The phosphoinositide (PI) 3-kinase family
Fiona M. Foster,
Colin J. Traer,
Siemon M. Abraham and
Michael J. Fry*
School of Animal and Microbial Sciences, University of Reading,
Whiteknights, PO Box 228, Reading RG6 6AJ, UK
*
Author for correspondence (e-mail:
m.j.fry{at}reading.ac.uk)
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Introduction
|
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Phosphoinositide (PI) 3-kinase was first observed in 1984 as a minor
inositol lipid kinase activity associated with immunoprecipitated oncogene
products (e.g. Src, Abl and polyoma mT antigen) and present in activated
growth factor receptor complexes (e.g. PDGF receptor). In 1988, the enzyme
associated with this activity was found to have the novel ability to
phosphorylate the 3 position hydroxyl group of the inositol ring (see poster)
of phosphatidylinositol (PtdIns). PI 3-kinase activities have been
subsequently found in all eukaryotic cell types examined
(Fry, 1994
;
Katso et al., 2001
) and are
linked to an incredibly diverse set of key cellular functions, including cell
growth, proliferation, motility, differentiation, survival and intracellular
trafficking (Fry, 1994
;
Rameh and Cantley, 1999
;
Fry, 2001
;
Katso et al., 2001
). The
emerging links between PI 3-kinase activity and many human maladies, including
allergy, inflammation, heart disease and cancer, has made them the focus of
intense study, and inhibitors of these enzymes are considered potential
therapeutic agents (Stein and Waterfield,
2000
).
Although the majority of published studies have focused on the classical
p110-p85 (now known as class I) PI 3-kinases, it has emerged over the past 10
years that the PI 3-kinase superfamily (EC 2.7.1.137) is made up of a large
family of structurally related enzymes, with differing PI substrate
requirements and modes of regulation, which probably accounts for the reported
diversity of function (Rameh and Cantley,
1999
; Fry, 2001
;
Katso et al., 2001
). PCR
cloning strategies and data mining of genome sequencing projects would seem to
set the family limit at eight distinct PI 3-kinase catalytic subunits that are
capable of phosphorylating inositol lipids. These eight isoforms have been
divided into three functional classes on the basis of their protein domain
structure, lipid substrate specificity and associated regulatory subunits:
namely, the class I enzymes, p110
, p110ß, p110
and
p110
; the class II enzymes, PI3K-C2
, PI3K-C2ß and
PI3K-C2
; and the sole class III enzyme, Vps34
(Fry, 2001
;
Katso et al., 2001
).
The relationships between the kinase domains of the different enzymes
identified in the human, fly, worm and yeast genomes are indicated in the
poster by the non-rooted phylogenetic tree, which was prepared using ClustalX
(hs, Homo sapien; dm, Drosophila melanogaster; ce,
Caenorhabditis elegans; sp, Schizosaccharomyces pombe; sc,
Saccharomyces cerivisiae). Simple eukaryotes, such as yeasts, and all
plant species investigated to date seem to possess only a single class III PI
3-kinase. Multicellular invertebrate organisms, exemplified by C.
elegans and D. melanogaster, have a single representative member
of each of the three functional classes. Vertebrate genomes (human, mouse)
contain eight distinct PI 3-kinase genes, with some of the family members
being widely or ubiquitously expressed (e.g. p110
, p110ß,
PI3K-C2
, PI3K-C2ß and Vps34), while others are more restricted to
specific cell and tissue types (e.g. p110
, p110
and
PI3K-C2
). To add to this complexity there is a class IV group of
PI-3-kinase-related protein serine/threonine kinases found in all eukaryotes
(Kastan and Lim, 2000
).
Mammals have four such protein kinases: TOR (the target of the drug
rapamycin), ATM (Ataxia telangiectasia mutated), ATR (Ataxia telangiectasia
mutated related) and DNA-PK (DNA-dependent protein kinase). Here, we focus
solely on the three classes of true PI 3-kinase.
