Macromolecules can be transported into the cells by endocytosis, either by phagocytosis or by pinocytosis. Typically, phagocytosis involves the uptake of solid large particles mediated by cell-surface receptors, whereas pinocytosis takes up fluid and solutes. The synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 plays fundamental roles in all forms of endocytosis. Curiously, almost all eukaryotic cells have multiple isoforms of the kinases that synthesize these critical phosphatidylinositols. In this issue of the Biochemical Journal, Namiko Tamura, Osamu Hazeki and co-workers report that the subunit p110α of the type I PI3K (phosphoinositide 3-kinase) is implicated in the phagocytosis and the pinocytosis of large molecules, whereas the receptor-mediated pinocytosis and micropinocytosis of small molecules do not seem to be controlled by this mechanism. The present commentary discusses recent literature that has begun to unravel why cells need so many phosphatidylinositol kinase isoforms, which were previously believed to be redundant.
- Fcγ receptor (FcγR)
- phosphoinositide 3-kinase α (P13Kα)
Eukaryotic cells are able to internalize small and large molecules through their plasma membranes via different mechanisms of endocytosis . The classification of endocytosis may be based on morphological and molecular differences. The best-established pathway for endocytosis utilizes clathrin. However, endocytosis can also occur by utilizing several clathrin-independent pathways. These include the caveolin-dependent, CLIC/GEEC-type, IL2Rβ (interleukin-2 receptor β chain), Arf6-dependent, flotin-associated, phagocytosis, macropinocytosis, circular dorsal ruffles and entosis pathways, among others. Large particles such as pathogens and apoptotic remnants are transported by phagocytosis. This process occurs exclusively in specialized scavenger cells, such as monocytes, neutrophils and macrophages. In contrast, fluid-phase large molecules are transported by macropinocytosis. This latter process occurs in all cells. Despite the morphological differences among the different types of endocytosis, all endocytic processes seem to share many intracellular signalling cascades. For example, both phagocytosis and pinocytosis require reorganization of the actin cytoskeleton, and both also require the synthesis of specific polyphosphoinositides.
KINASES AND PHOSPHATASES REGULATE PHOSPHATIDYLINOSITOL SIGNALLING
Phospholipid kinases are a group of enzymes that phosphorylate the inositol ring of phosphatidylinositol (see Figure 1). PIP5KI (phosphatidylinositol 4-phosphate 5-kinase I) and PI3K (phosphoinositide 3-kinase) are two phospholipid kinases that have been found to play a critical role in endocytosis . The three isoforms of PIP5KI (α, β and γ) all phosphorylate the D-5 position of the inositol ring to generate phosphatidylinositol 4,5-bisphosphates [PtdIns(4,5)P2, or PIP2]. PtdIns(4,5)P2 is the substrate used to generate the platelet second messengers Ins(1,4,5)P3, diacylglycerol and PtdIns(3,4,5)P3. It has also been shown that PtdIns(4,5)P2 in vitro binds to, and thereby regulates, a wide variety of actin-binding proteins.
PI3Ks are a group of enzymes that phosphorylate the D-3 position of the inositol ring of phosphatidylinositol. This enzymatic step contributes to intracellular signalling in processes as diverse as cell growth, cell cycle control, cell metabolism, cell survival and cell migration. Defects in the PI3K pathway contribute to the development of cancer and diabetes in humans . The mammalian PI3K isoforms have been categorized further into several classes that are ordered according to their preferred substrate. It is the class I PI3K enzymes that are primarily responsible for the phosphorylation of PtdIns(4,5)P2 at the D-3 position of the inositol head group, which produces PtdIns(3,4,5)P3. Family members of this class of PI3K are heterodimers composed of a 110 kDa catalytic subunit (p110) coupled to a regulatory subunit. Members of this class of PI3K can be further divided into two groups. Class IA PI3Ks are heterodimers composed of a catalytic subunit (p110α, p110β, or p110δ) bound to one of the several SH2 (Src homology 2)-motif-containing regulatory subunits. The SH2 motif regulates the PI3K complex by binding specific tyrosine-phosphorylated proteins. Class IB PI3K is a category of PI3K that has only a single member, and is a heterodimer composed of the p110γ catalytic subunit and the p101 regulatory subunit. This p110γ–p101 complex becomes activated after binding the Gβγ heterodimers associated with G-protein-coupled receptors. Genetically modified mice lacking either p110α or p110β die in utero, whereas mice lacking either p110δ or p110γ have a variety of haematopoietic defects.
