From the Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706
The importance of phosphoinositides as lipid
signaling molecules in eucaryotic cells was first recognized by Lowell
and Mabel Hokin in the 1950s (who also discovered the enzyme activities that phosphorylate phosphatidylinositol
(PI)1) (1-5). Since those
early years, PI signaling pathways have expanded both in importance and
complexity. The classical pathway transforms PI to
PI-4,5-P2 by the successive actions of PI 4-kinases and
PI-4-P 5-kinases. PI-4,5-P2 is the precursor for second
messengers and also acts directly to modify effectors, for example
actin-binding proteins (6-9). Significant roles for other
phosphoinositide lipid products in signaling, combined with recently
identified lipid kinase activities, are illuminating the many
mechanisms by which cells use lipid messengers (10-13). This review
will focus on the phosphatidylinositol-phosphate kinase (PIPK) family,
which has the ability to synthesize all known PIP2 isomers
and PIP3.
Historically, in PI signaling as we understood it a few years ago,
the PIPKs synthesize PI-4,5-P2 by phosphorylating the fifth hydroxyl of PI-4-P (6). Two isoforms of PIPKs were characterized from
erythrocytes; these were denoted type I and II PI-4-P 5-kinase (PIPKI
and PIPKII) based their biochemical properties (14-16). Both enzymes
synthesize PI-4,5-P2 in vitro, although by
different mechanisms (see below).
Isolation and analysis of PIP5KI and PIP5KII cDNA sequences
demonstrated that the PIPKs constitute a novel family of kinases (17,
18). Surprisingly, the PIPKs do not share any statistically significant
identity to other known lipid or protein kinases. Known PI 4-kinases
and PI 3-kinases share sequence homology over distinct domains, and
some of these domains are related to those found in protein kinases
(19-24), suggesting that these enzymes have a similar
phosphotransferase mechanism.
Initially, the only homologs of PIPKs were the Saccharomyces
cerevisiae gene products, Mss4p and Fab1p (17, 25, 26). Since
then, the number of PIPK homologs in the sequence data base has mounted
to over 20. PIPKI and PIPKII homologs are now found throughout the
animal and plant kingdoms suggesting that their signaling functions are
ubiquitous and conserved. In mammalian cells, three isoforms of each
PIPKI and PIPKII subfamily, encoded by distinct genes, have been
characterized and named The regions of sequence homology are found within the C terminus. The
identity between PIPKs can be as low as 27% (17). However, the regions
of sequence identity are clustered, reminiscent of that between diverse
protein kinases and phosphatidylinositol 3- and 4-kinases (20, 24). We
now know these conserved domains represent the catalytic core of the
kinases. The kinase domain, except in Fab1p homologs, is separated by
an insert region. Recently, PIPKs in Arabidopsis
thaliana have been characterized and based on their sequence
appear to form a unique subfamily of enzymes (65). In Fig.
1, the subfamilies of PIPK have been
summarized and the regions of identity in the kinase domains aligned
and shown as a similarity plot.
INTRODUCTION
Novel Kinases
,
, and
(27-30), and additional
PIPKs likely remain to be discovered.
View larger version (35K):
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Fig. 1.
A model comparing currently known PIPK
subfamilies and a similarity plot comparing regions of identity between
all known PIPK isoforms in the kinase domain. Invariant sequence
motifs are defined and structural regions are labeled.
Although statistically there is no homology with other kinases, there
are similarities in the conserved sequence motifs. For example, a
glycine-rich motif (GXSGS) in the homologs resembles the
phosphate-binding loop of protein kinases and other ATP-binding proteins (24, 31). It is also similar to GTP-binding sequences, which
is consistent with the ability of PIPKs to use both ATP and GTP as
phosphate donors (15, 32). An invariant lysine (IIK sequence) that is
C-terminal of the glycine-rich region resembles the conserved lysine
found in protein kinases that binds the -phosphate of ATP (24,
25).