The various 3-phosphorylated lipid products that are produced by these
enzymes [PtdIns(3)P, PtdIns(3,4)P2,
PtdIns(3,5)P2 and PtdIns(3,4,5)P3]
function as part of the mechanism by which a diverse set of signalling
molecules, containing pleckstrin homology (PH), FYVE, Phox (PX) and other
lipid-binding domains, are recruited to various cellular membranes
(Rameh and Cantley, 1999
;
Wurmser et al., 1999
;
Ellson et al., 2002
). Cellular
PI 3-kinase activities are balanced by phosphoinositide 3-phosphatase
activities, found in the tumour suppressor protein PTEN and in members of the
myotubularin (MTM) family (Maehama et al.,
2001
).
 |
Functional analysis of PI 3-kinases
|
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Two selective, chemically unrelated PI 3-kinase inhibitors are in wide use:
namely, wortmannin and LY294002. Unfortunately, the use of these inhibitors is
limited by the fact that they inhibit all known PI 3-kinase isoforms,
including the class IV PI-3-kinase-like enzymes. This has led to many cellular
processes being linked with PI 3-kinase activity, but considerable confusion
as to which PI 3-kinase isoforms control specific cellular functions. In
mammalian cells, wortmannin suppresses class I, class II PI3K-C2ß and
PI3K-C2
, and class III PI 3-kinase activity with an IC50 in
the 1-10 nM range, while it inhibits the class II PI3K-C2
isoform with
an approximate IC50 of 400 nM, and class IV PI-3-kinase-related
enzymes in the 100-300 nM range (data summarising many sources). Similarly,
LY294002 inhibits all PI 3-kinases with an IC50 in the 1-50 µM
range. Thus, at low concentrations, these two inhibitors (preferably used in
parallel) can implicate a PI 3-kinase activity in a cellular process of
interest, but are not suitable for dissecting the involvement of individual PI
3-kinase species. Many pharmaceutical companies are working on
isoform-specific PI 3-kinase inhibitors and it is hoped that in time these
will become available to the research community. Currently alternative
approaches, such as the use of dominant negative mutants, knockout mice or RNA
interference, are necessary to attribute a function to a specific PI 3-kinase
class or isoform unambiguously.
 |
Class I PI 3-kinases
|
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The class I family of PI 3-kinase enzymes in vertebrates comprises four
distinct protein species of approximately 110 kDa (p110
, p110ß,
p110
and p110
). All class I enzymes share the majority of their
structural features and a common substrate specificity
(Rameh and Cantley, 1999
;
Fry, 2001
;
Katso et al., 2001
). In vitro,
all class I PI 3-kinases are capable of phosphorylating PtdIns to
PtdIns(3)P, PtdIns(4)P to PtdIns(3,4)P2
and PtdIns(4,5)P2 to PtdIns(3,4,5)P3,
with PtdIns(4,5)P2 being considered the preferred lipid
substrate in vivo. Class I PI 3-kinases are largely cytosolic in resting
cells, but upon stimulation are recruited to membranes via interactions with
receptors or adaptor proteins. They are thought to function primarily at the
plasma membrane, but there have been reports of class I PI 3-kinases
associated with vesicular and nuclear membranes
(Rameh and Cantley, 1999
;
Fry, 2001
;
Katso et al., 2001
). The
cellular roles of class I PI 3-kinases are diverse, with evidence linking them
to cell size, motility, survival and proliferation in response to numerous
signalling systems in many different cell types
(Fry, 2001
;
Katso et al., 2001
). The class
I family is further subdivided into two groups on the basis of their
regulatory partners and mechanisms of activation.
