet Dev
(2004) - et al.
Nuclear inositol lipid signaling and its potential involvement in malignant transformation
Biochim Biophys Acta
(2002) - et al.
Nuclear inositides: facts and perspectives
Pharmacol Ther
(2004) - et al.
The nuclear phosphoinositide 3-kinase/AKT pathway: a new second messenger system
Biochim Biophys Acta
(2002) - et al.
Phosphoinositides: key players in cell signalling, in time and space
Cell Signal
(2001) - et al.
Synthesis of l-α-phosphatidyl-d-myo-inositol 5-phosphate and l-α-phosphatidyl-d-myo-inositol 3,5-bisphosphate
Tetrahedron Lett
(1998)
In situ detection of phospholipid and phosphoinositide metabolism
Adv Enzyme Regul
Phosphatidylinositol-5-phosphate activation and conserved substrate specificity of the myotubularin phosphatidylinositol 3-phosphatases
Curr Biol
Phosphatidylinositol (3,4,5)P3 is essential but not sufficient for protein kinase B (PKB) activation; phosphatidylinositol (3,4)P2 is required for PKB phosphorylation at Ser-473: studies using cells from SH2-containing inositol-5-phosphatase knockout mice
Diacylglycerol kinase-theta is localized in the speckle domains of the nucleus
Exp Cell Res
Evidence that a phosphatidylinositol 3,4,5-trisphosphate-binding protein can function in nucleus
J Biol Chem
Pathways for phosphoinositide synthesis
Chem Phys Lipids
Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi
Cell
Pike. A nuclear GTPase that enhances PI3kinase activity and is regulated by protein 4.1N
Cell
Evidence that 3′-phosphorylated polyphosphoinositides are generated at the nuclear surface: use of immunostaining technique with monoclonal antibodies specific for PI 3,4-P(2)
FEBS Lett
Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling
Cell
Phosphoinositide signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA processing factors
Mol Biol Cell
Allosteric activation of PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate
J Biol Chem
The phosphoinositide 3-kinase pathway
Science
A monoclonal antibody to visualize PtdIns(3,4,5)P(3) in cells
J Histochem Cytochem
SHIP-2 and PTEN are expressed and active in vascular smooth muscle cell nuclei, but only SHIP-2 is associated with nuclear speckles
J Biol Chem
Membrane recognition and targeting by lipid-binding domains
Sci STKE
Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate
Science
PIK1, an essential phosphatidylinositol 4-kinase associated with the yeast nucleus
Embo J
Cited by (13)
Polyphosphoinositides in the nucleus: Roadmap of their effectors and mechanisms of interaction
2019, Advances in Biological RegulationCitation Excerpt :As mentioned earlier, decreased levels of PtdIns5P releases ING2 from the chromatin leading to its translocation to the cytoplasm, suggesting that the localisation of ING2 is dependent upon PtdIns5P (Gozani et al., 2003; Jones et al., 2006). This was further substantiated by the effect of synthetic PtdIns5P, as well as other PPIn, on the sub-nuclear targeting of fluorescently labelled NLS-PHD finger of ING2, which was altered in the wild type but not in the PPIn binding mutant (Gozani et al., 2005). Similarly, PIP3BP, which harbours two PH domains, both necessary for PtdIns(3,4,5)P3 binding, is translocated from the nucleus to the cytoplasm upon the introduction of constitutively active PI3K, which is prevented by loss of the PH domain binding to PPIn (Tanaka et al., 1997, 1999).
Subcellular localizations of Arabidopsis myotubularins MTM1 and MTM2 suggest possible functions in vesicular trafficking between ER and cis-Golgi
2016, Journal of Plant PhysiologyCitation Excerpt :ATX1 and elevated PtdIns5 P (either exogenously provided or resulting from the over-expression of Arabidopsis MYOTUBULARIN1, AtMTM1) oppositely affected the expression from an overlapping set of dehydration stress responding genes suggesting counteracting roles in a common lipid-signaling pathway (Alvarez-Venegas et al., 2006a,b; Ding et al., 2009,2012). Despite its potential to regulate both cytoplasmic and nuclear functions (Gozani et al., 2003, 2005; Jones and Divecha, 2004; Jones et al., 2006), PtdIns5 P remains the least-characterized and the most enigmatic member of the PtdIns family. Its localization and molecular functions in either mammalian or plant cells remains elusive.
SLy2 targets the nuclear SAP30/HDAC1 complex
2010, International Journal of Biochemistry and Cell BiologyThe polybasic region that follows the plant homeodomain zinc finger 1 of Pf1 is necessary and sufficient for specific phosphoinositide binding
2006, Journal of Biological ChemistryCitation Excerpt :The PHD zinc finger is a broadly expressed domain conserved across phyla that appears to have a predominant role in transcriptional regulation (9). One reported function of the PHD zinc finger is the binding of PIs, suggesting that PHD-containing proteins are responsive to PI-dependent signaling pathways (8, 15). We show here that a polybasic region that follows some, but not all, PHD domains is necessary and sufficient for PI binding.
LincRNA-p21 acts as a mediator of ING1b-induced apoptosis
2015, Cell Death and DiseaseING1 and ING2: Multifaceted tumor suppressor genes
2013, Cellular and Molecular Life Sciences