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Molecular and Cellular Signaling - Martin Beckerman

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8.4 Generation of Lipid Second Messengers from PIP2

165

TABLE 8.2. Phosphoinositide nomenclature.

 

 

 

 

 

 

Phosphoinositide

Members

Designation

 

 

 

 

 

D3

Phosphatidylinositol-3 phosphate

PtdIns(3)P

 

 

Phosphatidylinositol-3,4 biphosphate

PtdIns(3,4)P2

 

 

Phosphatidylinositol-3,5 biphosphate

PtdIns(3,5)P2

 

 

Phosphatidylinositol-3,4,5 triphosphate

PtdIns(3,4,5)P3 or PIP3

D4

Phosphatidylinositol-4 phosphate

PtdIns(4)P

 

 

Phosphatidylinositol-4,5 biphosphate

PtdIns(4,5)P2 or PIP2

D5

Phosphatidylinositol-5 phosphate

PtdIns(5)P

 

 

 

 

 

also facilitate the formation of signaling complexes and the initiation of signaling by them.

8.3Lipid Kinases Phosphorylate Plasma Membrane Phosphoglycerides

The plasma membrane phosphoglyceride known as phosphatidylinositol plays an important role in signaling, cytoskeleton regulation, and membrane trafficking. The inositol ring of the phosphatidylinositol molecule contains a phosphoryl group at position 1 that is tied to the glycerol backbone. All other OH groups of the inositol ring can be phosplorylated except those at positions 2 and 6. Just as protein kinases catalyze the transfer of phosphoryl groups to selected amino acid residues, lipid kinases catalyze the transfer of phosphoryl groups to specific sites on lipids. Several lipid kinases catalyze the phosphorylation of phosphatidylinositol.

The lipid kinase phosphoinositide-3-OH kinase (PI3K) catalyzes the transfer of a phosphoryl group from an ATP molecule to the OH group at position 3 of the inositol ring of the lipid. Other lipid kinases, PI4K and PI5K, catalyze the transfer of phosphoryl groups to the other available sites, positions 4 and 5, on the ring. An entire ensemble of phosphoinositides can be produced through the addition and subtraction of phosploryl groups from positions 3, 4, and 5 of the inositol rings. These phosphorylated lipid products, their placement into D3, D4, and D5 phosphoinositide groups, and their common abbreviations are listed in Table 8.2.

8.4 Generation of Lipid Second Messengers from PIP2

Two lipid second messengers are generated from PIP2 by phospholipase C. Phosphatidylinositol 4,5 biphosphate (PIP2) serves as the source of two lipid second messengers: diacylglycerol (DAG) and inositol 1,4,5- triphosphate (designated Ins(1,4,5)P3 or IP3). The plasma membrane functions as a cellular repository for the PIP2 and other phosphoinositides.

166 8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

FIGURE 8.3. Structure of phosphatidylinositol 4,5 biphosphate (PIP2): Cleavage of this molecule by PLC generates the lipid second messengers diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). The insert shows in block form the organization of the molecule into tail, backbone, linker phosphoryl group, and inositol head groups.

Phospholipase C (PLC) cleaves the membrane-situated PIP2 into DAG— which contains the acyl chains plus the glycerol backbone—and IP3—which contains the rest of the head group (Figure 8.3).

A large number of plasma membrane receptors use PLC as an intermediary to signal and activate downstream kinases. Prominent among these are G protein-coupled receptors (GPCRs) and growth factor receptors. There are three PLC subtypes, designed as PLCb, PLCg, and PLCd. These enzymes have a modular organization that supports their (a) localization at the plasma membrane, (b) activation by upstream receptor signals, and

(c) catalytic activities (Figure 8.4). The Pleckstrin homology domain and the C-terminus SH2 domain mediate binding to the plasma membrane PtdIns. The upstream, signaling elements such as G protein subunits bind to and activate PLC, and the SH3 and N-terminal SH2 domain mediates interactions with upstream growth factor receptors. Once formed by PLC acting on PIP2, IP3 diffuses to intracellular stores located in the endoplasmic reticulum (ER) and triggers the release of Ca2+. The calcium ions together with DAG activate protein kinase C, the main downstream target of PLC signaling (Figure 8.5).

