Quantification of PtdIns(3,4,5)<i>P</i>3 dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods

Yip, Shu Chin; Eddy, Robert J.; Branch, Angie M.; Pang, Huan; Wu, Haiyan; Yan, Ying; Drees, Beth E.; Neilsen, Paul O.; Condeelis, John S.; Backer, Jonathan M.

doi:10.1042/bj20071179

Cited by 37 publications

(49 citation statements)

References 35 publications

(246 reference statements)

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“…3 A and B). Unlike PIP3 staining at the cell periphery, the diffuse staining in the center of the cell is nonspecific, as it has been previously be shown to be resistant to wortmannin (38).…”

Section: P85 Truncations and Mutations Of The Ish2-c2 Contact Are Notmentioning

confidence: 99%

“…S2B). To evaluate the effects on intracellular PIP3 production, we stained quiescent cells expressing wild-type or mutant p85ni and stained cells with an anti-PIP3 antibody that we have previously characterized (38). Quiescent cells expressing mutant p85ni showed increased PIP3 production at the cell edge as compared to cells expressing wild-type p85ni, and no additively between the truncation and C2/iSH2 interface mutants was seen (Fig.…”

Section: P85 Truncations and Mutations Of The Ish2-c2 Contact Are Notmentioning

confidence: 99%

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Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Shekar

Flinn

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110␣ requires (i) an inhibitory contact between the p85 nSH2 domain and the p110␣ helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110␣. We proposed that oncogenic truncations of p85 fail to inhibit p110 due to a loss of the iSH2-nSH2 contact. However, we now find that within the context of a minimal regulatory fragment of p85 (the nSH2-iSH2 fragment, termed p85ni), the nSH2 domain rotates much more freely ( c Ϸ12.7 ns) than it could if it were interacting rigidly with the iSH2 domain. These data are not compatible with our previous model. We therefore tested an alternative model in which oncogenic p85 truncations destabilize an interface between the p110␣ C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110␣, similar to the truncated p85ni-572 STOP . Conversely, wild-type p85ni poorly inhibits p110␣N345K. Strikingly, the p110␣N345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110␣-N345 is not additive with the p85ni-572 STOP mutation. Similarly, the D560K/N564K mutation is not additive with the p85ni-572 STOP mutant for downstream signaling or cellular transformation. Thus, our data suggests that mutations at the C2-iSH2 domain contact and truncations of the iSH2 domain, which are found in human tumors, both act by disrupting the C2-iSH2 domain interface.cancer ͉ glioblastoma ͉ phosphoinositide 3-kinase ͉ PIK3CA P I 3-kinases are important cellular regulators of growth, survival, and motility, and deregulation of PI 3-kinase signaling contributes to cancer and other human diseases (1). Class IA PI 3-kinases, which produce PI[3,4,5]P3 in intact cells (2), are obligate heterodimers of a regulatory subunit (p85␣, p85␤, p55␣, p50␣, or p55␥) and a catalytic subunit (p110␣, p110␤, or p110␦) (reviewed in ref.3). The regulatory subunits have two major functions: they stabilize the catalytic subunits against thermal denaturation, and they maintain the catalytic subunit in an inhibited, low activity state (4, 5).p85 and p110 are both multidomain proteins that bind to each other and to upstream activators such as Rac and Cdc42, Ras, and tyrosine phosphorylated receptors and adapters (reviewed in ref. 6). p85 contains an SH3 domain, a Rac/Cdc42-binding domain homologous to a GAP domain in the BCR gene product, and two SH2 domains that flank an antiparallel coiled coil domain (the iSH2 domain). While NMR, EPR, and crystal structures have been obtained for the individual domains (7-15), there are currently no structures that define how these domains are arranged in space. The p110␣ catalytic subunit has been better defined, with structures of the N-terminal adapter-binding domain (ABD) or the entire p110␣ bound to the coiled coil (iSH2) domain of p85 (15,16). Like the related Class IB catalytic subunit p110␥ (17), p110␣ contains Ras-binding, C2, ...