PTEN (phosphatase and tensin homologue deleted on chromosome 10) and SHIP-1 (SH2-domain-containing inositol phosphatase 1) are inositol phosphatases that also play an important role in the regulation of phosphoinositides during endocytosis. These enzymes dephosphorylate PtdIns(3,4,5)P3, and thereby counteract the activity of PI3K. PtdIns(3,4,5)P3 undergoes dephosphorylation to either PtdIns(4,5)P2 by PTEN or to PtdIns(3,4)P2 by SHIP. The complex orchestration of phosphatidylinositols by PI3K, PIP5KI, PTEN and SHIP is critical for the cells to carry out the equally complex process of endocytosis.
ENDOCYTOSIS AND THE ACTIN SIGNALLING PATHWAY
In spite of the morphological variants of endocytosis, some pathways, such as phagocytosis and macropinocytosis, seem to share many regulatory signalling mechanisms that are involved in actin dynamics . Phagocytosis involves several well-co-ordinated steps in the plasma membrane (Figure 2). These sequential steps are: particle recognition, engulfment and phagosome maturation. In macrophages, particle recognition can be mediated by FcγR binding the particles through opsonins, such as complement proteins or γ-immunoglobulins. However, other scavenger receptors can also bind directly to the ligands. This recognition is followed by the micro-clustering of receptors. This induces the Src-family tyrosine kinases to phosphorylate the ITAM (immunoreceptor tyrosine-based activation motif) domain within these receptors. Tyrosine phosphorylation of the receptor generates docking sites for the SH2-domain-containing proteins, such as Syk tyrosine kinase, phospholipase Cγ1, the adaptor proteins GRB2 (growth-factor-receptor-bound protein 2) and GAB2 (GRB2-associated binding protein 2), and class IA PI3K.
The engulfment step requires the recruitment of phospholipid kinases and phosphatases into the phagocytic cup that changes the local concentrations of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 (see Figure 2) [5,6]. PtdIns(4,5)P2 controls the fission reaction by directly binding endocytic clathrin adaptors [AP-2 (adaptor protein 2), AP180 (assembly protein 180), CALM (clathrin assembly lymphoid myeloid leukaemia protein) and epsin] and many other endocytic factors, such as dynamin. PtdIns(4,5)P2 is also necessary for the selective recruitment and regulation of critical small GTPases (such as Rac1, CDC42 and ARF6), which localize at, and regulate the advancing edges of, the phagosomal cups. For example, GTP-bound Cdc42 directly associates with PtdIns(4,5)P2 and WASP (Wiskott–Aldrich syndrome protein), and together they stimulate the Arp2/3 complex (actin-related protein 2/3 complex) to induce actin assembly. The local production of PtdIns(3,4,5)P3 by PI3K within the nascent phagosomes also contributes to the actin nucleation that is necessary for the formation of cell membrane protrusions. Once formed, these pseudopods engulf and seal the particle. PtdIns(3,4,5)P3 plays another role by recruiting myosin X into the region, which then contracts the phagocytic cups.
Equally important to the synthesis of these phospholipids is their catabolism. Consumption of PtdIns(4,5)P2 is required to seal off the phagocytic cup, and the degradation of PtdIns(3,4,5)P3 is required to shed actin and fuse the phagosomes within the endosome/lysosome compartment. Thus the early phases of phagocytosis, including actin polymerization necessary for the extension of the phagosome cup, require mostly the PtdIns(4,5)P2 synthesis by PIP5KI. In contrast, later phases of phagocytosis, including contraction of the cup's distal margin, as well as fusion of membrane vesicles with cup membranes, depend on PtdIns(3,4,5)P3 synthesis by PI3K. In macropinocytosis, similar signalling cascades are activated during the different stages of ruffle formation, ruffle closure and vesicle formation.
INDIVIDUAL ISOFORMS OF PHOSPHATIDYLINOSITOL KINASES CONTRIBUTE TO SPECIFIC ASPECTS OF PHAGOCYTOSIS
Genetically modified mice have been useful in dissecting the unique roles of the different isoforms of phospholipid kinases. Mao et al.  have analysed the phagocytic function of macrophages containing loss-of-function mutations within either the PIP5KI-α or PIP5KI-γ gene. Although both PIP5KI isoforms are recruited to the phagocytic cup, and they both are capable of synthesizing PtdIns(4,5)P2, loss of individual isoforms indicates that they fulfil specific functions during phagocytosis. PIP5K-γ-mediated PtdIns(4,5)P2 synthesis is critical for the early stage of this process. Loss of PIP5K-γ specifically impairs actin depolymerization and FcγR micro-clustering. This demonstrates that PtdIns(4,5)P2 synthesis mediated solely by PIP5KI-γ is essential for particle attachment. This contrasts with the finding in macrophages lacking PIP5K-α that have no defect in particle attachment, but instead fail to engulf. In this stage of phagocytosis, PIP5K-α-mediated PtdIns(4,5)P2 synthesis sequentially activates CDC42, WASP and Arp2/3 complex. This ultimately induces actin polymerization at the nascent phagocytic cup and particle ingestion. This demonstrates that, even though the different PIP5KI isoforms may all synthesize PtdIns(4,5)P2, the individual phosphatidylinositol isoforms have unique roles.