8.5 Regulation of Cellular Processes by PI3K

167

FIGURE 8.4. Organization of phospholipase C: The domain structure of the three classes of PLC isozymes is shown. Each type of PLC has a Pleckstrin homology (PH) domain in its N-terminus. PH domains bind to plasma membrane PtdIns proteins. The PH domains are not all the same; they vary their binding affinities among the three classes. The X and Y domains form the catalytic domain of the enzyme.

FIGURE 8.5. Signaling through PLC: Activation of PLC by ligand-GPCR binding stimulates dissociation of the G protein subunits, which then activate PLC. The PLC proteins tether to the plasma membrane by binding PIP3 lipids, and cleave PIP2 into DAG and IP3. The latter translocates to the intracellular stores (IS) triggering release of calcium ions, which along with DAG bind to and stimulate protein kinase C activity.

8.5 Regulation of Cellular Processes by PI3K

Phosphoinositide-3-OH kinase (PI3K) helps regulate a variety of cellular processes. PI3Ks mediate cellular responses to GPCRs, growth factors and insulin, activation by cell adhesion molecules called integrins, and leukocyte (white blood cell) function. An important characteristic of the proteins that interact with lipid second messengers is the presence of one or more lipid-

168 8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

FIGURE 8.6. PI3 kinase domain structure: (a) Classes of catalytic subunits. (b) Examples of regulatory subunits that associate with Class IA subunits of PI3 kinases. Abbreviations: Adapter-binding (AB) domain; Ras-binding (RB) domain; Src homology domain-3 (SH3); Src homology domain-2 (SH2); BCR-homology GTPase activating (BH) domain.

binding domains in their regulatory regions. Several kinds of lipid-binding modules—PH domains, C2 domains, and FYVE domains—mediate the binding of lipid second messengers to their downstream targets.

There are three classes of mammalian PI3Ks (Figure 8.6). Class I PI3Ks are heterodimers composed of a 100-kDa catalytic subunit and an 85-kDa or 55-kDa joint regulatory/adapter subunit. There are two kinds of Class I PI3Ks, determined by the presence or absence of an adapter-binding (AB) domain in its N-terminal region (Figure 8.4a) and by kinds of receptor binding events that activate them. The adapter for the Class IA PI3Ks binds to growth factor receptors, while Class IB PI3Ks are activated primarily by G protein coupled receptors operating through the associated Gbg subunits. Class II PI3Ks contain a C2 domain in their C-terminal region. Class III PI3Ks may be constitutively active in the cell and help regulate membrane trafficking and vesicle formation, two housekeeping activities carried out all the time. As shown in Figure 8.7, PI3K functions as a key intermediary to activate protein kinase B.

8.6 PIPs Regulate Lipid Signaling

There is a corresponding set of lipid phosphatases that catalyzes the removal of phosphoryl groups from inositol rings. Perhaps the most prominent of these is PTEN (phosphatase and tensin homolog deleted on chromosome 10). PTEN acts in opposition to PI3K and catalyzes the removal

8.7 Role of Lipid-Binding Domains

169

FIGURE 8.7. Signaling through PI3K: Ligand growth factor receptor binding stimulates the dissociation of the regulatory and catalytic subunits of PI3K from each other. The catalytic subunit phosphorylates the PIP2 proteins at the 3¢ position, thereby making PIP3, which then diffuses to and binds PDK1 and protein kinase B.

of phosphoryl groups from position 3 on inositol rings. It acts on PIP3 to return it to a PIP2 form, thereby reversing the catalytic effects of PI3K.