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Section: P85 Truncations and Mutations Of The Ish2-c2 Contact Are Notmentioning

confidence: 99%

Section: P85 Truncations and Mutations Of The Ish2-c2 Contact Are Notmentioning

confidence: 99%

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Shekar

Flinn

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…General protocols for VASP, lamellipodin, and phalloidin staining have been previously described (11). For PI(3,4)P 2 staining, we followed a previous protocol (28). Briefly, cells, fixed/permeabilized by 3.7% formaldehyde/0.1% glutaraldehyde in 0.15 mg/mL saponin solution for 1 h at 37°C, were stained sequentially with a monoclonal mouse anti-PI (3,4)P 2 antibody (Echelon Biosciences; 1:200 dilution in 5% BSA/PBS) for 1 h and a rhodamine-conjugated secondary antibody for 45 min by standard methods.…”

Section: Methodsmentioning

confidence: 99%

Profilin1 regulates PI(3,4)P ₂ and lamellipodin accumulation at the leading edge thus influencing motility of MDA-MB-231 cells

Bae

Ding

Das

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

109

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Profilin1, a ubiquitously expressed actin-binding protein, plays a critical role in cell migration through actin cytoskeletal regulation. Given the traditional view of profilin1 as a promigratory molecule, it is difficult to reconcile observations that profilin1 is down-regulated in various invasive adenocarcinomas and that reduced profilin1 expression actually confers increased motility to certain adenocarcinoma cells. In this study, we show that profilin1 negatively regulates lamellipodin targeting to the leading edge in MDA-MB-231 breast cancer cells and normal cells; profilin1 depletion increases lamellipodin concentration at the lamellipodial tip (where it binds Ena/VASP), and this mediates the hypermotility. We report that the molecular mechanism underlying profilin1's modulation of lamellipodin localization relates to phosphoinositide control. Specifically, we show that phosphoinositide binding of profilin1 inhibits the motility of MDA-MB-231 cells by negatively regulating PI(3,4)P 2 at the membrane and thereby limiting recruitment of lamellipodin [a PI(3,4)P 2 -binding protein] and Ena/ VASP to the leading edge. In summary, this study uncovers a unique biological consequence of profilin1-phosphoinositide interaction, thus providing direct evidence of profilin1's regulation of cell migration independent of its actin-related activity.T he ubiquitously expressed cytoskeleton-modulating protein profilin1 influences multiple processes involved in cell motility, making it a challenge to elucidate the exact molecular mechanism that controls migration. At least one major function of profilin1 is to regulate actin polymerization. Profilin1 regenerates actin monomers from disassembling filament networks by facilitating the exchange of ATP for ADP on G-actin. By further inhibiting spontaneous nucleation of G-actin, profilin1 causes an accumulation of profilin1/ATP-G-actin pool available for polymerization. Because profilin1 also has an affinity for poly-Lproline sequences, it binds to almost all major actin nucleating and F-actin elongating proteins that contain proline-rich domains [e.g., N-WASP (neuronal Wiskott-Aldrich syndrome protein), Ena (enabled)/VASP (vasodilator stimulated phosphoprotein), and formins], and this allows profilin1-mediated recruitment of ATP-G-actin to these proteins, enhancing actin polymerization (1, 2). In addition, profilin1 binds to plasma membrane presumably through its interactions with various phosphoinositides (3). Profilin1 binds to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], phosphatidylinositol-3,4-bisphosphate [PI(3,4)P 2 ], and phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5) P 3 ]), at least in vitro (4). Based on PI(4,5)P 2 binding, it has been proposed that the phosphoinositide binding site of profilin1 overlaps with its actin-binding site (5), and to some extent spans a second region neighboring the polyproline binding site (6). This has prompted speculation that the major role of phosphoinositide binding of profilin1 would be to inhibit its interaction with act...