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Novel Lipid Substrates and Products |
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The diversity of PIPKs may, in part, be because of their unexpected ability to utilize different substrates and to generate multiple signaling molecules. PIPKI isoforms preferentially phosphorylate PI-4-P to PI-4,5-P2. However, at least in vitro, they also phosphorylate PI-3-P on both the 4- and 5-hydroxyls forming PI-3,4-P2 and PI-3,5-P2 (33-35). In addition, they remarkably generate PI-3,4,5-P3 directly from PI-3-P in a concerted reaction (33). Some PIPKI isoforms weakly phosphorylate PI forming PI-5-P (35). Thus, PIPKIs are dual specificity kinases phosphorylating the 4- and 5-hydroxyls of inositol.
Because of their sequence identity to PIPKIs, PIPKII isoforms were thought to have similar substrate specificity. It therefore came as a surprise when the preferred substrate of PIPKII was identified as PI-5-P rather than PI-4-P (34). The product of PI-5-P phosphorylation remains PI-4,5-P2 (17, 33-35). The earlier assumption that PIPKIIs are PI-4-P 5-kinase appears to be because of putative contamination of PI-4-P preparations with PI-5-P and the difficulty to distinguish PI-5-P from PI-4-P chromatographically. PIPKII isoforms also use PI-3-P as a substrate, and the product of this reaction is PI-3,4-P2. Therefore, in vitro the PIPKIIs are predominantly 4-kinases and appear to be less promiscuous in their substrate usage than PIPKIs. In summary, the in vitro substrate preference of the PIPKII isoforms is PI-5-P > PI-3-P > PI-4-P,2 whereas the efficacy of substrate usage for characterized PIPKI isoforms is PI-4-P > PI-3-P > PI-3,4-P2 > PI-5-P = PI (14-18, 33-38). The spectrum of messengers generated and substrates used by specific kinases in vivo is presumably tightly regulated. Nevertheless, these activities position PIPKs as potential participants in the generation of most known polyphosphoinositide signaling molecules. These kinase activities are remarkable and demonstrate the widest specificity of any family of signal-generating enzymes.
An indication that the in vitro activities of PIPKs may
reflect their in vivo functions is the finding that PI-5-P
and PI-3,5-P2 have been recently identified in living cells
(34, 36). However, in both cases, the route of synthesis of these novel
PIs remains to be elucidated. In vitro data show that
certain PIPKI isoforms synthesize PI-5-P from PI (35). Whether PIPKI
activity accounts for the low level of PI-5-P detected in NIH-3T3
fibroblasts or whether an as yet unidentified kinase or phosphatase is
involved in its generation is unknown. Recently, Dove et al.
(36) elegantly demonstrated that PI-3,5-P2 is generated
in vivo in both yeast and mammalian cells in response to
osmotic stress. Yeast Mss4p is reported to produce only
PI-4,5-P2 and PI-3,4-P2 (37, 38). Thus, Fab1p,
the only other PIPK homolog in yeast, is likely responsible for
PI-3,5-P2 production (see below).
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PIP Kinase Structure |
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Recently, the structure of the first PIPK, PIPKII, has been
solved (39). The tertiary structure of PIPKII
consists of two
identical subunits that interact at the N terminus (Fig.
2). This structure is consistent with the
oligomeric state of the protein in solution (14, 15, 39). At the
dimerization site a combination of
sheets and
helices
participates to form a clasp-like interface. The two
sheets at the
dimer interface form a flat surface area that extends beyond the two
sheets to the C terminus of the PIPKII
(Fig. 2, top).
Thus, although the subunits are globular the dimer is an elongated flat
disc-like structure. One face of this disc is highly basic, containing
10 lysine, 4 histidine, and 4 arginine residues, with a net positive charge of +10 (39). The charge and the flatness suggest that this
region functions as an interface for membrane association. To
illustrate this point the PIPKII
structure was modeled onto a
phosphatidylcholine bilayer structure (Fig. 2, bottom). This demonstrated that the PIPKII
interface region contacts the bilayer through the phospholipid headgroups without penetration into
hydrophobic membrane regions, suggesting that only electrostatic
interactions are involved in docking. The reliability of this model is
supported by orientation of the catalytic sites at the membrane
interface.
