From the Department of Biochemistry, University of
Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands and
§ Institute of Interdisciplinary Research, Free University
of Brussels, Campus Erasme, Building C, 808 Route de Lennik, B-1070
Brussels, Belgium
Received for publication, August 16, 2002, and in revised form, November 26, 2002
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ABSTRACT |
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Inositol phosphate-containing molecules play an
important role in a broad range of cellular processes. Inositol
5-phosphatases participate in the regulation of these signaling
molecules. We have identified four inositol 5-phosphatases in
Dictyostelium discoideum, Dd5P1-4, showing a high
diversity in domain composition. Dd5P1 possesses only a inositol
5-phosphatase catalytic domain. An unique domain composition is present
in Dd5P2 containing a RCC1-like domain. RCC1 has a seven-bladed
propeller structure and interacts with G-proteins. Dd5P3 and Dd5P4 have
a domain composition similar to human Synaptojanin with a
SacI domain and OCRL with a RhoGAP domain,
respectively. We have expressed the catalytic domains and show that
these inositol 5-phosphatases have different substrate preferences.
Single and double gene inactivation suggest a functional redundancy for
Dd5P1, Dd5P2, and Dd5P3. Inactivation of the gene coding for
Dd5P4 leads to defects in growth and development. These defects are
restored by the expression of the complete protein but not by the
5-phosphatase catalytic domain.
Inositol phosphates play a role in a variety of eukaryotic
cellular processes, including chemotaxis and membrane trafficking. They
are regulated by a number of enzymes. The group of phosphatidylinositol 3-kinases (PI3K)1
phosphorylates the lipid substrates PI, PI(4)P, and
PI(4,5)P2 at the 3-position of the inositol ring (1). The
lipid product PI(3,4,5)P3 has been strongly implicated to
be important in chemotaxis in neutrophils and fibroblasts (2, 3).
PTEN, identified as a tumor suppressor gene (4), reverses the
action of PI3K by dephosphorylation of PI(3,4,5)P3 and
PI(3,4)P2 at the 3-position (5). Another group of enzymes,
the inositol 5-phosphatases, can remove the phosphate group at the
5-position of the inositol ring (6-8). The importance of inositol
5-phosphatase activity in PI(3,4,5)P3 regulation is
demonstrated by SHIP1. In stimulated B-cells, SHIP1 accounts for the
major phosphatase activity toward PI(3,4,5)P3, and
inactivation of SHIP1 leads to an increased and prolonged
PI(3,4,5)P3 production (9). Other inositol 5-phosphatases have been shown to play important roles in a number of cellular processes. Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome in human (10), and deletion of the presynaptic inositol 5-phosphatase Synaptojanin leads to neurological abnormalities and early death of mice (11).
In the social amoeba, Dictyostelium discoideum chemotaxis
toward folic acid and cAMP is an essential strategy for survival (12).
Several observations suggest that phosphatidylinositol phosphates
mediate chemotaxis and, in particular, the localization of the signal
inside D. discoideum cells. The PH domains of a number of
proteins involved in chemotaxis, including CRAC, Akt/PKB, and
PhdA, have been shown to transiently localize at the leading edge of cells moving in a chemotactic gradient (13-15). As these PH
domains bind to PI(3,4,5)P3 and PI(3,4)P2, an
asymmetric lipid distribution is implicated by these observations. In
pi3k1/2-null cells, a strain with two putative PI3Ks
inactivated (16), the transient localization of PhdA can no
longer be observed and cells show reduced chemotaxis (15). On the other
hand, in PTEN-null cells, a strain in which a putative
3-phosphatase is inactivated, the localization of PH-domains is
prolonged and broadened and chemotaxis is also reduced (17, 18).
Inositol 5-phosphatases may play an important role in the regulation of
the phosphoinositides. As this group of enzymes leads to the
degradation of PI(3,4,5)P3 and at the same time formation
of PI(3,4)P2, another PH-binding molecule, they can be
central players in the metabolic route of these signaling molecules.
