Download Lipids as hormones and second messengers

Lipids as hormones and second messengers
Alfred H. Merrill Jr and Dennis C. Liotta
Emory University, Atlanta, Georgia, USA
Some lipids act as hormones to induce responses in target tissues whereas
others influence the behavior of cell-surface receptors and/or serve as
second messengers; i.e. they are formed as part of the cellular response
to extracellular agonists, and directly affect protein kinases, ion channels
and other systems that govern cell behavior. Our understanding of these
functions on a structural level is improving, but this is still a relatively new,
and an extremely complex, area of investigation.
Current Opinion in Structural Biology 1991, 1:516-521
Introduction
Lipids participate in cellular structure and function at essentiaUy all levels, which probably accounts for the diversity of the lipid classes and subtypes. As stated by Thudichum [1] over a century ago, 'Their physical properties are, viewed from a teleological point of standing, eminently adapted to their functions.' This has proven to
be true at at least three levels. Firstly, the molecular features of lipids allow them to define membranes, lipoproteins and other biological structures, as discussed elsewhere in this issue by Reiss-Husson (pp 506-509). Secondly, many lipids also embody structural domains that
interact with receptors, enzymes and other components
of cell membranes as well as with extrinsic cytoskeletal proteins, antibodies, microbial toxins, etc. (some aspects of this topic are discussed in this issue by Ferguson,
pp 522-529). Finally, a growing number of lipids can be
converted to products that are highly bioactive as hormones and lipid second messengers. When one considers the iterative fine-tuning that proteins have undergone
over the course of evolution, it should not be surprising
that the lipid components of biological membranes have
also evolved to exhibit similar sophisticated properties.
Overview of lipid hormones and
second-messenger systems
As shown in Fig. 1, there are a large number of compounds with proposed functions as lipid second messengers, and we probably know only a fraction of the
intraceIIular systems that are controlled by these types
of mechanisms. In most instances, the formation of
the lipid hormones (e.g. platelet-activating factor, the
eicosanoids, etc.) and lipid second messengers (e.g. diacylglycerols (DAGs), phosphatidic acid (PA), lysophospholipids, ceramides, etc.) proceeds from the cleavage
of more complex membrane lipids by phospholipases
[2 oo] that have been activated by the relevant agonist.
Nearly all of the possible products of these cleavage
reactions (i.e. phospholipid headgroups, lysophospholipids, DAGs, PA and fatty acids) have been implicated
in signal transduction. For example, release of lysophospholipids and fatty acids via phospholipase A2 provides
compounds that serve as precursors for platelet-activating factor, prostaglandins, leukotrienes, thromboxanes
and other eicosanoids, and as possible modulators of
the activity of protein kinase C (PKC). Phospholipases C
and D produce DAGs, alkylacylglycerols and PA from diverse phospholipid precursors (including phosphatidylinositols (PIs), phosphaticlylcholines (PCs) and PI glycanlinked proteins inter alia). These products can have multiple intracellular targets, such as. the activation of PKC
by DAGs and the stimulation of Ca2 + release from intracellular stores by inositol triphosphate, as shown in
Fig. 1. Bioactive lipids can be made in multiple intracellular sites; in Fig. 1, this is illustrated by the formation of
steroid hormones but this can also occur, at least theoretically, for most of the other types of lipid second
messengers. In addition to these phospholipases for glycerolipids, the hydrolysis of sphingomyelin to ceramides
has recently been proposed as another lipid second-messenger cycle [2",3]. Other hydrolysis products of sphingolipids (e.g. sphingosine and some lysosphingolipids)
are potent inhibitors of PKC [4°], Na+/K + -ATPase, PA
phosphohydrolase, and other phospholipases, as well as
activators of the epidermal growth factor receptor; hence,
the hydrolysis of sphingolipids to bioactive constituents
may prove to be widespread and important [5°].
The multiplicity of these pathways, and the fact that most
of the enzymes, substrates and products are membraneassociated, make it difficult to understand these systems
on a molecular level. Nonetheless, recent findings have
provided a more detailed picture of the interactions between lipid second messengers and their targets, and
some of these lessons will be discussed using PKC as
a model.
