One lipid, multiple functions: how various pools of PI(4,5)P(2) are created in the plasma membrane

Abstract

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] is a minor lipid of the inner leaflet of the plasma membrane that controls the activity of numerous proteins and serves as a source of second messengers. This multifunctionality of PI(4,5)P(2) relies on mechanisms ensuring transient appearance of PI(4,5)P(2) clusters in the plasma membrane. One such mechanism involves phosphorylation of PI(4)P to PI(4,5)P(2) by the type I phosphatidylinositol-4-phosphate 5-kinases (PIP5KI) at discrete membrane locations coupled with PI(4)P delivery/synthesis at the plasma membrane. Simultaneously, both PI(4)P and PI(4,5)P(2) participate in anchoring PIP5KI at the plasma membrane via electrostatic bonds. PIP5KI isoforms are also selectively recruited and activated at the plasma membrane by Rac1, talin, or AP-2 to generate PI(4,5)P(2) in ruffles and lamellipodia, focal contacts, and clathrin-coated pits. In addition, PI(4,5)P(2) can accumulate at sphingolipid/cholesterol-based rafts following activation of distinct membrane receptors or be sequestered in a reversible manner due to electrostatic constrains posed by proteins like MARCKS.

Fig. 1

PI(4,5)P₂ metabolism and activity. PI(4,5)₂ is generated mainly by phosphorylation of PI(4)P by PIP5KI (the pathway indicated by thicker arrows) and, to lower extend, by PIP4KII-catalyzed phosphorylation of PI(5)P. PI(4,5)₂ directly affects activity of numerous proteins, serves as a source of second messengers: IP₃, DAG, and PI(3,4,5)P_3. The lipid can be also dephosphorylated at the D-4 and D-5 positions of the inositol ring

Fig. 2

Organization of PIP5KI molecule. a Amino acid sequence of activation loop regions of human PIP5KIα, human Iβ, and murine Iγ (upper panel) and human PIP4KIIβ (lower panel). The first and the last amino acid of the regions as well as the total number of amino acids composing the proteins are indicated. Dark gray background marks amino acids conserved between PIP5KI and PIP4KII subfamilies; amino acids conserved only within the PIP5KI and PIP4KII subfamilies, but different between them are shown by the light gray background [22, 23, 59]. Bar below the alignment indicates regions exchanged between PIP5KIβ and PIP4KIIβ by Kunz et al. [56]. Asterisks indicate two lysine residues participating in membrane binding of PIPKs while glutamic acid residue and alanine residue determining substrate specificity of PIP5KI and PIP4KII, respectively, are marked by boxes. b A schematic representation of murine PIP5KIγa (661 amino acids) and PIP5KIγb (635 amino acids). PIP5KIγc of rodents is composed of 687 amino acids and contains a 26-amino-acid insert at Ph635 [23, 24]. AL, activation loop. In the lower panel, the amino acid composition of 26-amino-acid tail of murine PIP5KIγa is shown. The motifs engaged in the binding of talin, AP-2β and AP-2μ, are marked by bars. In human PIP5KIγa, two amino acids are inserted between Arg652 and Pro653 with the Arg652 exchanged for Glu652 [132]

Fig. 3

Rac1 forms multimolecular complex engaging PIP5KI. a In resting cells, PIP5KIα/β and DAGKζ are bound to the C-terminus of GDP-loaded Rac1. The interaction of Rac1 with the plasma membrane is blocked by RhoGDI, which masks the prenyl group attached to the C-terminal CAAX motif of Rac1. b Upon cell activation, PLC produces DAG. c DAG is subsequently phosphorylated by DAGKζ to PA. The PA activates PAK1, which phosphorylates RhoGDI at two sites. Phosphorylated RhoGDI dissociates from Rac1 exposing its prenyl group. d Rac1 binds to the membrane via the prenyl group and adjacent polybasic region and this facilitates replacement of GDP with GTP by membrane-bound exchange factors. PIP5KIα/β recruited to the plasma membrane together with Rac1 is activated by PA and phosphorylates PI(4)P to PI(4,5)P₂

Fig. 4

Talin and PIP5KIγa/c interact during focal contact assembly. Upon cell activation, PIP5KIγa/c undergoes phosphorylation on Tyr644 by Src kinase, which also associates with it. Both events are positively regulated by FAK kinase. When phosphorylated, PIP5KIγa/c binds to the FERM domain located in the globular N-terminal part of talin. The kinase produces PI(4,5)P₂, which binds to the FERM domain of talin and relieves autoinhibitory interaction of the talin head with rod. At these conditions, talin is able to bind β-integrins, inducing additionally their aggregation and activation. Intracellularly, talin binds actin filaments, which enables the formation of stress fibers. The Tyr644 can be dephosphorylated by Shp-1, which associates with PIP5KIγ. The activity of Shp-1 is inhibited by PI(4,5)P₂ favoring the lipid accumulation. On the other hand, the binding of β-integrin to talin can be inhibited by phosphorylation of β-integrin by Src kinase

Fig. 5

PIP5KIγa interacts with AP-2 at the onset of receptor endocytosis. a, b Initially, GTP-loaded ARF6 activates PIP5KIγa leading to synthesis of PI(4,5)P₂. The lipid provides an anchor for the α subunit of the AP-2 complex. Concomitant dephosphorylation of Ser645 of PIPKIγa by calcineurin (CalN) is required for binding of the kinase C-terminus to the β2 appendage of the AP-2 complex. c Conformational changes of the μ subunit of the AP-2 complex allow recognition of a cargo receptor. Besides the binding of the cargo, μ2 also interacts with PIPKIγa catalytic core, activating the kinase and leading to PI(4,5)P₂ production. The lipid serves as an anchor for the μ2 chain of the AP-2 complex. d The C-terminus of the kinase can also shift from β2 to the μ2 and binds to the μ2 chain via the ⁶⁴⁴YSPL⁶⁴⁷ motif. This interaction promotes the recognition of cargo receptors devoid of the canonical YXXØ motif; it also activates PIP5KIγa facilitating local PI(4,5)P₂ accumulation