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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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PIP5KIγ RNAi. (A) PIP5KIγ siRNA design. Pan siRNA is directed against both isoforms. (B) PIP5KIγ protein knockdown. Effect of PIP5Kγ RNAi on protein expression of the targeted and nontargeted PIP5KIs. Western blots were probed with isoform specific antibodies. Additional data are provided in Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200408008/DC1. (C) Quantitative real-time PCR. PCR primers were used to quantitate PIP5KIγpan and PIP5KIγ90 mRNA and PIP5KIγ87 mRNA was calculated from the difference. Numbers indicate the amounts of each isoform relative to PIP5KIγ90 in control cells. Data are the average of duplicate RNAi samples from a single experiment. Similar results were obtained from another experiment. (D) PIP5KIγ is enriched in the PM. Endogenous PIP5KIγ was detected with anti-PIP5KIγpan antibody, and overexpressed HA-PIP5KIγ87 (in cDNA-transfected cells) were stained with anti-HA. Arrows indicate PM. Bars, 50 μm. (E) Differential PIP5KI membrane association. Fractions obtained after sequential sedimentation were loaded equivalently, except for the cytosol fraction (CYT), which was loaded 10 times less. Western blot band intensity was determined by quantitative densitometry, and expressed as a percent of total recovered, after correcting for differences in fraction of sample loaded.
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fig1: PIP5KIγ RNAi. (A) PIP5KIγ siRNA design. Pan siRNA is directed against both isoforms. (B) PIP5KIγ protein knockdown. Effect of PIP5Kγ RNAi on protein expression of the targeted and nontargeted PIP5KIs. Western blots were probed with isoform specific antibodies. Additional data are provided in Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200408008/DC1. (C) Quantitative real-time PCR. PCR primers were used to quantitate PIP5KIγpan and PIP5KIγ90 mRNA and PIP5KIγ87 mRNA was calculated from the difference. Numbers indicate the amounts of each isoform relative to PIP5KIγ90 in control cells. Data are the average of duplicate RNAi samples from a single experiment. Similar results were obtained from another experiment. (D) PIP5KIγ is enriched in the PM. Endogenous PIP5KIγ was detected with anti-PIP5KIγpan antibody, and overexpressed HA-PIP5KIγ87 (in cDNA-transfected cells) were stained with anti-HA. Arrows indicate PM. Bars, 50 μm. (E) Differential PIP5KI membrane association. Fractions obtained after sequential sedimentation were loaded equivalently, except for the cytosol fraction (CYT), which was loaded 10 times less. Western blot band intensity was determined by quantitative densitometry, and expressed as a percent of total recovered, after correcting for differences in fraction of sample loaded.

Mentions: PIP5KIγ has two splice variants (PIP5KIγ87 and 90) that are distinguished by a 28–amino acid extension at the COOH terminus of PIP5KIγ90 (Di Paolo et al., 2002; Ling et al., 2002; Fig. 1 A). PIP5KIγ90 is particularly enriched in neurons (Wenk et al., 2001); it is the major PIP2 synthesizing enzyme at the synapse, where it has been implicated in the regulation of clathrin coat recruitment, actin dynamics (Wenk et al., 2001) and focal adhesion formation (Di Paolo et al., 2002; Ling et al., 2002). In contrast, PIP5KIγ87 is not involved in focal adhesion formation or clathrin-mediated endocytosis (in HeLa cells; Padron et al., 2003).


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)

PIP5KIγ RNAi. (A) PIP5KIγ siRNA design. Pan siRNA is directed against both isoforms. (B) PIP5KIγ protein knockdown. Effect of PIP5Kγ RNAi on protein expression of the targeted and nontargeted PIP5KIs. Western blots were probed with isoform specific antibodies. Additional data are provided in Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200408008/DC1. (C) Quantitative real-time PCR. PCR primers were used to quantitate PIP5KIγpan and PIP5KIγ90 mRNA and PIP5KIγ87 mRNA was calculated from the difference. Numbers indicate the amounts of each isoform relative to PIP5KIγ90 in control cells. Data are the average of duplicate RNAi samples from a single experiment. Similar results were obtained from another experiment. (D) PIP5KIγ is enriched in the PM. Endogenous PIP5KIγ was detected with anti-PIP5KIγpan antibody, and overexpressed HA-PIP5KIγ87 (in cDNA-transfected cells) were stained with anti-HA. Arrows indicate PM. Bars, 50 μm. (E) Differential PIP5KI membrane association. Fractions obtained after sequential sedimentation were loaded equivalently, except for the cytosol fraction (CYT), which was loaded 10 times less. Western blot band intensity was determined by quantitative densitometry, and expressed as a percent of total recovered, after correcting for differences in fraction of sample loaded.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172614&req=5

fig1: PIP5KIγ RNAi. (A) PIP5KIγ siRNA design. Pan siRNA is directed against both isoforms. (B) PIP5KIγ protein knockdown. Effect of PIP5Kγ RNAi on protein expression of the targeted and nontargeted PIP5KIs. Western blots were probed with isoform specific antibodies. Additional data are provided in Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200408008/DC1. (C) Quantitative real-time PCR. PCR primers were used to quantitate PIP5KIγpan and PIP5KIγ90 mRNA and PIP5KIγ87 mRNA was calculated from the difference. Numbers indicate the amounts of each isoform relative to PIP5KIγ90 in control cells. Data are the average of duplicate RNAi samples from a single experiment. Similar results were obtained from another experiment. (D) PIP5KIγ is enriched in the PM. Endogenous PIP5KIγ was detected with anti-PIP5KIγpan antibody, and overexpressed HA-PIP5KIγ87 (in cDNA-transfected cells) were stained with anti-HA. Arrows indicate PM. Bars, 50 μm. (E) Differential PIP5KI membrane association. Fractions obtained after sequential sedimentation were loaded equivalently, except for the cytosol fraction (CYT), which was loaded 10 times less. Western blot band intensity was determined by quantitative densitometry, and expressed as a percent of total recovered, after correcting for differences in fraction of sample loaded.
Mentions: PIP5KIγ has two splice variants (PIP5KIγ87 and 90) that are distinguished by a 28–amino acid extension at the COOH terminus of PIP5KIγ90 (Di Paolo et al., 2002; Ling et al., 2002; Fig. 1 A). PIP5KIγ90 is particularly enriched in neurons (Wenk et al., 2001); it is the major PIP2 synthesizing enzyme at the synapse, where it has been implicated in the regulation of clathrin coat recruitment, actin dynamics (Wenk et al., 2001) and focal adhesion formation (Di Paolo et al., 2002; Ling et al., 2002). In contrast, PIP5KIγ87 is not involved in focal adhesion formation or clathrin-mediated endocytosis (in HeLa cells; Padron et al., 2003).

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