The importance of dephosphorylating actions is made apparent by the high frequency of either mutated or missing forms of PTEN in at least one kind of brain cancer (glioblastoma), in prostate cancer, and in endometrial (uterine) cancer. The major downstream target of the PIP3 lipids is protein kinase B (Figure 8.7). This kinase supplies what may best be termed a “survival” signal in response to growth factor-binding to receptors such as the insulin receptor. In the absence of growth signals such as insulin, plateletderived growth factor, and neural growth factor, the levels of activated protein kinase B remain low. They increase in response to the just mentioned growth signals. When PTEN does not throttle back the survival signaling to a baseline level by dephosphorylating the lipid second messengers, the cells undergo uncontrolled growth and proliferation. The actions taken by PTEN tend to suppress the cancer-promoting actions of overly active protein kinase B. For this reason PTEN is referred to as a tumor suppressor.

8.7 Role of Lipid-Binding Domains

Lipid-binding domains facilitate the interactions between proteins and lipids. As has been discussed in the last few sections, a number of different lipid-binding domains mediate the interactions between proteins and lipid bilayers. Four of these domains—the PH, C1, C2, and FYVE domains— are especially prominent. They are found in hundreds of proteins and mediate protein recruitment to lipid membranes. These domains and their properties are summarized in Table 8.3. As indicated in the table, the PH domain also mediates protein-protein interactions. Two modules that appear in Table 8.3—the SH2 and PTB domains—are primarily known as

170 8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

TABLE 8.3. Lipid-binding domains found on proteins.

Designation

Domain name

Description

C1

Protein kinase C homology-1 ~50 amino acid residues; binds DAG

C2

Protein kinase C homology-2 ~130 amino acid residues; binds acidic

 

 

lipids

FYVE

Fab1p, YOTB, Vac1p, Eea1

~70 amino acid residues; binds PI3P

PH

Pleckstrin homology

~120 amino acid residues, binds PIP3

 

 

headgroup, PIP2 and its headgroup; binds

 

 

proteins

PTB

Phosphotyrosine-binding

~100 amino acid residues; binds

 

 

phosphorylated tyrosine residues; binds

 

 

phospholipids in a weak nonspecific manner

SH2

Src homology-2

~100 amino acid residues; binds

 

 

phosphorylated tyrosine residues; binds

 

 

phospholipids

 

 

 

phosphotyrosine binding domains. These domains possess phospholipidbinding properties, and for that reason have been included in Table 8.3.

8.8 Role of Intracellular Calcium Level Elevations

Transient elevations in intracellular calcium levels serve as a second messenger. As is characteristic of a second messenger, local increases in intracellular calcium concentration are triggered by the binding of signal proteins to receptors embedded in the plasma membrane. In the absence of triggering signals, intracellular calcium levels are maintained at a low level, no more than 0.1 mM in some cell types. Unlike cAMP and cellular lipids, calcium is not synthesizesd by cells. Instead, there are two reservoirs of calcium—the extracellular spaces outside the cell and the calcium stores located within the cell. The calcium concentration in the extracellular spaces is on the order of 2 mM, some 20,000 times greater than the resting levels within the cell. Extracellular calcium enters a cell through ion channels located in the plasma membrane. Calcium is sequestered within the cell in intracellular stores (IS), regions enriched in calcium buffers located in the lumen of the endoplasmic reticulum, the matrix of mitochondria, and in the Golgi. In response to the appropriate signals, calcium is released into the cytosol from the stores.

Signals that trigger the entry of extracellular calcium through ion channels and the release of intracellular calcium from stores are sent through two kinds of receptors embedded in the plasma membrane. The first kind of receptor is the voltage-gated ion channel. These are opened and closed, or gated, through changes in membrane voltage. These channels are found

8.9 Role of Calmodulin in Signaling

171

in cells whose membranes are excitable. Whenever the membrane is depolarized the ion channels open allowing calcium ions from the extracellular spaces to diffuse through and enter the cell. The second kind of membrane signal molecule is the ligand-gated receptor such as the G protein-coupled receptor. When a ligand binds the GPCR receptors, phospholipase C is activated. As discussed earlier in this chapter, PLC hydrolyzes PIP2 to IP3 and DAG. IP3 diffuses over to, and binds to, IP3 receptors located in the ER. This event serves as a release signal, resulting in the movement of Ca2+ out of the stores and into the cytosol.

