Membrane-Permeant Phosphoinositide Derivatives as Modulators of Growth Factor Signaling and Neurite Outgrowth

Abstract

Phosphoinositides are important signaling molecules that govern a large number of cellular processes such as proliferation, differentiation, membrane remodeling, and survival. Here we introduce a fully synthetic membrane-permeant derivative of a novel, easily accessible, and very potent phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] mimic: phosphatidylinositol 3,4,5,6-tetrakisphosphate [PtdIns(3,4,5,6)P(4)]. The membrane-permeant PtdIns(3,4,5,6)P(4) derivative activated pathways downstream of phosphatidylinositol 3-kinase (PI3K), including protein kinase B, p70S6K, mitogen-activated protein kinase, and protein kinase C, more potently than similar membrane-permeant PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) derivatives in the absence of receptor stimulation. In addition, we demonstrate that treatment of PC12 cells with the membrane-permeant PtdIns(3,4)P(2), PtdIns(3,4,5)P(3), and PtdIns(3,4,5,6)P(4) derivatives increases the number of neurites per cell in the presence of NGF. This work establishes membrane-permeant phosphoinositides as powerful tools to study PI3K signaling and directly demonstrates that 3-phosphorylated phosphoinositides are instrumental for neurite initiation.

Phosphoinositides in endocytosis

“…For example, internalization of β2-adrenergic receptors has been reported to be stimulated by class I PI3K p110γ-mediated PI(3,4,5)P 3 formation [66]. An elegant study using membrane-permeable PI derivatives [101] showed that acute increases of PI(3,4,5)P 3 in the absence of ligand-induced receptor activation trigger the internalization of receptor tyrosine kinases (RTKs). The epidermal growth factor receptor (EGFR) as well as the ephrinA4 receptor, but not GPCRs or the transferrin receptor, was internalized when the cellular concentration of PI(3,4,5)P 3 was increased by either administration of exogenous PI(3,4,5)P 3 or acute recruitment of class I PI3Ks to the plasma membrane [102].…”

Detection and manipulation of phosphoinositides

“…However, membrane permeable analogs of several PIs have been synthesized and successfully used. Using these compounds it was proven that PI(3,4)P 2 and PI(3,4,5)P 3 directly stimulate the formation of cell protrusions, tyrosine kinase receptor recycling and clathrin-mediated endocytosis [89–91]. More recently, these lipid analogs have been employed to show a role of PIs in actomyosin contraction in the developing Drosophila larvae, proving their applicability in in vivo settings [92].…”

Lipid Sorting and Multivesicular Endosome Biogenesis

“…For example, the addition of aminophospholipid increases endocytosis in living cells (Farge et al 1999) and adding the biologically active isoform, but not other isoforms, of LBPA reduces cholesterol overload in NPC cells (Chevallier et al 2008). Finally, a promising approach has been elaborated by Carsten Schultz and colleagues that developed very elegant strategies to introduce membrane-permeant versions of phosphoinositides into cells so that the lipid is released upon cleavage by cytosolic esterases (Laketa et al 2009;Mentel et al 2011). Such strategies, together with the development of novel chemical biology tools, offer promising new ways to study the function of membrane lipids (Riezman and Johnsson 2011;Wymann and Schultz 2012).…”