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Although the PIPKs are not statistically homologous to protein kinases,
the structure does contain a region of roughly 80 amino acid residues
in each subunit of the dimer that could be superimposed on the ATP
binding and catalytic residues of protein kinase structures (39). Three
catalytic site residues are absolutely conserved for PIPKs and have
counterparts in protein kinases, namely Lys-150 (in the IIK
sequence), Asp-278 (MDYSL), and Asp-369 (IID) of
PIPKII (Fig. 1 and Ref. 36). The corresponding residues in protein
kinase A (PKA) are Lys-72, Asp-166, and Asp-184 (40). The conserved
lysine in PKA coordinates with the
-phosphate of ATP whereas Asp-184
binds Mg2+ or Mn2+ ions (40). Asp-166 appears
to have a role in general base catalysis. Mutation of Lys-72 in protein
kinases destroys activity, and this is also the case when Lys-150 is
mutated in PIPKII
.2 Asp-166 in PKA is in the
"RDLK" motif conserved in protein kinases. The PIPKs
contain a similar conserved sequence, the "DLKGS" motif,
that had been suggested as a counterpart (25). Although mutation of
Asp-216 in the DLKGS destroys activity,2 the
structure of PIPKII
demonstrated that these two motifs are not
equivalent. The counterpart of the PKA Asp-166, in the RDLK, is Asp-278 in the PIPKII
structure. The analog of the Asp-184 in
protein kinases is Asp-369 in the PIPKs.
PIPKs have a glycine-rich loop or G-loop with the consensus sequence of
GXSGS. This region corresponds topologically to the GXGXXG loop of protein kinases. In both kinase
types this loop aligns consecutive peptide residues so that their amide
groups interact with the phosphate triester backbone of ATP/GTP acting as a "phosphate anchor." The modeling of ATP into the binding pocket of PIPKII showed that the adenine moiety fits into a
conserved hydrophobic pocket in each monomer. The phosphate triester
backbone is positioned to interact with the G-loop and Lys-150, and the
-phosphate is pointed toward the membrane interface. The specificity of PIPKII
was analyzed by modeling PI-5-P and ATP together into the
structure. In this model, the 5-phosphate interacts with a cluster of
partially conserved basic side chains in a shallow binding pocket. The
openness of the putative catalytic site suggests that the
phosphoinositol head group can freely rotate such that PI-3-P or PI-5-P
could occupy the same binding site, consistent with the specificity of
the kinases. In both cases, the 4-hydroxyl of inositol retains its
position in line with the
-phosphate. A disordered loop in the
structure (residues 373-391) was referred to as the "activation
loop" because it topologically corresponds to the activation loop in
protein kinases and possibly PI kinases (20, 39, 41). This loop is
anchored on each side of the substrate binding site and thus has the
potential to fold around the substrate and modulate both kinase
specificity and activity. Taken together, the flatness of the membrane
binding interface and the proximity of the substrate binding site to
this interface indicate that the PIPKII dimer was elegantly designed to
phosphorylate substrates at the membrane interface.
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Cellular Functions and Signaling Mechanisms |
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Many of the phosphoinositide signaling enzymes directly associate with membrane receptors; the most characterized interactions are with tyrosine kinase receptors (6-13). These signaling enzymes include isoforms of PI 3-kinase and phospholipase C. They associate at least transiently with receptors, and their activities are tightly coupled to agonist activation of receptors. Phospholipase C family members utilize PI-4,5-P2 for second messenger production. Upon agonist stimulation, PI-4,5-P2 is also observed to be a substrate for the agonist-activated PI 3-kinases (11), producing PI-3,4,5-P3. PI-3,4-P2 can then be subsequently produced by the activity of 5-phosphatase (42). Polyphosphoinositide 5-phosphatases also associate with receptors (see the next minireview in this series (Majerus et al. (42)).
Within this same theme, there is also evidence that PIPKs directly
associate with receptors. These include the p55 tumor necrosis factor
(TNF) receptor and the epidermal growth factor (EGF) receptor (27, 43).
In the case of the p55 TNF receptor, only PIPKII associates with the
receptor; the highly homologous PIPKII
does not (27). The EGF
receptor is reported to associate with both PI 4-kinase and PIP
5-kinase activities, although the kinase isoforms have not been defined
(43). Interestingly, both the TNF and EGF receptors interact with PIPKs
through their juxtamembrane domains within sequences of ~20 residues
extending from the membrane interface into the cytoplasm. The site on
PIPKII
required for association is within the N-terminal 100 amino
acids close to the dimerization interface. The PIPKII
association
with receptors may facilitate dimer formation and specificity of
membrane assembly. The functional significance of these interactions
remains to be demonstrated, but these signaling enzymes are likely to
contribute to second messenger production or regulation of receptor
function. In platelets, there is also evidence for stimulation of a
PI-3-P 4-kinase activity by thrombin and protein kinase C (44, 45). PIPKII
is present in platelets and as the only currently identified PI-3-P 4-kinase in platelets is a candidate for that PI-3-P 4-kinase activity (46, 47). Finally, there is evidence that the newest PIPKII
isoform, the PIPKII
, can be phosphorylated in response to mitogenic
stimulation (30).