Abbreviations
DAG~cliacylglycerol; PA~hosphatidic acid; PC--phosphatidylcholine; PG~phosphatidylglycerol; PKC--protein kinase C;
Pl--phosphatidylinositol; PMA--phorbol 12-myristate,13-acetate; PS~phosphoatidylserine.
516
(~) Current Biology Lid ISSN 0959-440X
tipids as hormones and second messengers Merrill Jr and Liotta
PI-glycanlinkedproteins
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/N~ DAG ) ) ~
~?EDAAG(~G
)) _.~_._
!O~
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hidr~Og~j
ipisd
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~
Arachidonic
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Prostagl.~ins'etc"~2"
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oe ,eo Cholesterol
Ceua
responses
Fig. 1. Overview of hormone and signal-transduction pathways involving lipid mediators. Binding of stimuli (hormones, growth and
differentiation factors, etc.) to membrane receptors triggers a sequence of events that usually involves cleavage of a membrane lipid
to a bioactive fragment or derivative. Examples of the lipases that are involved in these events are as follows. Phospholipase A 2 (PLA 2)
releases a (usually polyunsaturated) fatty acid (FA, which may proceed to other bioactive products such as prostaglandins, etc.) from
the 2-position of a phosphoglycerolipid; in some cells, lysoalkylphosphatidylcholine (lysoalkyI-PC) undergoes reacylation to produce
platelet-activating factors (PAF). Phospholipase C (PLC) cleaves phosphoglycerolipids (such as phosphatidylinositol 4,5-bisphosphate, PIP2)
to diacylglycerol (DAG) or 1-alkyl- or 1-alkenyl-2-acylglycerols (EAG); DAG activates protein kinase C (PKC) and EAG may activate or
inhibit this enzyme. When the substrate is PIP2, release of inositol triphosphate (IP3) increases cytosolic Ca2+. Phospholipase D (PLD)
cleaves phosphoglycerolipids to phosphatidic acid (PA) which may act directly, or be converted to DAG to stimulate cell responses.
Other classes of PLC and PLD act on phosphatidylinositol-glycan-linked proteins to release the protein and DAG or EAG. Membranes
also contain sphingolipid (SL) hydrolases, such as sphingomyelinase and ceramidase that can liberate ceramide (Cer) and/or sphingosine
(So). Other signal transduction elements in cells include the enzymes that hydrolyse cholesterol esters to cholesterol then.to various
steroid hormones. In some instances, the lipids act on systems in the same cell (i.e., are second messengers); in others (which have not
been distinguished for simplicity), they act on targets elsewhere and are termed hormones. Other abbreviations: PS, phosphatidylserine.
Protein kinase C as a model for lipid
modulation of a signal-transduction system
Protein ldnase C is actually a family of enzymes, therefore caution is to be recommended in making generalizing statements about this system. Nonetheless, the studies
reported thus far illustrate many structural considerations
that should be pertinent to this and other lipid-modulated signal-transduction systems [6°°]. Firstly, the activity is affected both by phospholipids (phosphatidylserine (PS)) and by phospholipase cleavage products acting as activators (DAGs and unsaturated fatty acids) and
inhibitors. Secondly, the phorbol-ester-binding domain
(regarded as the lipid-activating domain of PKC) is located in the amino-terminal region - - hence, activation is
thought to involve a conformation change that displaces a
pseudosubstrate from the active site. Thirdly, PKC occurs
in multiple intracellular sites so that the localized formation of activators (and inhibitors) and enzyme translocation may be important factors in the biological responses.
Finally, PKC activity is also influenced by other factors
such as the availability of Ca 2 +, the extent of oxidation
of key sulfhydryl groups, the conformation of PKC upon
insertion into membranes (some forms are less sensitive
to lipid modulators), down-regulation by proteases, and
many other factors.
