Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biosenzitivní techniky MeSH
• buněčná membrána metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfatidylinositoly metabolismus   MeSH
• fosfolipasy typu C genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• intracelulární membrány metabolismus   MeSH
• kinetika MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• systémy druhého messengeru MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fosfatidylinositolfosfáty MeSH
• fosfatidylinositoly MeSH
• fosfolipasy typu C MeSH
• luminescentní proteiny MeSH
• rekombinantní fúzní proteiny MeSH

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIβ (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.

• Klíčová slova
• Hedgehog signaling, PI4KB, biological activity, inhibitors,
• MeSH
• buněčné linie MeSH
• cilie metabolismus   MeSH
• exprese genu účinky léků   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem antagonisté a inhibitory   genetika   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• morfoliny farmakologie   MeSH
• myši MeSH
• osteogeneze účinky léků   MeSH
• proteiny hedgehog antagonisté a inhibitory   genetika   metabolismus   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• puriny farmakologie   MeSH
• receptor Smoothened genetika   metabolismus   MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• thiofeny chemie   farmakologie   MeSH
• vedlejší histokompatibilní antigeny genetika   metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• malá interferující RNA MeSH
• morfoliny MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Prohlížeč
• proteiny hedgehog MeSH
• protinádorové látky MeSH
• puriny MeSH
• purmorphamine MeSH Prohlížeč
• receptor Smoothened MeSH
• thiofeny MeSH
• vedlejší histokompatibilní antigeny MeSH

Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.

• Klíčová slova
• PI4K2A, PtdIns4P, SNARE, Sorting, VAMP3, Vesicle fusion,
• MeSH
• buněčná membrána metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• endozomy metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus   MeSH
• fúze membrán fyziologie   MeSH
• lidé MeSH
• membránový protein 3 asociovaný s vezikuly metabolismus   MeSH
• proteiny SNARE metabolismus   MeSH
• receptory transferinu metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• vedlejší histokompatibilní antigeny MeSH
• vezikulární transportní proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• membránový protein 3 asociovaný s vezikuly MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Prohlížeč
• proteiny SNARE MeSH
• receptory transferinu MeSH
• vedlejší histokompatibilní antigeny MeSH
• vezikulární transportní proteiny MeSH