3. Inositol lipids and phosphates in signaling
One of the most essential characteristics of cells is their ability to respond with changes in their behavior to chemical changes in their environment. Many of these extracellular chemical signals cannot penetrate the plasma membrane, and so the cell surface receptors that respond to them couple to transmembrane signal transduction mechanisms that pass information to the cell interior. Inositol lipids play essential roles as the metabolic substrates and/or the output signals of at least two, and maybe three or more, widespread cell signaling systems.
3.1. Signaling through Phospholipase C Activation
The best characterized of these signaling pathways uses receptor activation of PtdIns(4,5) P2 hydrolysis by phosphoinositidase C (phospholipase C, PLC) to make the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5) P3) and 1,2-diacylglycerol, as outlined in Fig 3 (top right).
Despite extensive early work by the Hokins (3), only in the 1970s was it realized that inositol lipid hydrolysis has the properties of a coupling reaction that links cell-surface receptors to the intracellular mobilization of Ca (2) (see Calcium Signaling). PtdIns(4,5)P2 hydrolysis was then identified as the primary stimulated reaction (10). Meanwhile, Nishizuka defined the polyunsaturated 1,2-diacylglyerol (1,2-DG) liberated from PtdIns(4,5)P2 as a second messenger that activates many members of the protein kinase C family (11). Finally, Streb et al. showed that Ins(1,4,5) P3 released Ca from an intracellular store (12). We now know that Ins(1,4,5)P3 triggers intracellular Ca release through the intrinsic cation channels of at least four subtypes of Ins(1,4,5)P3 "receptor" that are expressed primarily in various elements of the endoplasmic reticulum (13).
These revelations established two new principles. First, chemical species very different from nucleotide cyclic phosphates (see -cyclic AMP, cAMP); -Monophosphate, cGMP)) and the Ca ion, the prototypic intracellular messenger molecules that were recognized between the mid-1950s and the mid-1970s, can function as "second messengers." Second, receptor-generated intracellular messenger molecules need not be water-soluble and thus mobile within the cell. Instead, protein kinase C homes to the 1,2-DAG that is formed at the plasma membrane as a consequence of PtdIns (4,5)P2 hydrolysis. There it is activated by simultaneous interactions with 1,2-DAG, anionic aminophospholipids, notably phosphatidylserine, in the inner leaflet of the plasma membrane, and the ambient concentration of cytoplasmic Ca2 .
There are numerous excellent reviews of the PLC signaling pathway and the extraordinary variety of receptors that harness it (eg, refs. 11-15; and see Phospholipases C): some of the important receptors that harness this signaling pathway are listed in Table 1(see also Acetylcholine Receptor, Rhodopsin). There is a substantial family of PLCs that can hydrolyze PtdIns(4,5)P2, and these are activated by two or more fundamentally different mechanisms (16). PLCs of the b-family are activated by some subfamilies of receptors in the 7-span or "serpentine" superfamily. Different serpentine receptors activate PLC-bs, either through the GTP-ligated a-subunits of heterotrimeric G proteins of the G q subfamily (pertussis toxin-insensitive) or bg-subunit dimers that are liberated following activation of G proteins in the G^G o subfamily (pertussis toxin-sensitive).
PLCs of the g-subfamily are attracted to the cytoplasmic face of the plasma membrane by certain phosphotyrosine-containing peptide motifs that are made by activated tyrosine kinases, and there they are tyrosine-phosphorylated: these two events cooperate to activate PtdIns(4,5)P2 hydrolysis by the PLCgs (16). PLCds exist alongside PLCbs and PLCgs in animal cells, but only PLCds seem to be present in yeasts and plants. To some degree, the PLCds remain a puzzle, particularly because we still do not fully understand how they are activated. They can be activated by increases in cytosolic (Ca ) that are fairly large but physiologically plausible, but there may be other activating factors yet to be discovered.