517
518 Upids
Structure-function studies of lipid modulators of PKC
have adopted two broad approaches. The first has compared the structures of analogs of phorbol esters, DAGs
and other potent activators (and inhibitors) of PKC. A
major conclusion of these studies has been that DAGs
overlay well with phorbol 12-myristate,13-acetate (PMA),
with properly orientated oxygen atoms located at sites
corresponding to C-4, C-9 and C-20 of PMA, and a
hydrophobic component located in the proper region of
space, i.e. R and R' of PMA and DAG respectively [7,8], as
illustrated in Fig. 2. By reducing the alkyt-chain length of
phorbol esters (to phorbol dibutyrate), it is possible to
measure binding to PKC directly, and KdS of the nanomolar order are typically obtained. A similar strategy has led
to the development of short-chain analogs of DAGs (1oleoyl,2-acetylglycerol and, more usefully, m,2-dioctanoylglycerol) as more water-soluble activators of PKC [9].
However, attempts to mimic the potent activation and
binding by phorbol esters with glyceride-type molecules
have, thus far, been fairly unsuccessful, indicating that the
interactions between phorbol esters and PKC go beyond
the molecular features of DAGs that are important for biological activity [10].
o
°'W
,3
H
o
|
R'~
O
O
O~
O
"R'
OH
Fig. 2. Comparison of the presumed bioactive conformation of a
diacylglycerol (DAG) with phorbol 12-myristate,13-acetate (PMA).
The second approach has been to elucidate the molecular details of PKC activation by lipid mediators encountered under more physiological conditions. This is much
more complicated because it requires knowledge of not
only the structural features of the lipids (e.g. stereochemistry, alkyt-chain composition, etc.) but also of the physical state of these molecules. The use of mixed micelles
by Bell and colleagues [6"'] has simplified these analyses
considerably. Figure 3 attempts to illustrate some of the
features that are important in understanding PKC activation at this level.
Structural requirements of lipid modulators of
protein kinase C
A series of elegant investigations have identified the structural specificity of PKC for DAGs and PSs (reviewed in
[6"'] ). Activation requires ester-linked fatty acids at positions 1 and 2 of sn-l,2-diacylglycerols (1,3- and 2,3isomers are not active) and a free hydroxyl group at position 3. The nature of the alkyt chains is less important,
and shorter-chain DAGs will activate if they are sufficiently
hydrophobic to be membrane bound [9]; for this reason,
a fairly close and specific interaction between PKC and
the glycerol moiety has been depicted in Fig. 3.
The major structural requirements for PS binding to PKC
appears to be the L-serine headgroup as other naturally
occurring phospholipids, and synthetic phospholipids
made from serine analogs, are not as effective [6..,11].
In contrast, there is little specificity inherent to the glycerol backbone or the fatty acyl chains, and 1,3-diacyl-PS
is able to activate PKC despite its considerable deviation
from the usual 1,2-diacyt configuration [6..,11]. The exact stoichiometry of the interaction between PKC and PS
is not known; however, on the basis of the co-operativity of activation in a mixed micelle system [12] and
similar studies with vesicles [13], at least four PSs appear to be involved. Hence, several interactions between
PKC and the headgroups of PSs have been depicted in
Fig. 3. Flexibility has been left in the diagram in this
region, nonetheless, because some activation can be
achieved by other acidic lipids [6..,14.]. In the case of
PKC activation by PI 4,5-bisphosphate, the effect was to
reduce the concentration of PS required for activation
[14°]. This may indicate that there are a few sites that
are highly specific for PS and others that accommodate
other acidic lipids more readily. Not depicted in Fig. 3
is the ability of certain lipids, especially unsaturated fatty
acids [15 o] but perhaps other lipids as well [16°], to interact with cytosolic PKC.
Physical properties of lipids related to their
effects on protein kinase C
What is the actual nature of the interactions between PKC
and these lipid activators, Ca 2+ , the protein substrate,
and other factors that can influence the activity of this
enzyme? One difficulty in envisioning the lipid modulation of PKC and other membrane-associated processes
is the necessity for diagrammatically depicting them in
two dimensions and in a static state. Membrane phospholipids typically have a high degree of mobility (inplane exchange rates for phospholipids are of the order
of 107s -1 [17°']) and dipalmitoyl-PC, for example, has
been estimated to have 436 755 rotational isomeric configurations [18"]. A 'snapshot' of the membrane such as
that shown in Fig. 3 clearly poses some impediment to
fully appreciating the behavior of these molecules. The
membrane surface that modulates the activity of PKC
should be viewed, to paraphrase Roux and Bloom [19"'],
Lipids as hormones and second messengers Merrill Jr and Liotta
Fig. 3. Hypothetical depiction of the
PS
PS
PS PS
DAG
as a three-dimensional region of space whose limits can
be broadly defined by the polar moieties of the phospholipids, the contiguous water layers (particularly the first
layer containing orientated water molecules) and associated metallic cations adsorbed at the membrane-solution
interface.
