Rapid Chemically Induced Changes of PtdIns(4,5)P
            <sub>2</sub>
            Gate KCNQ Ion Channels

Suh, Byung Chang; Inoue, Takanari; Meyer, Tobias; Hille, Bertil

doi:10.1126/science.1131163

Cited by 467 publications

(600 citation statements)

References 26 publications

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“…HEK293 cells were transfected with mRFP-PH(PLC␦), Lyn11-FRB, together with either CFP-FKBP-Inp54p(Inp54p) or the phosphatase-dead mutant Inp54p(D281A). Addition of rapamycin induced a rapid translocation of mRFP-PH(PLC␦) from the plasma membrane to the cytosol within 3 min in cells transfected with Inp54p (Supplemental Figure S4) as described previously (Suh et al, 2006), confirming the PI(4,5)P2 depletion. In contrast, mRFP-PH(PLC␦) did not detach from the plasma membrane upon the addition of rapamycin in cells expressing Inp54p(D281A).…”

Section: Marcks Increased Filopodia Motility In a Pi(45)p2-dependentsupporting

confidence: 83%

“…Thus, we used a more specific method to deplete PI(4,5)P2: rapamycin-induced membrane translocation of Inp54p, a yeast inositol polyphosphate 5-phosphatase that specifically cleaves the phosphate at the 5-position of PI(4,5)P2 (Suh et al, 2006). HEK293 cells were transfected with mRFP-PH(PLC␦), Lyn11-FRB, together with either CFP-FKBP-Inp54p(Inp54p) or the phosphatase-dead mutant Inp54p(D281A).…”

Section: Marcks Increased Filopodia Motility In a Pi(45)p2-dependentmentioning

confidence: 99%

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Actin Filament Assembly by Myristoylated, Alanine-rich C Kinase Substrate–Phosphatidylinositol-4,5-diphosphate Signaling Is Critical for Dendrite Branching

Chen

Zhou

et al. 2008

MBoC

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Dendrites undergo extensive growth and branching at early stages, but relatively little is known about the molecular mechanisms underlying these processes. Here, we show that increasing the level of myristoylated, alanine-rich C kinase substrate (MARCKS), a prominent substrate of protein kinase C and a phosphatidylinositol-4,5-diphosphate [PI(4,5)P2] sequestration protein highly expressed in the brain, enhanced branching and growth of dendrites both in vitro and in vivo. Conversely, knockdown of endogenous MARCKS by RNA interference reduced dendritic arborization. Results from expression of different mutants indicated that membrane binding is essential for MARCKS-induced dendritic morphogenesis. Furthermore, MARCKS increased the number and length of filamentous actin-based filopodia along neurites, as well as the motility of filopodia, in a PI(4,5)P2-dependent manner. Time-lapse imaging showed that MARCKS increased frequency of filopodia initiation but did not affect filopodia longevity, suggesting that MARCKS may increase dendritic branching through its action on filopodia initiation. These findings demonstrate a critical role for MARCKS-PI(4,5)P2 signaling in regulating dendrite development.

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Section: Marcks Increased Filopodia Motility In a Pi(45)p2-dependentsupporting

confidence: 83%

Section: Marcks Increased Filopodia Motility In a Pi(45)p2-dependentmentioning

confidence: 99%

Actin Filament Assembly by Myristoylated, Alanine-rich C Kinase Substrate–Phosphatidylinositol-4,5-diphosphate Signaling Is Critical for Dendrite Branching

Chen

Zhou

et al. 2008

MBoC

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“…To build up membrane PIP 2 , we inhibited PLC (25 μ M U73122; Wilkerson et al, 2006 ), the major PIP 2 -metabolizing enzyme ( Tolloczko et al, 2002 ). This treatment, however, can reroute PIP 2 toward formation of PI3ki-nase-mediated PIP 3 ( Fruman et al, 1998 ;Suh et al, 2006 ), a powerful BK channel activator ( Fig. 2 ).…”

Section: Pip 2 Regulates Cerebrovascular Tone Via Bk Channelsmentioning

confidence: 99%

Direct Regulation of BK Channels by Phosphatidylinositol 4,5-Bisphosphate as a Novel Signaling Pathway

Vaithianathan¹,

Bukiya²,

Liu³

et al. 2008

J Cell Biol

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“…Conditional regulation of protein localization has been achieved using the FKBP⅐rapamycin⅐FRB complex (7)(8)(9)(10)(11)(12). With this method a protein of interest is fused to either FKBP, the 12-kDa FK506-and rapamycin-binding protein or FRB, the FKBP-rapamycin binding domain of the protein kinase, mTOR.…”

mentioning

confidence: 99%

The Rapamycin-binding Domain of the Protein Kinase Mammalian Target of Rapamycin Is a Destabilizing Domain

Edwards

Wandless

2007

Journal of Biological Chemistry

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Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506-and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.A common approach for studying complex biological processes is to perturb a protein of interest and observe subsequent changes to the biological system. The classical approach of disrupting a gene and studying the resultant phenotype is problematic for examining essential genes. As a result, several conditional techniques have been developed including the tetracycline/doxycycline and Cre recombinase/lox P systems, where control is exerted at the transcriptional level (1, 2). At the post-transcriptional level, RNA interference (RNAi) 2 is becoming rapidly used as a method of gene silencing (3), but RNAi lacks tunability and does not affect existing protein levels. An ideal perturbation method would directly affect the protein with a cell-permeable probe that rapidly and reversibly targets a specific protein of interest.Small molecules have been very useful as conditional perturbants; however, the lack of specificity of many of these reagents can make it difficult to unambiguously interpret results. One approach to ensure specificity is to chemically modify a small molecule with a large substituent, and this technique has been widely used to interrogate specific protein tyrosine kinases that possess a complementary cavity-forming mutation (4, 5). An alternative system has recently been developed using a cellperm...

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Rapid Chemically Induced Changes of PtdIns(4,5)P ₂ Gate KCNQ Ion Channels

Cited by 467 publications

References 26 publications

Actin Filament Assembly by Myristoylated, Alanine-rich C Kinase Substrate–Phosphatidylinositol-4,5-diphosphate Signaling Is Critical for Dendrite Branching

Actin Filament Assembly by Myristoylated, Alanine-rich C Kinase Substrate–Phosphatidylinositol-4,5-diphosphate Signaling Is Critical for Dendrite Branching

Direct Regulation of BK Channels by Phosphatidylinositol 4,5-Bisphosphate as a Novel Signaling Pathway

The Rapamycin-binding Domain of the Protein Kinase Mammalian Target of Rapamycin Is a Destabilizing Domain

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Rapid Chemically Induced Changes of PtdIns(4,5)P 2 Gate KCNQ Ion Channels

Cited by 467 publications

References 26 publications

Actin Filament Assembly by Myristoylated, Alanine-rich C Kinase Substrate–Phosphatidylinositol-4,5-diphosphate Signaling Is Critical for Dendrite Branching

Actin Filament Assembly by Myristoylated, Alanine-rich C Kinase Substrate–Phosphatidylinositol-4,5-diphosphate Signaling Is Critical for Dendrite Branching

Direct Regulation of BK Channels by Phosphatidylinositol 4,5-Bisphosphate as a Novel Signaling Pathway

The Rapamycin-binding Domain of the Protein Kinase Mammalian Target of Rapamycin Is a Destabilizing Domain

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Rapid Chemically Induced Changes of PtdIns(4,5)P ₂ Gate KCNQ Ion Channels