Phosphoinositides have also been implicated in endocytosis in D. discoideum. Pi3k1/2-null cells are affected with
respect to pinocytosis (19), suggesting a role for
PI(3,4,5)P3 in this process. The inhibitors of
phospholipase C, an enzyme converting PI(4,5)P2 into
Ins(1,4,5)P3 and diacylglycerol, reduce the rate of
phagocytosis (20, 21). Because inositol 5-phosphatases can act on
PI(4,5)P2, PI(3,4,5)P3, and
Ins(1,4,5)P3, they are probably important in the
endocytotic pathway.
To investigate the role of inositol 5-phosphatases in chemotaxis and
endocytosis, we cloned and characterized four D. discoideum inositol 5-phosphatases. Catalytic activity was
determined, indicating that they act as inositol 5-phosphatases. Single
and double gene disruptants were obtained and growth, chemotaxis, and
development were studied in these knock-out strains.
Identification and Sequence Analysis--
The first putative
inositol 5-phosphatase sequence was obtained using degenerated primers
complementary to the conserved motifs I and II present in inositol
5-phosphatases (see "Results"), and the PCR product was used to
screen a cDNA library kindly provided by Dr. R. A. Firtel. The
D. discoideum genomic (www.sdsc.edu/mpr/dicty) and cDNA
databases (www.csm.biol.tsukuba.ac.jp) were screened for other
putative inositol 5-phosphatases using either the conserved motif I or
II. Using Seqman from Lasergene (DNAstar), contigs were formed.
Additional sequences were obtained by screening a cDNA library,
kindly provided by Dr. R. H. Gomer, with a PCR fragment containing
part of the catalytic domain (used primers: Dd5P2: 5P2S1 + 5P2R1;
Dd5P3: 5P5S1 + 5P5R1; and Dd5P4: 5P4S1 + 5P4R1, see "Appendix
A"). The longest clones obtained for Dd5P3 (4212 bp) and Dd5P4
(2795 bp) encode the complete open reading frame of 1377 and 787 amino
acids, respectively. For Dd5P2, the longest clone only encoded for the
amino acids 1-599. In combination with data from the genomic data
base, a complete open reading frame of 1794 amino acids was
constructed. The longest clone obtained for Dd5P1 encodes for amino
acids 118-678; the missing part of the 5'-open reading frame was
obtained from the data base. A comparison of the sequence obtained from
cDNA clones with the genomic data base sequences revealed the
presence of one intron in each inositol 5-phosphatase gene (Dd5P1:
nucleotides 397-499; Dd5P2: nucleotides 53-187; Dd5P3: nucleotides
241-384; and Dd5P4: nucleotides 1357-1622) (GenBankTM accession numbers AY184992, AY184993, AY184994,
and AY184995, respectively). BLAST, Smart, Pfam, and Expasy
programs were used to analyze the obtained sequences. Alignments are
made using Megalign (Lasergene) and Genedoc followed by optimizing by eye.
Strains and Growth Conditions--
The D. discoideum
strains AX3 (wild type), DH1 (uracil auxotroph wild type), and the
mutant strains described were grown in HG5 medium supplemented
with 10 µg/ml blasticidin S or G418 when necessary. Selection for
presence of the pyr5/6 cassette was
obtained using the uracil-deficient FM medium. When grown in shaking
culture, the cell density was kept below 6.106 cells/ml.
Growth on bacterial lawns was studied using Klebsiella aerogens grown on 3 × LP plates (8.3 mM
lactose, 2 mM KH2PO4, 2 mM
Na2HPO4, 3 g/liter bactopeptone, 15 g/liter agar). To
determine whether cells were capable to aggregate properly, they were
put on non-nutrient agar plates (11 mM KH2PO4,
2.8 mM Na2HPO4, 15 g/liter agar) at three
different cell densities (2 × 106, 4 × 105, and 8 × 104 cells/cm2).
Chemotaxis toward cAMP was studied using the small-droplet chemotaxis
assay (22).