The duration of a calcium signal is short. Intracellular calcium levels are restored to their base values fairly rapidly. Buffering agents bind calcium ions before they can diffuse appreciably from their entry point. Free calcium path lengths, the distance traveled by calcium ions before being bound, average less than 0.5 m, which is far smaller than the linear dimensions, 10 to 30 m, of typical eukaryotic cells. In addition to being buffered, ATP-driven calcium pumps located in the plasma membrane rapidly remove calcium ions from the cell, and other ATP-driven pumps transport calcium back into the intracellular stores. The take-up of calcium by buffers along with its rapid pumping out of the cytosol and into the stores produces a sharp localization of the signaling both in space and time.

8.9 Role of Calmodulin in Signaling

Calmodulin is a calcium sensor involved in activating many signaling pathways. Calmodulin is an abundant protein, consisting of 0.1% of all the protein present at any given time in the cell. It functions as a calcium sensor, and to carry out this role it is distributed throughout the cytosol and nucleus. Calcium is an extremely important regulator of cells in the brain, and the cytosolic concentrations of calmodulin in neurons may reach 2%.

Calmodulin is a small protein, consisting of only 148 amino acid residues. It is organized into two lobes connected by a flexible helix giving it a fairly elongated dumbbell shape. As shown in Figure 8.8, calmodulin has four calcium-binding sites; two are in the N-terminal lobe and two are in the C- terminal lobe. Calcium binding produces a shift in the population of equilibrium states from a fairly closed to a more open elongated structure. The shift in population exposes a number of hydrophobic patches that serve as attachment sites to downstream signaling partners.

Calmodulin serves as a key intermediary in a number of signaling pathways. When bound to calcium it promotes the activity of PI3K, nucleotide phosphodiesterases and adenylyl cyclases (both to be discussed shortly), protein kinases such as multifunctional CaM-dependent protein kinase II (CaMKII), protein phosphatases such as CaM-dependent protein phosphatase 2B(PP2B), and a number of cytoskeleton regulators.

172 8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

FIGURE 8.8. Solution NMR structure of calmodulin, free and bound to calcium:

(a) Calcium-free calmodulin consisting of an (upper) N-terminal domain and a (lower) C-terminal domain. (b) Ca2+4-bound calmodulin. The four calcium ions are depicted as dark gray spheres. The two prominent hydrophobic patches that are exposed in this more open conformation are bound by W7 molecules shown in a space-filled model. The figure was prepared using Protein Explorer with atomic coordinates deposited in the PDB under accession numbers 1dmo (a) and 1mux (b).

8.10Adenylyl Cyclases and Phosphodiesterases Produce and Regulate cAMP Second Messengers

Adenylyl cyclase is an integral membrane enzyme that catalyzes the conversion of intracellular ATP into cyclic adenosine monophosphate (cyclic AMP or cAMP). The organization of the adenylyl cyclase molecule is depicted in Figure 8.9. As can be seen there are two transmembrane (TM) regions (M1 and M2) and two large cytoplasmic regions (C1 and C2). Each transmembrane region consists of six highly hydrophobic membranespanning helices connected by short loops. One of the cytoplasmic regions lies topologically in-between the TM regions. The other cytoplasmic region is a situated C-terminal to the second membrane-spanning region. The overall structure of the adenylyl cyclase molecule resembles a dimer, each unit consisting of a TM region followed by a cytoplasmic region. However, monomer-like structures are not functional. The cytoplasmic regions together form the catalytic core of the molecule. The relative orientation of C1 relative to C2 is important, and both C1 and C2 are required for binding and catalysis.