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Secretion/Vesicle Trafficking |
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Phosphoinositide signaling has been implicated in secretion and vesicular trafficking and has been reviewed recently (48). Fab1p was the first PIPK homolog to have an implied role in vesicular trafficking (17, 25). Mutation or deletion of Fab1p results in formation of aploid and binucleate cells and a defect in vacuole function and morphology (25). The vacuole defect is manifested by enlarged vacuoles that fill the cytoplasm; other phenotypes appear to be secondary. Recently, a link has developed between FAB1 and VPS34. VPS34 encodes the only yeast PI 3-kinase, and it is required for protein sorting to the vacuole (49). When yeast were osmotically stressed the synthesis of PI-3,5-P2 was stimulated, and this was dependent upon Vps34p function (36). Thus, PI-3-P generated by Vps34p is likely a substrate for a PIPK, and the most likely candidate is Fab1p. Fab1p mutants show defects in vacuolar function and may share some of the phenotypes of Vps34p mutants. Thus, Fab1p may form a functional alliance with Vps34p to synthesize PI-3,5-P2 (see the second minireview in this series (49)).
A role for PIPK and PI-4,5-P2 has been reported for
catecholamine secretion (50). The process that required PIPK activity is the ATP-dependent priming step. This step is required
for the subsequent Ca2+-dependent triggering of
secretion. The PIPKs required for ATP priming were specific for PIPKI
enzymes. In light of the specificity of the PIPKs, this in retrospect
may not be surprising. The enzymes that are implicated in priming,
PIPKI, -
, and/or -
, have at least one property in common,
stimulation by phosphatidic acid (PA) (16, 28, 29). Phospholipase D
(PLD), which synthesizes PA, has also been implicated in vesicular
trafficking and possibly secretion (51). In fact, mammalian PLDs
require PI-4,5-P2 for robust enzymatic activity (52).
Hence, the PIPKI enzymes may be functionally regulated by PA, and the
PIP2 produced may stimulate PLD activity. This would result
in a positive stimulatory loop that would generate both PA and
PIP2 at specific sites within cells. In the case of
Ca2+-stimulated secretion, the PIP2 and PA
synthesis may be spatially segregated to the secretory vesicle. In
other signaling pathways, the synthesis of PA and PIP2
could be spatially and temporally regulated to modulate other functions
such as actin assembly.
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Actin Assembly |
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For two decades evidence has accumulated that PI-4,5-P2 can modulate the activity of proteins which regulate the assembly or disassembly of actin filaments in vitro (53). Recently, it was reported that overexpression of PIPKI isoforms in COS cells induced dramatic reorganization of actin in vivo (54). However, surprisingly a mutant of the PIPKI which lacked activity also induced actin reorganization (29). As a result, there remains some doubt as to whether or not PIP2 generation is required for PIPKI modulation of actin assembly in vivo. Nevertheless, if the PIPKs indeed modulate actin assembly by synthesis of PIP2 this would presumably require syntheses of PI-4,5-P2 to be spatially and temporally regulated. If PIPKI activity is required for regulation of actin assembly, then a role for PLD and PA is also conceivable. Activation of PLD is mediated by many stimuli that also induce actin reorganization; in particular PLD is stimulated by the small G-protein Rho (55, 56). Rac and Rho modulate actin assembly (55) and have also been reported to activate or associate with PIPKI isoforms (57-60). These results again illustrate the potential for a set of ordered interactions between the PIPKs and other signaling molecules that in turn modulate PIP2 production.