Table 1. Stimuli that Transmit Signals into Target Cells at Least in Part Through Phosphoinositide-Based Signaling Pathways
PLC-Catalyzed PtdIns(4,5)P2 Hydrolysis
|
Platelet-derived growth factor |
Angiotensin I (AT^eceptor) |
|
(PDGF) |
Endothelins (3 receptors) |
|
Adrenergic (a1receptor) |
Rhodopsin (invertebrates only) |
|
Histamine (Hj receptor) |
|
|
Acetylcholine (muscarinic, 2 subtypes) Thromboxane A2 |
|
|
Interleukin 8 |
|
|
Vasopressin (V1receptor) |
Glutamate (metabotropic receptors) |
|
Oxytocin |
|
|
Substance P and other tachykinins |
Some taste receptors? |
|
PtdIns(3,4,5)^3 Synthesis Catalyzed by Type I Phosphoinositide3-Kinases |
|
|
Platelet-derived growth factor |
The ab/CD3 antigen receptor |
|
(PDGF) |
complex of T lymphocytes |
|
Insulin |
|
|
IGF-1 |
fMetLeuPhe |
|
Colony-stimulating factor-1 |
|
|
(CSF-1) |
|
|
PtdIns3.P 5-Kinase-Catalyzed PtdIns(3,5)P2 Synthesis |
|
|
Hyperosmotic stress (yeast) |
|
A particularly clear illustration of the essential nature of the phospholipase C signaling pathway has emerged from genetic studies of the visual systems of insects (eg, Drosophila), which use rhodopsin-linked phospholipase C activation as their primary signaling pathway in response to the light activation of rhodopsin (17). Fly retinas show a striking set of genetic defects in this signaling pathway. They do not function properly if they lack: (1) a receptor-coupled phosphoinositidase C; (2) a diacylglycerol kinase and CDP-diacylglycerol synthase necessary for the resynthesis of PtdIns from the hydrolyzed PtdIns(4,5)P2; or (3) a PtdIns transfer protein that is assumed somehow to maintain the PtdIns supply to the photoreceptor membranes (see Table 2and ref. 5).
Table 2. Biological Effects for Eye Function in Drosophila of Mutations in, or Changes in, the Expression Level of Proteins Involved in Phosphoinositide-Based Signaling
|
Gene or Protein |
Target Protein |
Phenotype |
|
NorpA |
PLC-b, coupled to light- activated rhodopsin |
Eye elements fail to generate a light- triggered receptor potential |
|
RdgA |
Diacylglycerol kinase |
Photoreceptors degenerate |
|
RdgB |
PtdIns transfer protein |
Light-induced degeneration of photoreceptors |
|
Cds |
CDP-diacylglycerol |
Reduced signal gain |
|
synthase (a step in |
during |
|
|
PtdIns biosynthesis) |
photoreception; |
|
|
light-dependent degeneration of photoreceptors |
||
|
Type-I |
Organ-targeted |
Eye size correlates |
|
Type I PI3K |
positively with the |
|
|
PtdIns(4,5)P2 3- |
concentration of expressed |
|
|
kinase |
PI3K |
3.2. Signaling through 3-Phosphorylation of PtdIns(4,5)P2 to Phosphatidyl-inositol 3,4,5-Trisphosphate (PtdIns(3,4,5)P3)
In the late 1980s, eukaryote cells yielded up both PtdIns3 P and phosphoinositide 3-kinases that can make PtdIns3 P from PtdIns (generically abbreviated below as PI3Ks). Soon, however, it was found that some PI3Ks can use PtdIns, PtdIns4P, and PtdIns(4,5)P2 as substrates and so can also make two other novel lipids: PtdIns(3,4)P2 and PtdIns(3,4,5)P3. Moreover, neutrophils stimulated by the chemotactic attractant fMetLeuPhe (see Chemotaxis) were seen to make a lipid with the charge of a phosphatidylinositol trisphosphate (PtdIns P3): this proved to be PtdIns(3,4,5)P3.
Soon came recognition that many activated receptor tyrosine kinases (see Growth Factors; Platelet-Derived Growth Factor; Insulin) and a few G-protein-coupled serpentine receptors (especially those in hematopoietic cells) can very rapidly (within seconds) stimulate a subset of PI3Ks that phosphorylate PtdIns(4,5)P2 to PtdIns(3,4,5)P3 (see Table 1). These are now known as the Type I subfamily (18).
