PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

Phospholipase D signaling: orchestration by PIP2 and small GTPases

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions

The multifaceted role of PIP2 in leukocyte biology

Phosphatidylinositol 4,5-bisphosphate (PIP2) represents about 1 % of plasma membrane phospholipids and behaves as a pleiotropic regulator of a striking number of fundamental cellular processes. In recent years, an increasing body of literature has highlighted an essential role of PIP2 in multiple aspects of leukocyte biology. In this emerging picture, PIP2 is envisaged as a signalling intermediate itself and as a membrane-bound regulator and a scaffold of proteins with specific PIP2 binding domains. Indeed PIP2 plays a key role in several functions. These include directional migration in neutrophils, integrin-dependent adhesion in T lymphocytes, phagocytosis in macrophages, lysosomes secretion and trafficking at immune synapse in cytolytic effectors and secretory cells, calcium signals and gene transcription in B lymphocytes, natural killer cells and mast cells. The coordination of these different aspects relies on the spatio-temporal organisation of distinct PIP2 pools, generated by the main PIP2 generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K). Three different isoforms of PIP5K, named α, β and γ, and different splice variants have been described in leukocyte populations. The isoform-specific coupling of specific isoforms of PIP5K to different families of activating receptors, including integrins, Fc receptors, toll-like receptors and chemokine receptors, is starting to be reported. Furthermore, PIP2 is turned over by multiple metabolising enzymes including phospholipase C (PLC) γ and phosphatidylinositol 3-kinase (PI3K) which, along with Rho family small G proteins, is widely involved in strategic functions within the immune system. The interplay between PIP2, lipid-modifying enzymes and small G protein-regulated signals is also discussed