The MSS4-encoded PIPK is important for actin assembly in
yeast (36, 37). Yeast lacking Mss4p do not have the ability to form
normal actin filaments and to properly localize their actin cytoskeleton during polarized cell growth. Interestingly,
overexpression of Rho2p, a Rho GTPase, restored growth and polarized
actin assembly (36). Yeast lacking Mss4p are rescued by a PIPKI
isoform, and the enzymatic properties of Mss4p, including PA
stimulation, are most similar to PIPKI isoforms (37).
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Nuclear Phosphoinositide Signaling |
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Classical PI signaling pathways are based at the plasma membrane and linked to receptor activation by agonists. However, a distinct PI cycle exists in the nucleus and is regulated independently from the plasma membrane PI cycle (see for review see Ref. 61). Within nuclei the spatial organization of these signaling pathways has been vague. Biochemical evidence suggests that a fraction of the enzymes and phosphoinositides are retained in highly purified nuclei that have been stripped of their nuclear envelope with detergent (62). The explanation is that the enzymes and phosphoinositides are not associated with membrane structures within the nucleus.
Some PIPKI and PIPKII isoforms are concentrated in nuclei, spatially
organized to "nuclear speckles" that are separated from known
membrane structures (63). These speckle structures also contain enzymes
required for mRNA processing and transcription (Fig.
3). These same speckles are reported to
contain polyphosphoinositide(s), suggesting that the PIPKs generate
PIP2 in speckles (63). Nuclear speckles are highly dynamic,
and their morphology is tightly linked to the state of mRNA
transcription. Inhibition of mRNA transcription induces these
structures to become larger and fewer in number; the PIPKs and
PIP2 reorganize identically. Although the function of the
PIPKs at speckles is not known, this is additional evidence for spatial
organization of the PIPKs that presumably are linked functionally to
processes modulated by the polyphosphoinositide messengers that they
generate. There is also evidence for the regulated localization of
diacylglycerol kinase to nuclei (64). Consequently, PA generated by
diacylglycerol kinase could activate nuclear localized PIPKI
isoforms.
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Prospectus and Future Directions |
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These combined data suggest a model of how the PIPKs may assemble
in cells at the membrane, in vesicles, on receptors, or within nuclei.
The in vitro data demonstrate that PIPK substrate specificities are broad, suggesting that the PIPK family may generate all possible combinations of polyphosphoinositide signaling molecules. In vivo, the specificity for polyphosphoinositide substrates
and the generation of different products by these kinases must be tightly regulated. One mechanism by which to regulate PIPK specificity is to channel phosphoinositide substrates to these kinases. This could
occur by assembling both PI and PIPKs together into a macromolecular complex or within a cellular compartment. In this model, PI kinases would synthesize a specific PIP isomer, which would then be a substrate
for the contiguous PIPKs (Fig. 4).
PI-4,5-P2 concentrations within cells are reported to not
fluctuate greatly. Thus, it is difficult to understand how effectors of
PI-4,5-P2 can be modulated in vivo. The model
suggests that PIPKs spatially organize within cells and that the
spatial and temporal generation of PI-4,5-P2 at these
cellular sites or compartments is regulated and coupled to effectors
also at these sites. As such, PIP2 concentrations would
vary at specific cellular compartments but not globally within cells.
Clearly such a model needs refinement, but the concept illustrates the
paths that can be taken.
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FOOTNOTES |
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* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This is the third article of five in "A Thematic Series on Kinases and Phosphatases That Regulate Lipid Signaling."
To whom correspondence should be addressed: Molecular and Cellular
Pharmacology Program, Dept. of Pharmacology, University of Wisconsin
Medical School, 1300 University Ave., Madison, WI 53706. Tel.:
608-262-3753; Fax: 608-262-1257; E-mail:
raanders{at}facstaff.wisc.edu.
2 I. V. Boronenkov, J. C. Loijens, J. Kunz, and R. A. Anderson, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PI, phosphatidylinositol; PIPK, phosphatidylinositol-phosphate kinase; PIPKI, type I phosphatidylinositol-phosphate kinase; PIPKII, type II phosphatidylinositol-phosphate kinase; phosphatidylinositol 4-phosphate, PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol trisphosphate; PKA, cyclic adenosine monophosphate-dependent protein kinase catalytic subunit; TNF, tumor necrosis factor; EGF, epidermal growth factor; PA, phosphatidic acid; PLD, phospholipase D.
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REFERENCES |
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