The PtdIns(3,4,5)P3 generated in response to receptor stimulation, which is the most highly charged membrane phosphoinositide present in cells, is presumed to be located in the inner leaflet of the plasma membrane. This identified it as another potential receptor-generated signaling molecule. Several key observations soon combined to confirm this view (18-20). First, the PtdIns (4,5)P2 3-kinase responsible for PtdIns(3,4,5) P3 synthesis in response to activated tyrosine kinases is a heterodimer of a catalytic subunit (p110) and a SH2 domain-containing regulatory subunit (p85). Immediately after the activation of some growth factor receptors, this p110/p85 PI3K becomes physically complexed with the activated receptor tyrosine kinase. This binding is mediated by the SH2 domain of p85, which recognizes a particular phosphotyrosine-containing motif (typically PTyr-Met-Asp/Pro-Met) in the cytoplasmic "tail" of the activated receptor tyrosine kinase. Interaction with this motif localizes the kinase to the inner surface of the plasma membrane, where it becomes tyrosine-phosphorylated and is activated. Second, mutations of the "kinase insert" sequence of the platelet-derived growth factor (PDGF) receptor abolish this PTyr-containing motif and reduce the growth-stimulating and survival-promoting activity of PDGF. Third, the tyrosine kinase activities of the activated insulin and insulin-like growth factor I receptors phosphorylate insulin receptor protein 1 (IRS-1), generating multiple PI3K-activating motifs. This activation of PI3K activity is essential both for the survival-promoting activities of these hormones and for a number of their classical actions on carbohydrate metabolism (19, 20). Fourth, overexpressed or constitutively active PI3Ks are growth-promoting and can be oncogenic (18). Finally, neutrophils contain a different Type I PtdIns(4,5)P2-directed PI3K, which is activated by the G protein bg-subunit complexes that are released following activation of some G protein-coupled receptors (eg, the fMetLeuPhe receptor) (21).
The fungal metabolite wortmannin A was a key reagent in many of these studies. Interest in this compound was aroused when it was found to inhibit multiple, and apparently unrelated, cell responses to external stimulation. These included:
1. The rapid oxidative burst characteristic of stimulated neutrophils;
2. Insulin-stimulated glucose uptake into target tissues, notably adipose and skeletal muscle, which is achieved primarily by fusion of vesicles enriched in glucose-permeable channels with the plasma membrane;
3. Maintenance of cell survival in response to "growth/survival factors" such as IGF1; and
4. Activation of the formation of lamellipodia at the leading edge of migrating cells (see Cytoskeleton).
Once it was realized that wortmannin is a very potent inhibitor of the Type-I PI3Ks that make PtdIns(3,4,5)P 3, this became an obvious candidate as the immediate activator of some or all of these responses. Although subsequent studies showed that wortmannin can be a less than scrupulously precise pharmacological reagent, other evidence has subsequently confirmed that many of the biological responses that are most sensitive to wortmannin inhibition are indeed consequences of PI3K activation. Used carefully, therefore, this compound has remained valuable for the initial identification of responses that might be consequences of receptor-stimulated PtdIns (3,4,5)P3 synthesis.
Some details of how cells sense and respond to a PI3K-driven rise in PtdIns(3,4,5)P3 concentration as a regulatory signal have been elucidated. Most notably, accumulation of PtdIns(3,4,5)P3 very quickly leads to activation of protein kinase B (PKB, also known as c-Akt), a serine-threonine protein kinase and the product of a proto-oncogene (22). To become fully activated, cytosolic PKB must become firmly localized to the inner face of the plasma membrane and must be phosphorylated on two widely separated residues (Thr473 and Ser308). PKB physically associates with the plasma membrane, primarily through a direct interaction between its pleckstrin homology (PH) domain and the normal PtdIns(4,5)P2 complement of that membrane, but this association may be strengthened by newly formed PtdIns(3,4,5)P3.
