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A dPIP5K dependent pool of phosphatidylinositol 4,5 bisphosphate (PIP2) is required for G-protein coupled signal transduction in Drosophila photoreceptors.

Chakrabarti P, Kolay S, Yadav S, Kumari K, Nair A, Trivedi D, Raghu P - PLoS Genet. (2015)

Bottom Line: Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane.These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction.Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

View Article: PubMed Central - PubMed

Affiliation: Inositide Laboratory, Babraham Institute, Cambridge, United Kingdom.

ABSTRACT
Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

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Related in: MedlinePlus

PIP kinase genes in Drosophila genome.A) Multiple alignment of the protein sequences of PIP4K and PIP5K genes. The sequence around the activation loop is presented with the region indicated by a grey line. Amino acid residues common between PIP5K and PIP4K proteins are marked in blue; residues unique to PIP5K are marked in yellow and unique residues for PIP4K in grey. The red arrow indicates the single residue described as responsible for the unique substrate specificity of PIP4K and PIP5K. Notations used for gene names are; hs-Homo sapiens; a-alpha; b-beta; g-gamma; CG17471-Drosophila PIP4K. B) RNA expression pattern of PIP kinases in various fly tissues: Qualitative RT (reverse transcription) PCR analysis with RNA extracted from various fly tissues. The tissue sources are labeled above the lanes. ‘+’ denotes +RT and ‘−’ denotes −RT. The corresponding gene names are indicated on the left side of the agarose gel picture. C) Comparative real time PCR analysis showing eye enrichment of dPIP5K and dPIP4K; the X-axis indicates gene names and the Y-axis represents transcript level expression in arbitrary units (A.U). White bars represent expression levels from cDNA samples of wild type fly heads and black bars represent samples from heads of soD (mutants that lack eyes). Values shown are the means ± S.D of three independent samples. p values between wild type and soD samples were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05)
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pgen.1004948.g001: PIP kinase genes in Drosophila genome.A) Multiple alignment of the protein sequences of PIP4K and PIP5K genes. The sequence around the activation loop is presented with the region indicated by a grey line. Amino acid residues common between PIP5K and PIP4K proteins are marked in blue; residues unique to PIP5K are marked in yellow and unique residues for PIP4K in grey. The red arrow indicates the single residue described as responsible for the unique substrate specificity of PIP4K and PIP5K. Notations used for gene names are; hs-Homo sapiens; a-alpha; b-beta; g-gamma; CG17471-Drosophila PIP4K. B) RNA expression pattern of PIP kinases in various fly tissues: Qualitative RT (reverse transcription) PCR analysis with RNA extracted from various fly tissues. The tissue sources are labeled above the lanes. ‘+’ denotes +RT and ‘−’ denotes −RT. The corresponding gene names are indicated on the left side of the agarose gel picture. C) Comparative real time PCR analysis showing eye enrichment of dPIP5K and dPIP4K; the X-axis indicates gene names and the Y-axis represents transcript level expression in arbitrary units (A.U). White bars represent expression levels from cDNA samples of wild type fly heads and black bars represent samples from heads of soD (mutants that lack eyes). Values shown are the means ± S.D of three independent samples. p values between wild type and soD samples were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05)

Mentions: In silico analysis of the Drosophila genome sequence revealed that there are four distinct genes that encode open reading frames that include the Interpro domain IPR00002498 which is the “PIP kinase catalytic domain”. These include CG6355, CG3682, CG9985 and CG17471. Of these CG6355 encodes a FYVE domain containing protein that is the single ortholog of yeast Fab1, a protein with 1-phosphatidylinositol 3—phosphate 5 kinase activity [12][13]. CG17471 (dPIP4K) has recently been shown to encode a PIP4K activity that can generate PIP2 by 4 kinase activity using PI5P as a substrate [14]. The remaining two genes namely CG9985 (sktl) and CG3682 could encode putative PIP5K activity. sktl has been proposed to encode a Drosophila PIP5K [15][16]. CG3682 is an independent gene that also encodes a putative PIP5K activity. Previous studies have shown that the activation loop region of PIPKs contains specific residues that are conserved among PIP5K and are distinct from PIP4K enzymes [8]. A multiple alignment of PIP5K and PIP4K proteins from mammals and Drosophila reveals that sktl and CG3682 have activation loop residues that are highly diagnostic of those seen in mammalian PIP5K enzymes (Fig. 1A). Both SKTL and dPIP5K show high level of sequence similarity with all the isoforms of mammalian PIP5K. In catalytic domain the identity is more than 80%, whereas the overall sequence homology is from 55–65% with different mammalian isoforms. SKTL is ubiquitously expressed in all organs (Fig. 1B) suggesting its function in many/all cell types. In mammals this kind of expression pattern is evident for α and β isoforms of the PIP5K [17]. By contrast, the γ isoform of PIP5K is mostly expressed in neuronal tissues [18,19] an expression pattern recapitulated by dPIP5K. In addition dPIP5K has multiple splice variants with a conserved catalytic domain and variable C-terminal extensions [19]. The splicing pattern and protein isoforms of dPIP5K so generated as well as its expression pattern (enriched in the adult head Fig. 1B) recapitulates that seen for mammalian PIP5Kγ. The functional significance of the splice variants of dPIP5K remains to be established. In summary it is very likely that the Drosophila genome contains two genes that encode PIP5K activity namely sktl and CG3682. We have named CG3682 as dPIP5K. Thus, collectively there are three phosphoinositide kinases (PIPK), sktl, dPIP5K and dPIP4K all of which could generate PIP2.