In this issue of the Biochemical Journal, Tamura et al.  report that the p110α isoform of PI3K subtype IA plays a specific role in the regulation of phagocytosis and macropinocytosis in macrophages. By using RNA interference to lower the levels of individual PI3K isoforms within Raw 264.7 macrophage cells, these investigators were able to analyse the specific contribution of p110α, p110β, p110δ and p110γ to the uptake of various particles and solutes. They found that only the p110α-deficient Raw 264.7 cells had decreased phagocytosis of IgG-opsonized, C3bi-opsonized and non-opsonized zymosans in comparison with the control cells. In contrast, Raw 264.7 cells deficient in other PI3K isoforms (p110β, p110δ and p110γ) had normal phagocytosis of these particles. This finding suggests that p110α is required for a specific function within Raw 264.7 cells. However, the relative amounts of each of the PI3K isoforms will need to be determined in future studies to definitively support this conclusion.
Previous studies have shown that the different PI3K type I isoforms can have redundant and non-redundant functions . The unique functions of each isoform might depend on the cell type and the relative expression in the cell. Papakonstanti et al.  have recently compared the roles of the class IA PI3K isoforms in the signalling and biological activities in the primary and immortalized macrophages. They showed that, in primary macrophages, all class IA PI3K isoforms participated in the regulation of Rac1, whereas p110δ selectively controlled the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. In immortalized macrophages, the CSF1R also engaged p110α in signalling cascades that include Akt phosphorylation and regulation of DNA synthesis. In these cells, migration depended mainly on another PI3K isoform, p110γ. Interestingly, these isoform-specific roles of the PI3K also varied depending on the triggering ligand or the cell context.
HOW DO THE INDIVIDUAL ISOFORMS OF PHOSPHATIDYLINOSITOL KINASES FULFIL UNIQUE ROLES?
Since the different isoforms of PIP5KI all synthesize PtdIns(4,5)P2, and the isoforms of class I PI3K all generate PtdIns(3,4,5)P3, why do they have non-redundant functions? The literature suggests several possible explanations. First, activation of specific phosphatidylinositol kinase isoforms could be regulated differently. This explanation might be true for PIP5KI isoforms that appear to be structurally dissimilar. However, this explanation is difficult to account for the different roles played by individual PI3K isoforms. The p110α, p110β and p110γ isoforms all bind the same regulatory subunits, and are therefore believed to be regulated in an identical fashion.
An alternative explanation to explain the unique roles of the different isoforms of a phosphatidylinositol kinase is that individual isoforms are localized and activated within different subdomains of cells. In this model, individual isoforms generate compartmentalized pools of phosphatidylinositols within discrete regions of the cell that fulfil specific functions within that microdomain. In order for this model to be correct, the newly synthesized phosphatidylinositol would need to be used immediately after synthesis before it can diffuse away. Alternatively, another molecule could prevent the rapid diffusion of the phosphatidylinositol out of the microdomain. The PIPmodulins are examples of proteins speculated to constrain the diffusion of phosphoinositols. The role of PIPmodulins, or other proteins that restrict phosphatidylinositol diffusion, in phagocytosis remains to be elucidated.
Even though different isoforms of phosphatidylinositol kinases synthesize the same product, the study by Tamura et al.  helps to explain why there are so many of them in cells. Their work confirms that individual phosphatidylinositol kinase isoforms have overlapping, but not completely redundant, functions within cells. Future studies of these processes should provide greater insight and understanding of both fundamental phospholipid biochemistry and cell biology.
Abbreviations: Arp2/3 complex, actin-related protein 2/3 complex; PIP5KI, phosphatidylinositol 4-phosphate 5-kinase I; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SH2, Src homology 2; SHIP-1, SH2-domain-containing inositol phosphatase 1; WASP, Wiskott–Aldrich syndrome protein
- © The Authors Journal compilation © 2009 Biochemical Society