 |
Class IA
|
---|
There are three class IA catalytic subunits: p110
(human gene
designation PIK3CA all subsequent gene names and chromosomal locations
listed refer to humans), which maps to chromosome 3 at 3q26.3; p110ß
(PIK3CB at 3q23); and p110
(PIK3CD at 1p36.2). All of these PI
3-kinases physically interact with a family of Src homology 2
(SH2)-domain-containing regulatory adaptor proteins. Three distinct genes
encode the p85
(PIK3R1 at 5q12-q13), p85ß (PIK3R2 at 19q13.2q13.4)
and p55
(PIK3R3 at 1p34.1) adaptors, each with a number of possible
splice variants. All members of this `p85' family of adaptors bind to the
N-terminal 100 amino acids (shown in purple) of the class IA PI 3-kinases and
mediate their activation by growth factor receptors (mainly of the
protein-tyrosine kinase family) through the two SH2 domains that bind to
sequence-specific phosphorylated tyrosine residues either on
autophosphorylated receptors or on substrate adaptor proteins. Some of the
p85
and p85ß splice forms also possess an SH3 domain, which can
bind to proline-rich ligands in other proteins instead of, or in addition to,
SH2-domain-mediated recruitment (Fry,
1994
; Katso et al.,
2001
). A class IA PI 3-kinase was the first to be identified and
cloned and thus this class are best understood. Links between interesting
biological questions and class I PI 3-kinases are starting to emerge
suggesting that the individual isoforms have distinct, but possibly
overlapping, roles, which may vary between cell types. Class I PI 3-kinases
are linked to cell size in Drosophila and in mammals the p110
isoform has been shown to regulate the size of the adult heart
(Crackower et al., 2002
).
Murine knockouts of the p110
and p110ß genes result in embryonic
lethality, pointing to their essential nature. Reports suggest that
p110
may play a role in cell survival, whereas p110ß may be more
important in promoting cell proliferation
(Benistant et al., 2000
). Data
implicating p110
in numerous cancers continues to mount
(Fry, 2001
), while studies with
knock-in mice bearing catalytically inactive p110
would seem to suggest
that this isoform is critical for full B- and T-cell antigen receptor
signalling (Okkenhaug et al.,
2002
).
 |
Class IB
|
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The p110
(PIK3CG at 7q21.11) catalytic subunit is the sole class IB
member and differs from the class IA enzymes in its extreme N-terminus
(lacking a p85 binding site) and in its adaptor partner, p101 (PI 3-kinase
regulatory subunit gene, at 17p13.1), which lacks domains found in any other
proteins. Whereas class IA enzymes are preferentially activated by
tyrosine-kinase-mediated signals, the class IB enzyme is linked to
G-protein-coupled receptor (GPCR) systems. Activation of class IB seems to
predominantly involve interactions with Gß
subunits and also
possibly G
subunits (Katso et al.,
2001
). Recent results from knockout mouse models suggest that
p110
plays a key role as a modulator of inflammation and allergy
(Wymann et al., 2003
), and
also in the regulation of cardiac contractility
(Crackower et al., 2002
).
 |
PI 3-kinase core structure
|
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PI 3-kinase family classification is largely based on sequence alignments
that define four major blocks of sequence similarity in members of the class I
family, termed homology regions (HR) 1-4. X-ray crystal structures of these
four domains from p110
, alone and in complex with Ras or PI 3-kinase
inhibitors, have been determined. This provides a basis for understanding this
family of kinases at a molecular level
(Walker et al., 1999
;
Djordjevic and Driscoll, 2002
).
HR1 (shown in grey) is the kinase core domain and is the only domain that is
found in all four PI 3-kinase classes. This domain exhibits weak homology to
protein kinases. HR2 (also known as the PIK domain shown in green)
seems, from the available structural data, to play a scaffolding role for the
other domains. HR3 is a C2-like domain (small blue circle). C2 domains in
other proteins mediate interactions with lipids or with other proteins in
either a calcium-dependent or calcium-independent manner. The position of this
domain in the PI 3-kinase structure suggests a role in general membrane
binding and possibly substrate targeting
(Walker et al., 1999
). HR4
(shown in red) is a putative Ras-binding domain or small G-protein-binding
domain, which has only been found in class I and II PI 3-kinases. To date, the
HR4 domain has only been shown to have functional effects through interactions
with Ras in class I PI 3-kinases (Katso et
al., 2001
).
 |
Class II PI 3-kinases
|
---|
Class II PI 3-kinases were identified through homology and PCR cloning
approaches and for this reason are the least well understood class of PI
3-kinase (Fry, 2001
).