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“…Cells were fi xed and permeabilized at 37°C for 1 h with 3.7% (w/v) formaldehyde, 0.1% (w/v) glutaraldehyde, and 1.5 mg/ml saponin in buffer (5 mM KCl, 137 mM NaCl, 4 mM NaHCO 3 , 0.4 mM KH 2 PO 4 , 1.1 mM Na 2 HPO 4 , 2 mM MgCl 2 , 5 mM PIPES, pH 7.2, plus 2 mM EGTA and 5.5 mM glucose), as described previously ( 19 ). The cells were stained with Qdot-6Cys-GRP1-PH, Qdot-6Cys-PLC ␦ 1-PH, and Qdot-6Cys-TAPP1-2×PH domains and observed with an FV-1000 confocal microscope (Olympus, Tokyo, Japan).…”

Section: Phosphoinositide Extraction From Cellsmentioning

confidence: 99%

“…First, various concentrations of saponin for permeabilization were tested with a slight modifi cation as described previously ( 19 ). As shown in supplementary Fig.…”

Section: Visualization Of Pis In Insulin-stimulated Cho-ir Cellsmentioning

confidence: 99%

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes

Irino

Tokuda

Hasegawa

et al. 2012

Journal of Lipid Research

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This article is available online at http://www.jlr.org enzymes is associated with disorders such as diabetes and cancer, which suggests that cellular concentrations of each PI species is usually under strict control ( 4,5 ).A number of PI-binding domains have been identifi ed, including pleckstrin homology (PH); Phox (PX); Fab1, YOTB, Vac1p, and EEA1 (FYVE); Epsin N-terminal homology (ENTH); and Glucosyltransferase, Rab-like GTPase activator, and myotubularin (GRAM) domains ( 6, 7 ). Although most PH domains show little specifi city in the PIs they bind, several show relatively high specifi city; these include the PH domains of phospholipase C ␦ 1 (PLC ␦ 1) for PtdIns(4,5)P 2 and the general receptor for phosphoinositides-1 (GRP1) for PtdIns(3,4,5)P 3 ( 8, 9 ). In addition, the tandem-PH domain-containing protein-1 (TAPP1) and the four phosphate-adaptor protein-1 (FAPP1) PH domains specifi cally bind PtdIns(3,4)P 2 and PtdIns4P, respectively ( 10, 11 ). Other domains that bind specifi c PIs have also been described. The FYVE domains of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and early endosome antigen-1 (EEA1) specifi cally recognize PtdIns3P, which is primarily found in endosomes, multivesicular bodies, and phagosomes ( 12 ). The GRAM domains of myotubularin and myotubularin-related protein 2 (MTMR2) are involved in endosomal and vacuolar targeting through interaction with PtdIns(3,5)P 2 ( 13 ). Moreover, some PI-binding domains have functions beyond simply recruiting host proteins to the membrane surface. Such domains, including ENTH, Bin Amphiphysin Rvs (BAR), and FCH and BAR (F-BAR), bind PIs with little specifi city and participate in endocytosis, cytokinesis, and Press, February 3, 2012 DOI 10.1194 Quantifi cation and visualization of phosphoinositides by quantum dot-labeled specifi c binding-domain probes Abbreviations: CHO-IR , Chinese hamster ovary cells expressing the wild-type human insulin receptor; CHO-IR, GFP, green fl uorescent protein; GRP1, general receptor for phosphoinositides-1; PC, phosphatidylcholine; PDGF, platelet-derived growth factor; PE, phosphatidylethanolamine; PH, pleckstrin homology; PI, phosphoinositide; PI3K, PI 3-kinase; PLC ␦ 1, phospholipase C ␦ 1; Qdot, quantum dot; ROI, region of interest; TAPP-1, tandem-PH domain-containing protein-1. Published, JLR Papers in

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Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods

Cited by 37 publications

References 35 publications

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Profilin1 regulates PI(3,4)P ₂ and lamellipodin accumulation at the leading edge thus influencing motility of MDA-MB-231 cells

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes

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Quantification of PtdIns(3,4,5)P3 dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods

Cited by 37 publications

References 35 publications

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

Profilin1 regulates PI(3,4)P 2 and lamellipodin accumulation at the leading edge thus influencing motility of MDA-MB-231 cells

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes

Contact Info

Product

Resources

About

Profilin1 regulates PI(3,4)P ₂ and lamellipodin accumulation at the leading edge thus influencing motility of MDA-MB-231 cells