The magnitude and duration of cyclic nucleotide second messenger signaling is regulated by another class of enzymes, namely, nucleotide phosphodiesterases (PDEs). As shown in Figure 8.10, cAMP has a phosphate group attached to both the 3¢ carbon and 5¢ carbon of the ribose. PDEs are enzymes that catalyze the hydrolytic cleavage of 3¢ phosphodiester bonds

8.11 Second Messengers Activate Certain Serine/Threonine Kinases

173

FIGURE 8.9. Organization of adenylyl cyclase: The cylinders denote transmembrane segments. These are organized into a repeated set of six segments (M1 and M2). The cytoplasmic C1 and C2 catalytic domains consist of a compact region (C1a and C1b) and a broad loop (C1b and C2b).

FIGURE 8.10. Adenosine triphosphate and cyclic adenosine monophosphate:

(a) ATP molecule consisting of an adenine joined to a ribose to which are attached three phosphoryl groups, named in the manner shown. (b) cAMP showing the cyclic structure.

in cAMP resulting in its degradation to inert 5¢AMP. They also carry out the same operation in cGMP to yield inert 5¢GMP. The PDEs terminate second messenger signaling. They modulate these signals with regard to their amplitude and duration, and through rapid degradation restrict the spread of cAMP to other compartments in the cell.

8.11Second Messengers Activate Certain Serine/Threonine Kinases

Second messengers acting in the vicinity of the plasma membrane help organize the signaling pathways. They exert their influences by activating and regulating a large number of serine/threonine kinases, among which are

174 8. Organization of Signal Complexes by Lipids, Calcium, & Cyclic AMP

TABLE 8.4. Members of the AGC family of serine/threonine kinases: Different geneencoded isozymes and alternatively spliced isoforms are listed in column 3. Second messengers required for the activation of the kinases are listed in column 4.

AGC kinase family

Structure

Forms

Regulation

Protein kinase A

2 regulatory subunits;

RIa, RIb, RIIa, RIIb

cAMP

 

2 catalytic subunits

Ca, Cb, Cg

 

Protein kinase B

Single chain; PH,

a, b, g1, g2

PI3K lipid products

 

catalytic, regulatory

 

 

 

domains

 

Ca2+, DAG,

Protein kinase C

Single chain; catalytic,

PKC-a, PKC-b1,

Classical

regulatory domains

PKC-b2, PKC-g

phosphatidylserine

Novel

Single chain; catalytic,

PKC-d, PKC-e,

DAG,

 

regulatory domains

PKC-h, PKC-q

phosphatidylserine

Atypical

Single chain; catalytic,

PKC-z, PKC-i,

 

 

regulatory domains

PKC-l

 

 

 

 

 

those belonging to the AGC family. Three subfamilies of AGC kinases are included in Table 8.4 along with the second messengers involved in their activation. The kinases sequester their catalytic activities within a catalytic domain (or subunit) and similarly combine their regulatory activities into one or more regulatory domains (or subunits). Some of the kinases possess a separate lipid-binding PH (Pleckstrin homology) domain, while others incorporate lipid-binding structures such as C1 and C2 domains into their regulatory regions.

The kinases all have a common structure and similar modes of activation. Second messengers and upstream kinases activate them. Binding of the second messengers to PH domains and regulatory motifs induces the movements of the kinases to the plasma membrane near the sites of second messenger release. This step is followed by phosphorylation by an upstream kinase. In the case of protein kinase B and protein kinase C the upstream kinase kinase has been identified. It is called phosphoinositide-dependent kinase-1 (PDK1). This enzyme may also be the one responsible for activating protein kinase A. In all cases, the role of the upstream kinase is to catalyze the transfer of a phosphoryl group to a crucial residue situated in the activation loop of the AGC kinase. When this occurs the amino acid residues involved in catalysis are unblocked and can carry out their functions.

8.12 Lipids and Upstream Kinases Activate PKB

Protein kinase B is the primary target of signals relayed from membranebound signal receptors via lipid second messengers. It is activated in two stages. In the first stage the PI3K product PIP3 binds to PKB through its