Receptor-stimulated formation of PtdIns(3,4,5)P3 triggers phosphorylation of the membrane-bound PKB. In this process, one PtdIns(3,4,5)P 3-activated protein kinase (phosphoinositide-dependent kinase 1; PDK1) phosphorylates PKB on Thr308, and another may phosphorylate it on Thr473 (Fig. 2; refs. 23, 24 ). The interaction that activates PDK1 seems to be between the newly formed PtdIns(3,4,5)P3 and a PtdIns(3,4,5)P3-selective PH domain in PDK1.
A substantial number of other candidate target proteins avidly and selectively bind PtdIns(3,4,5)P3: these include the so-called centaurins (25, 26) and Bruton’s tyrosine kinase, an enzyme essential for normal B lymphocyte development (27). Which, if any, of these serve as directly-controlled regulatory targets of this receptor-responsive polyphosphoinositide remains to be determined (20).
4. Phosphoinositides and membrane trafficking
The earliest work on receptor-stimulated phosphoinositide turnover, in the 1950s and 1960s, focused primarily on its possible relationship to the triggering of secretory processes (3). The emphasis then moved to unraveling the central roles of phosphoinositides in signaling. Recently, however, there has been a remarkable resurgence of interest in how phosphoinositides contribute to intracellular membrane and protein trafficking (28).
4.1. PtdIns(4,5)P2 Synthesis and Exocytosis
It has long been known that secretory vesicles (eg, adrenal medullary chromaffin "granules") support rapid PtdIns4P biosynthesis, and recently it was recognized that exocytotic secretory processes in yeast and in mammalian cells fail in the absence of a functional cytosolic "PtdIns transfer protein" (PITP). In yeast, Sec14, one of many Sec genes implicated in secretory protein transit early in the pathway between the Golgi apparatus and the plasma membrane, encodes a PITP and, in permeabilized PC12 cells (which mimic catecholamine secretion by adrenal medulla), a PITP is among the proteins that are needed to reconstitute ATP-driven exocytotic secretion.
PtdIns is made at the endoplasmic reticulum but is involved in signaling at the plasma membrane. Soon after the discovery of PITPs in the 1970s, it was recognized that these molecules might serve as "ferries" that move PtdIns around cells (2), an idea that was recently validated (29, 30). PITPs bind PtdIns and/or phosphatidylcholine, one molecule at a time, and shuttle these lipids across the cytosol from membrane to membrane. Mammals have two very similar PITPs (a and b), either of which can support PtdIns(4,5)P2-dependent signaling and exocytosis. Although the yeast protein encoded by Sec14 is functionally quite similar, it has an unrelated amino acid sequence.
A second cytosolic protein that is essential for catecholamine secretion by PC12 cells is a PtdIns4P 5-kinase. Successful exocytosis, therefore, requires PtdIns to be delivered to the secretory vesicle membrane and phosphorylated to PtdIns(4,5)P2 (31). Accumulation of a substantial amount of PtdIns(4,5)P2 in the secretory vesicle membrane may somehow "prime" the vesicles, switching them to a state in which they can fuse with the plasma membrane in a subsequent Ca -activated exocytosis step.
4.2. PtdIns3P and Membrane Trafficking
PtdIns3P, by contrast, is somehow implicated in vesicular protein sorting to the vacuole of yeast and the lysosomes of mammalian cells (which are functionally homologous organelles), and also in membrane fusion between elements of a membrane vesicle compartment (the "early" endosomes) that plays a central role in the import into eukaryotic cells of macromolecules such as the iron-carrier protein transferrin. This first became apparent when the protein encoded by vps34, one of many genes whose disruption interferes with vesicular protein sorting in cells, was identified as a PtdIns-specific PI3K: Vps34p and other Vps34p-like PI3Ks make up the Type III PI3K subfamily. It is not clear how PtdIns3P contributes to the membrane fusion and fission events in these membrane trafficking processes. However, detailed studies of EEA1, a protein characteristic of early endosomes, have shown that a zinc fingerlike protein domain known as a "FYVE finger" serves as a highly selective PtdIns3P-binding domain; similar FYVE finger domains are present in several other mammalian and yeast proteins involved in membrane trafficking processes (32, 33).