Invertebrates have a single representative of this class of PI 3-kinase,
whereas mammals and fish (C.J.T. and M.J.F., unpublished) have three class II
PI 3-kinases. In humans these class II PI 3-kinases are termed PI3K-C2
(PIK3C2A at 11p15.5-p14), PI3K-C2ß (PIK3C2B at 1q32) and PI3K-C2
(PIK3C2G at 12p12). Whereas class I PI 3-kinases reside mainly in the
cytoplasm until recruited to active signalling complexes, the class II PI
3-kinases are largely constitutively associated with membrane structures,
including plasma membrane, intracellular membranes and somewhat surprisingly
with nuclei (Fry, 2001
).
Extracellular signals, including integrin engagement, growth factors (e.g.
insulin, EGF, SCF and HGF) and chemokines, have all been reported to stimulate
class II PI 3-kinase activity (Brown and
Shepherd, 2001
). No clear mechanism of activation has emerged,
with tyrosine phosphorylation, proteolysis and recruitment by adaptor proteins
(e.g. Grb2, clathrin) all suggested to play a role in different cell types or
receptor systems. Currently there is no clearly defined cellular role for
these enzymes or any clear consensus on their in vivo products. In vitro,
class II PI 3-kinases can phosphorylate PtdIns and PtdIns(4)P, but,
unlike class I PI 3-kinases, not PtdIns(4,5)P2. Class II
PI 3-kinases have not been isolated in association with a regulatory subunit,
but they possess extended N- and C-termini relative to the class I PI
3-kinases, which may serve this function. The N-terminus lacks defined
structural domains, but has both coiled-coil (pale blue) and proline-rich
(mauve) motifs, which may mediate protein-protein interactions. The extended
C-terminus has tandem PX (in yellow) and C2 (large blue circle) domains, the
functions of which have yet to be established in class II PI 3-kinases. Based
on structural and functional studies on their roles in other proteins, these
domains are likely to mediate binding to membrane lipids or protein-protein
interactions (Djordjevic and Driscoll,
2002
).
 |
Class III PI 3-kinases
|
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Class III PI 3-kinases are exemplified by the sole Saccharomyces
cerivisiae PI 3-kinase, Vps34 (Fry,
1994
; Wurmser et al.,
1999
; Katso et al.,
2001
). Unlike class I and II PI 3-kinases, this enzyme can
phosphorylate only PtdIns and is thus a PtdIns 3-kinase. All eukaryotes
investigated have a single Vps34 homologue. The human gene (PIK3C3) is located
on chromosome 18 at 18q12.3. A single 150 kDa regulatory subunit (in yeast
VPS15, in humans PIK3R4, located at 3q22.1) possesses intrinsic protein-serine
kinase activity that is required for Vps34 function
(Wurmser et al., 1999
).
Originally isolated as a mutant of vesicle-mediated vacuolar protein sorting
in yeast, class III PI 3-kinases are implicated in endosome fusion during
intracellular trafficking events and are located mainly on intracellular
membranes (Wurmser et al.,
1999
). Recent studies suggest that this class of PI 3-kinase is
involved in diverse intracellular trafficking events including autophagy
(Kihara et al., 2001
) and
phagosome formation (Vieira et al.,
2001
), internal vesicle formation within multivesicular endosomes
(Futter et al., 2001
),
retrograde endosome to golgi transport
(Burda et al., 2002
) and
transport at the nuclear membrane (Roggo
et al., 2002
).
This is an exciting time in PI 3-kinase research with various members of
this family being linked to key cellular processes and to many human diseases.
The major players are now all known and what remains is for us to tease out
the individual functions of the isoforms.
 |
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