A dPIP5K dependent pool of phosphatidylinositol 4,5 bisphosphate (PIP2) is required for G-protein coupled signal transduction in Drosophila photoreceptors.

Chakrabarti P, Kolay S, Yadav S, Kumari K, Nair A, Trivedi D, Raghu P - PLoS Genet. (2015)

PIP kinase genes in Drosophila genome.A) Multiple alignment of the protein sequences of PIP4K and PIP5K genes. The sequence around the activation loop is presented with the region indicated by a grey line. Amino acid residues common between PIP5K and PIP4K proteins are marked in blue; residues unique to PIP5K are marked in yellow and unique residues for PIP4K in grey. The red arrow indicates the single residue described as responsible for the unique substrate specificity of PIP4K and PIP5K. Notations used for gene names are; hs-Homo sapiens; a-alpha; b-beta; g-gamma; CG17471-Drosophila PIP4K. B) RNA expression pattern of PIP kinases in various fly tissues: Qualitative RT (reverse transcription) PCR analysis with RNA extracted from various fly tissues. The tissue sources are labeled above the lanes. ‘+’ denotes +RT and ‘−’ denotes −RT. The corresponding gene names are indicated on the left side of the agarose gel picture. C) Comparative real time PCR analysis showing eye enrichment of dPIP5K and dPIP4K; the X-axis indicates gene names and the Y-axis represents transcript level expression in arbitrary units (A.U). White bars represent expression levels from cDNA samples of wild type fly heads and black bars represent samples from heads of soD (mutants that lack eyes). Values shown are the means ± S.D of three independent samples. p values between wild type and soD samples were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05)
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Related In: Results  -  Collection

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pgen.1004948.g001: PIP kinase genes in Drosophila genome.A) Multiple alignment of the protein sequences of PIP4K and PIP5K genes. The sequence around the activation loop is presented with the region indicated by a grey line. Amino acid residues common between PIP5K and PIP4K proteins are marked in blue; residues unique to PIP5K are marked in yellow and unique residues for PIP4K in grey. The red arrow indicates the single residue described as responsible for the unique substrate specificity of PIP4K and PIP5K. Notations used for gene names are; hs-Homo sapiens; a-alpha; b-beta; g-gamma; CG17471-Drosophila PIP4K. B) RNA expression pattern of PIP kinases in various fly tissues: Qualitative RT (reverse transcription) PCR analysis with RNA extracted from various fly tissues. The tissue sources are labeled above the lanes. ‘+’ denotes +RT and ‘−’ denotes −RT. The corresponding gene names are indicated on the left side of the agarose gel picture. C) Comparative real time PCR analysis showing eye enrichment of dPIP5K and dPIP4K; the X-axis indicates gene names and the Y-axis represents transcript level expression in arbitrary units (A.U). White bars represent expression levels from cDNA samples of wild type fly heads and black bars represent samples from heads of soD (mutants that lack eyes). Values shown are the means ± S.D of three independent samples. p values between wild type and soD samples were determined using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05)
Mentions: In silico analysis of the Drosophila genome sequence revealed that there are four distinct genes that encode open reading frames that include the Interpro domain IPR00002498 which is the “PIP kinase catalytic domain”. These include CG6355, CG3682, CG9985 and CG17471. Of these CG6355 encodes a FYVE domain containing protein that is the single ortholog of yeast Fab1, a protein with 1-phosphatidylinositol 3—phosphate 5 kinase activity [12][13]. CG17471 (dPIP4K) has recently been shown to encode a PIP4K activity that can generate PIP2 by 4 kinase activity using PI5P as a substrate [14]. The remaining two genes namely CG9985 (sktl) and CG3682 could encode putative PIP5K activity. sktl has been proposed to encode a Drosophila PIP5K [15][16]. CG3682 is an independent gene that also encodes a putative PIP5K activity. Previous studies have shown that the activation loop region of PIPKs contains specific residues that are conserved among PIP5K and are distinct from PIP4K enzymes [8]. A multiple alignment of PIP5K and PIP4K proteins from mammals and Drosophila reveals that sktl and CG3682 have activation loop residues that are highly diagnostic of those seen in mammalian PIP5K enzymes (Fig. 1A). Both SKTL and dPIP5K show high level of sequence similarity with all the isoforms of mammalian PIP5K. In catalytic domain the identity is more than 80%, whereas the overall sequence homology is from 55–65% with different mammalian isoforms. SKTL is ubiquitously expressed in all organs (Fig. 1B) suggesting its function in many/all cell types. In mammals this kind of expression pattern is evident for α and β isoforms of the PIP5K [17]. By contrast, the γ isoform of PIP5K is mostly expressed in neuronal tissues [18,19] an expression pattern recapitulated by dPIP5K. In addition dPIP5K has multiple splice variants with a conserved catalytic domain and variable C-terminal extensions [19]. The splicing pattern and protein isoforms of dPIP5K so generated as well as its expression pattern (enriched in the adult head Fig. 1B) recapitulates that seen for mammalian PIP5Kγ. The functional significance of the splice variants of dPIP5K remains to be established. In summary it is very likely that the Drosophila genome contains two genes that encode PIP5K activity namely sktl and CG3682. We have named CG3682 as dPIP5K. Thus, collectively there are three phosphoinositide kinases (PIPK), sktl, dPIP5K and dPIP4K all of which could generate PIP2.

Bottom Line: Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane.These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction.Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

View Article: PubMed Central - PubMed

Affiliation: Inositide Laboratory, Babraham Institute, Cambridge, United Kingdom.

ABSTRACT
Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

Show MeSH
Related in: MedlinePlus