The importance of the 3-phosphorylation of PtdIns by Type III PI3Ks was further emphasized when it was discovered that the PtdIns3 P that Vps34p makes is the substrate for the synthesis of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2), the most recently discovered of the polyphosphoinositides (34, 35). Whereas PtdIns3P synthesis is a constitutive activity of unstressed cells—as it should be if it is involved in the continuous vesicle-mediated protein flows into cells from the exterior and from Golgi to lysosomes—PtdIns(3,5)P2 synthesis is acutely regulated. In both fission and budding yeasts, hyperosmotic stress provokes a striking acceleration of PtdIns (3,5)P 2 synthesis (35). The exact function of this striking response is yet to be defined.
5. Inositol lipid-binding domains in proteins
One common theme that has emerged particularly strongly from many recent studies of the functions of inositol lipids and phosphates is the involvement of stereospecific, high-affinity interactions between particular polyphosphorylated inositol lipid or phosphate species and protein domains whose primary function appears to be the selective recognition of these molecules. Many of these domains are members of a large family of so-called pleckstrin homology (or PH) domains (15, 36), but see also the mention above of ‘FYVE fingers.’
PH domains have a characteristic cluster of basic amino acid residues that, in each protein, ligates a particular subset of polyphosphorylated inositol derivatives [eg, (37)]. For example, the PH domains of PKB/Akt and of Bruton’s tyrosine kinase preferentially bind PtdIns(4,5)P2 and PtdIns (3,4,5)P3, respectively (23, 27). As the name suggests, the prototypic PH domain was found in pleckstrin: this is an abundant platelet cytosol protein that becomes heavily phosphorylated by protein kinase C downstream of receptors (eg, for thrombin ) that activate phospholipase C but whose function is still not entirely clear. A major function of PH domains is to mediate noncovalent binding of proteins to the cytoplasmic surfaces of polyphosphoinositide-containing membranes—notably the plasma membrane, but also other structures such as exocytotic secretory vesicles—so as to localize them at their functional sites. Some such effects are likely to be constitutive, as when a PH domain-containing protein homes to the steady-state complement of plasma membrane PtdIns(4,5) P^. In other circumstances, PH domains probably play a major regulatory role. For example, PtdIns(3,4,5) P3-binding PH domains will relocate proteins to the plasma membrane, often for activation, only after cells have been exposed to a PtdIns(3,4,5)P3-generating stimulus.
6. The Functions of inositol phosphates
Although the biological function of Ins(1,4,5)P3 is clear, why cells contain so many other inositol polyphosphates remains uncertain. That eukaryotic cells commit substantial genetic resources and metabolic energy to complex inositol polyphosphate interconversion pathways (see Fig. 4) surely indicates that at least some of these molecules have important cellular functions, but few of these are established with any certainty. Some of these are summarized in Table 3, and discussion of others can be found in recent reviews (5, 6, 15, 36, 42, 43).
Table 3. Defined and Probable Functions of Some Inositol Polyphosphates and Glycerophosphoinositol Polyphosphates.
|
Compound Ins(1,4,5)P3: formed by hydrolysis of PtdIns(4,5) P2 |
Function Signaling, through Ins(1,4,5)P3- 2+ stimulated Ca mobilization |
|
Ins(1,4,5,6)P 4: formed from Ins(1,4,5)P 3 by Ins (1,4,5)P 3 3-kinases |
O 1 Modulation of Ca entry following O 1 depletion of intracellular Ca stores? Modulation of GTPase-activating protein |
|
Ins(3,4,5,6)P 4 |
Slowly accumulates during stimulation of PLC-coupled receptors. 2+ Inhibits a Ca /calmodulin-dependent and kinase-regulated epithelial Cl-channel |
|
Ins(1,3,4,5,6)P5 (often |
Modulation of O2 binding to avian |
|
mimicked experimentally with InsP6) |
and reptilian hemoglobins |
|
GroPIns4 P (and/or GroPIns(4,5) P2) |
Accumulate in Ras-transformed cells, inhibit adenylate cyclase |
|
Several pyrophosphate derivatives of InsP6 |
Widespread, rapid metabolic turnover of pyrophosphate groups, high free |
|
(shown as InsP7 and InsP8 in Fig. 4) |
energy of hydrolysis, exact structures vary between cell types |