The requirement for PS and, in particular, for a fairly large
number of PSs for activation of PKC [12,13] has a number of structural implications. The headgroups of PSs are
thought to be somewhat more rigid than those of other
phospholipids [20], whereas ordering of the fatty awl
chain is slightly increased as a result of electrostatic repulsion [21o]. The ionization state of the carboxyl group of
PS, and of any other ionizable groups in this environment
(such as fatty acids [22], PA [23 °] or sphingosines [24],
inter alia), may be different to what one might expect
as it has been estimated that the local pH at the PS surface is approximately 3 pH units lower than the bulk pH
[25] (interestingly, micelles seem to behave differently
from membranes in this regard [26°]). The large proportions of negatively charged lipids on plasma membranes
give them a significant electrostatic potential that might
affect second-messenger systems [27,28°]. The electrostatic characteristics of the major anionic lipids (PS, phosphatidytglycerol (PG) and PI) can be described by the
simplest form of Gouy-Chapman-Stem theory, which assumes that the charges are smeared uniformly over the
surface [28°]. One possible implication of this behavior
is that one component of the membrane binding by PKC,
its substrates and other components of this signal-transduction pathway may be mediated by the specific molecular interactions implied by Fig. 3, whereas another (such
as the inhibition of PKC by cationic compounds) may be
described by electrostatic factors.
interactions of protein kinase C with
Ca2+, phosphatidylserine (PS) and diacylglycerol (DAG) at the membrane interface. The lipid components are represented using space-filling models which
were individually subjected to energy
minimization calculations. The lighter
shaded area at the membrane interface
symbolizes waters of hydration.
It appears that the initial interaction of PKC with the
membrane is mediated through its binding to PS and
Ca2+ [29o,30o], and that the Ca2+-binding site(s) a r e
generated at the interface between PKC and the membrane [30°]. Calcium is well known to form a complex
with PS [31,32°]; hence, a Ca2 + -binding site may be created by a mechanism like that depicted in Fig. 3. It is
thought that after the enzyme has bound PS and Ca2+ ,
it binds DAG (or phorbol esters) to become fully active [33]. Phosphatidylserine affects the specificity of PKC
such that substrate phosphorylation is favored over autophosphorylation [34°°], presumably through reorientation of amino acid groups coupled to the catalytic site of
the enzyme. The binding of PKC to membrane induces
an increase in surface pressure [29 ° ] that can be interpreted as a reorientation of the phospholipids and/or
possible insertion of a protein domain into the membrane (Fig. 3). The binding of PKC to PS is also expected to induce a reorientation of the lipid, as indicated
by model studies with amphophilic peptides [35°].
Elegant studies of the physical behavior of the 1,2-DAG
activators of PKC [36 °°] indicate that the sn-2 carbonyl
experiences a significantly greater degree of hydrogenbonding than the sn-1 carbonyl at the aqueous interface,
as indicated in Fig. 3. Furthermore, the transbilayer movement of 1,2-dilauroyt-sn-glycerol is extremely fast (tl/2 is
10 ms).
In addition to these physical considerations, it should be
remembered that biological membranes are undergoing
constant metabolism (albeit under careful metabolic control) and vesicular movement (it is estimated that the
equivalent of the entire surface of many cells is internalized every 20-30 min). There appear to be discrepancies in the ability of DAGs liberated from different phos-
519
520
Lipids
phoglycerolipids to activate PKC, and some, of these may
reflect intracenular compartmentalization [37°]. Furthermore, the asymmetry of PS in plasma membranes appears to be maintained by ATP-dependent 'aminophospholipid translocase(s)' [17..]. These issues are especially pertinent to PKC because many of the biological
processes regulated by this enzyme involve some form
of membrane movement and remodelling.
