Biochem. J. (2009) 423, e5–e8 (Printed in Great Britain)
e5
doi:10.1042/BJ20091274
COMMENTARY
Why do phosphatidylinositol kinases have so many isoforms?
Sang H. MIN and Charles S. ABRAMS1
Hematology-Oncology Division, University of Pennsylvania School of Medicine, 421 Curie Blvd., Biomedical Research Building II/III, #912, Philadelphia, PA 19104, U.S.A.
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 cellsurface 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.
INTRODUCTION
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
[3]. 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)-motifcontaining 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
Eukaryotic cells are able to internalize small and large molecules
through their plasma membranes via different mechanisms
of endocytosis [1]. The classification of endocytosis may be
based on morphological and molecular differences. The bestestablished pathway for endocytosis utilizes clathrin. However, endocytosis can also occur by utilizing several clathrinindependent pathways. These include the caveolin-dependent,
CLIC/GEEC-type, IL2Rβ (interleukin-2 receptor β chain), Arf6dependent, 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 [2]. 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
Key words: Fcγ receptor (Fcγ R), macrophage, phagocytosis,
phosphoinositide 3-kinase α (P13Kα), pinocytosis, zymosan.
Abbreviations used: Arp2/3 complex, actin-related protein 2/3 complex; PIP5KI, phosphatidylinositol 4-phosphate 5-kinase I; PI3K, phosphoinositide 3kinase; 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.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
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Figure 1
S. H. Min and C. S. Abrams
Phosphatidylinositol reactions
The number and positions of phosphates around the inositol head group determine differences between individual phosphatidylinositols. Shown are the metabolic relationships between mammalian
phosphatidylinositol species, kinases and phosphatases. The shaded area contains the phosphatidylinositides and enzymes demonstrated to directly contribute to phagocytosis.
Figure 2
Stages of phagocytosis and localization of phosphoinositides to the phagosome during engulfment
On the top is a schematic diagram showing the progressive stages of phagocytosis. Below is shown the time course of local synthesis and catabolism of PtdIns(4,5)P 2 , PtdIns(3,4)P 2 and
PtdIns(3,4,5)P 3 at the forming phagosomes as they correlate with the stages of phagocytosis. This correlation was originally described by Kamen et al. [6] and Botelho et al. [11].
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 [4]. Phagocytosis involves several
well-co-ordinated steps in the plasma membrane (Figure 2).
These sequential steps are: particle recognition, engulfment and
c The Authors Journal compilation c 2009 Biochemical Society
phagosome maturation. In macrophages, particle recognition can
be mediated by Fcγ R 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 Cγ 1, the
adaptor proteins GRB2 (growth-factor-receptor-bound protein 2)
and GAB2 (GRB2-associated binding protein 2), and class IA
PI3K.
Commentary
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. [7] 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 Fcγ R 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. [8]
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
e7
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, C3biopsonized 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 [9].
The unique functions of each isoform might depend on the
cell type and the relative expression in the cell. Papakonstanti
et al. [10] 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.
FINAL COMMENT
Even though different isoforms of phosphatidylinositol kinases
synthesize the same product, the study by Tamura et al. [10] helps
c The Authors Journal compilation c 2009 Biochemical Society
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S. H. Min and C. S. Abrams
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.
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Received 17 August 2009/24 August 2009; accepted 24 August 2009
Published on the Internet 14 September 2009, doi:10.1042/BJ20091274
c The Authors Journal compilation c 2009 Biochemical Society
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