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Critical role of PIP5KI{gamma}87 in InsP3-mediated Ca(2+) signaling.

Wang YJ, Li WH, Wang J, Xu K, Dong P, Luo X, Yin HL - J. Cell Biol. (2004)

Bottom Line: Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation.PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%.Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.

ABSTRACT
Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP(3) or IP(3)) and is therefore critical to intracellular Ca(2+) signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation. PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP(2) mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.

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Effect of PIP5KI RNAi on PIP2. (A) PIP2 mass (HPLC) and 32P-incorporation (TLC). Means ± SEM of independent experiments are shown. (B) PIP2 distribution as detected with anti-PIP2 and overexpressed GFP-PLCδ-PH. Cross-sectional plots of fluorescence intensity are shown next to the image. Bars, 50 μm. (C) Analysis of PIP2 quantitation. (Left) The average intensities of anti-PIP2 at the PM and inside the cell are expressed in arbitrary units (mean ± SEM). 10 cells were analyzed per RNAi condition. (Right) PM/cytoplasmic intensity ratios of anti-PIP2 and GFP-PLCδ-PH were shown. 10 cells were analyzed per label per RNAi condition.
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fig4: Effect of PIP5KI RNAi on PIP2. (A) PIP2 mass (HPLC) and 32P-incorporation (TLC). Means ± SEM of independent experiments are shown. (B) PIP2 distribution as detected with anti-PIP2 and overexpressed GFP-PLCδ-PH. Cross-sectional plots of fluorescence intensity are shown next to the image. Bars, 50 μm. (C) Analysis of PIP2 quantitation. (Left) The average intensities of anti-PIP2 at the PM and inside the cell are expressed in arbitrary units (mean ± SEM). 10 cells were analyzed per RNAi condition. (Right) PM/cytoplasmic intensity ratios of anti-PIP2 and GFP-PLCδ-PH were shown. 10 cells were analyzed per label per RNAi condition.

Mentions: To understand how PIP5KIγ87 uniquely contributes to GPCR mediated IP3 signaling, we estimated the size and location of its PIP2 pool relative to those of other PIP5KIs. PIP5KIγpan siRNA reduces PIP2 mass, determined by HPLC (Nasuhoglu et al., 2002), by 14% [from 377 ± 90 (n = 3) to 325 ± 95 (n = 3) pmol/mg protein] and 32P-incorporation into PIP2, determined by TLC, to a similar extent (Fig. 4 A). PIP5KIγ90 siRNA has no statistically significant effect. PIP5KIβ RNAi decreases PIP2 mass by 34% (Fig. 4 A), which is consistent with the large decrease in [32P]PIP2 reported previously (Padron et al., 2003). Paradoxically, although PIP5KIα RNAi does not decrease [32P]PIP2 (Padron et al., 2003), it decreases PIP2 mass by 33%. The difference between the TLC and HPLC estimates may be because they measure different parameters. 32P-labeling/TLC detects PIP2 that turns over during the labeling period, whereas the HPLC method does not involve radiolabeling (Nasuhoglu et al., 2002) and measures PIP2 mass. It is possible that the 4-h labeling interval we used was not long enough to completely equilibrate a particularly stable PIP2 pool, and therefore underestimates its size.


Critical role of PIP5KI{gamma}87 in InsP3-mediated Ca(2+) signaling.

Wang YJ, Li WH, Wang J, Xu K, Dong P, Luo X, Yin HL - J. Cell Biol. (2004)

Effect of PIP5KI RNAi on PIP2. (A) PIP2 mass (HPLC) and 32P-incorporation (TLC). Means ± SEM of independent experiments are shown. (B) PIP2 distribution as detected with anti-PIP2 and overexpressed GFP-PLCδ-PH. Cross-sectional plots of fluorescence intensity are shown next to the image. Bars, 50 μm. (C) Analysis of PIP2 quantitation. (Left) The average intensities of anti-PIP2 at the PM and inside the cell are expressed in arbitrary units (mean ± SEM). 10 cells were analyzed per RNAi condition. (Right) PM/cytoplasmic intensity ratios of anti-PIP2 and GFP-PLCδ-PH were shown. 10 cells were analyzed per label per RNAi condition.
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fig4: Effect of PIP5KI RNAi on PIP2. (A) PIP2 mass (HPLC) and 32P-incorporation (TLC). Means ± SEM of independent experiments are shown. (B) PIP2 distribution as detected with anti-PIP2 and overexpressed GFP-PLCδ-PH. Cross-sectional plots of fluorescence intensity are shown next to the image. Bars, 50 μm. (C) Analysis of PIP2 quantitation. (Left) The average intensities of anti-PIP2 at the PM and inside the cell are expressed in arbitrary units (mean ± SEM). 10 cells were analyzed per RNAi condition. (Right) PM/cytoplasmic intensity ratios of anti-PIP2 and GFP-PLCδ-PH were shown. 10 cells were analyzed per label per RNAi condition.
Mentions: To understand how PIP5KIγ87 uniquely contributes to GPCR mediated IP3 signaling, we estimated the size and location of its PIP2 pool relative to those of other PIP5KIs. PIP5KIγpan siRNA reduces PIP2 mass, determined by HPLC (Nasuhoglu et al., 2002), by 14% [from 377 ± 90 (n = 3) to 325 ± 95 (n = 3) pmol/mg protein] and 32P-incorporation into PIP2, determined by TLC, to a similar extent (Fig. 4 A). PIP5KIγ90 siRNA has no statistically significant effect. PIP5KIβ RNAi decreases PIP2 mass by 34% (Fig. 4 A), which is consistent with the large decrease in [32P]PIP2 reported previously (Padron et al., 2003). Paradoxically, although PIP5KIα RNAi does not decrease [32P]PIP2 (Padron et al., 2003), it decreases PIP2 mass by 33%. The difference between the TLC and HPLC estimates may be because they measure different parameters. 32P-labeling/TLC detects PIP2 that turns over during the labeling period, whereas the HPLC method does not involve radiolabeling (Nasuhoglu et al., 2002) and measures PIP2 mass. It is possible that the 4-h labeling interval we used was not long enough to completely equilibrate a particularly stable PIP2 pool, and therefore underestimates its size.

Bottom Line: Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation.PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%.Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.

ABSTRACT
Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP(3) or IP(3)) and is therefore critical to intracellular Ca(2+) signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation. PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP(2) mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.

Show MeSH
Related in: MedlinePlus