References and recommended reading
Papers of special interest, published within the annual period of review,
have been highlighted as:
•
of interest
••
of outstanding interest
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PKC activation is investigated using a Triton X-100 mixed-micelle assay.
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•
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••
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29.
•
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The association of PKC with membranes is assessed by the increase
in monolayer surface pressure using a mixed phospholipid film. The
addition of Ca 2+ results in an increase in the penetration of the kinase
(DAG and phorbol esters have no effect); hence, Ca 2 + may play a major
role in the translocation of PKC to membranes.
30.
•
BAZZIMD, NELSESTUENGk Protein Kinase C Interaction with
Calcium: a Phospholipid-Dependent Process. Biochemistry
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An analysis of Ca 2+ binding by PKC using equilibrium dialysis. Free
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the presence of acidic phospholipids, after correction for Ca 2+ binding by the phospholipids alone. These findings suggest that the Ca z +binding sites 'may be generated at the interface between PKC and the
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From these data, the authors calculate the excess partial molar free
energy to transfer 1-palmityol-2-oleoyt-PS from PS to PC as - 0 . 7 kcal
m o l - 1, and suggest that this arises in part from the transfer of negatively charged PS from an environment of negative charges to one of
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34.
••
NEWTONAC, KOSHLANDDE: Phosphatidylserine Affects Specificity of Protein Kinase C Substrate Phosphorylation and
Autophosphorylation. Biocheraistry 1990, 29:6656--6661.
An analysis of PKC activity using small unilamellar vesicles of varying
composition. Increasing the PS concentration (and the negative surface charge) favors substrate phosphorylation over autophosphorytation, whereas DAG increases both activities equally. Hence, PS affects
the specificity of PKC toward different substrates.
35.
•
DE KROON AIPM, KILLI.MqJA, DE GIER J, DE KRUIJFF B: The
Membrane Headgroup and Acyl Chain Order and Dynamics. Application of the 'Phospholipid Headgroup Electrometer' Concept to Phosphatidylserine. Biochemistry 1991,
30:1155-1162.
Amphiphilic peptides reduce the order parameter of both the dioleoylPS headgroup and acyl chains. The response of PS to the divalent peptides fits the electometer concept, i.e. the effective charge experienced
by PS is related to the distance between the charges. A divalent peptide
with a distance between the charges of ~ 10A. will have an effect similar to NaCI, which has a Debye length of 100 mM. Disruption of the
phospholipid acyLchain order appears to occur with cations that are
localized in the lipid-water interface and, in certain cases, where there
is a collapse of the integrity of the vesicles.
36.
••
HAMILTONJA, BHAMIDIPATISP, KODALIDR, SMALLDM: The Interfacial Conformation and Transbilayer Movement of Diacylglycerois in Phospholipid BRayers. J Biol Cbem 1991,
266:1177-1186.
The behavior of 1,2-dilauroyt-sn-gtycerol in PC is analysed by 31p and
13C NMR spectroscopy and other physical techniques. The NMR results
indicate that the sn-2 carbonyl experiences a significantly greater degree of hydrogen-binding than the sn-1 carbonyl at the aqueous interface. Hence, the orientation of the glycerol moiety is more like that of
PC, with the sn-2 carbonyl more accessible to solvent interactions. The
transbilayer movement of 1,2-dilauroyl-sn-glycerol is extremely fast (t]/2
of ~ 10 ms).
37.
LEACHKL, RUFF VA, WRIGHT TM, PESSlN MS, RABEN DM: Dis.
sociation of Protein Kinase C Activation and sn-l,2-Diacylglycerol Formation. Comparison of Phosphatidylinositoland Phosphatidylcholine-Derived Diglycerides in at-Throm
bin-Stimulated Fibroblasts. J Biol Chem 1991, 266:3215-3221.
Treatment of IIC9 fibroblasts with different amounts of ct-thrombin
stimulates differential formation of DAGs from P14,5-bisphosphate and
PC. The results indicate that DAGs from the former, but not the latter,
are able to activate PKC in this system.
All Merrill Jr, Department of Biochemistry, Emory University, Atlanta,
Georgia 30322, USA.
DC Liotta, Department of Chemistry, Emory University, Adanta, Georgia
30322, USA.
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