Northern Blot Analysis--
RNA was isolated from cells grown on
plates or starved on non-nutrient plates for 3 or 6 h or from
tight aggregates, slugs, or culminants using RNeasy Mini kit from
Qiagen. Equal amounts of RNA (~40 µg) were loaded on the gel. The
probes used to screen the cDNA library were used for hybridization
of inositol 5-phosphatase RNA. For Dd5P1, the PCR product of primer
6BIr + 6BIf was used. DNA was labeled with [ Gene Disruption--
Gene inactivation was obtained by
replacing part of the gene by the bsr or
pyr5/6 cassette. The cassettes were
inserted into NdeI/NsiI site of Dd5P1,
NdeI/EcoRI (nucleotide 1210) site of Dd5P2, AccI (nucleotide 1648)/BglII
site of Dd5P3, and NcoI (nucleotide 887)/NdeI site of Dd5P4. Orientation of the
cassette was opposite to the orientation of
Dd5P1(bsr and pyr),
Dd5P2(bsr), and Dd5P3(bsr) and similar to the orientation of Dd5P2(pyr) and
Dd5P4(bsr and pyr). The place
of insertion of the selection marker is indicated by an asterisk in
Fig. 1. Single gene inactivation was performed with AX3 cells, whereas
double gene inactivation was performed with DH1 cells. Potential
knock-outs were screened by PCR and confirmed by Southern and/or
Northern blots. As initiation from an internal ATG does not occur, at
the most the N-terminal part of the protein will be expressed. A
truncated RNA transcript could be observed for strains in which
Dd5P2, Dd5P3, or Dd5P4 was
inactivated. The size corresponded with the expected size for
transcription of the first part of the gene. Therefore, the catalytic
inositol 5-phosphatase domain is not transcribed and not expressed.
Expression of the Inositol 5-Phosphatase Catalytic Domains in
Escherichia coli--
The inositol 5-phosphatase catalytic domains
were cloned into the BamHI site (Dd5P1, Dd5P2, and Dd5P4) or
XhoI/PstI site (Dd5P3) of pRSETB (Invitrogen)
using primers shown in "Appendix A" (Dd5P1: 1OES2 + 1OER1; Dd5P2:
2OES1 + 2OER1; Dd5P3: 5OES2 + 5OER2; and Dd5P4: 4OES1 + 4OER1). The
catalytic domains were expressed in E. coli BL21DE3 (pLysS)
(Novagen). The expression of the proteins was induced at 22 °C using
1 mM isopropyl-1-thio- Western Blot Analysis--
The purified inositol 5-phosphatase
catalytic domains were analyzed by 10% SDS-PAGE followed by
immunostaining with Penta-His Antibody (Qiagen) and Anti-mouse IgG
peroxidase conjugate (Sigma). Bands were visualized using
chemiluminescence blotting substrate POD (Roche Molecular Biochemicals)
and 1-min exposure to film. Prestained Protein Marker, Broad Range (New
England Biolabs) was used to determine the size.
Expression of Proteins in D. discoideum--
To express proteins
in D. discoideum, the desired DNA fragment was
cloned into an extrachromosomal vector containing a neomycin cassette
(pMB74 or pAH2). This results in expressing the protein from actin 15 promoter. To express the full-length Dd5P4 in
Dd5P4 Activity Measurements--
Activity toward
Ins(1,4,5)P3 and Ins(1,3,4,5)P4 was
determined as described previously (23). 5 µl (diluted) of enzyme was added to the reaction mixture (50 mM HEPES, pH 7.4, 2 mM MgCl2, 48 mM
Activity toward PI(4,5)P2 and PI(3,4,5)P3 was
determined as described previously (24). 50 µg of phosphatidyl serine
and 10 µg of [32P]PI(3,4,5)P3 (see below
for preparation method) or 20 µg of phosphatidyl serine and 10 µg
of PI(4,5)P2/[3H]PI(4,5)P2 were
resuspended in 50 µl of 50 mM Tris-HCl, pH 7.4. Vesicles
were formed by sonication, and 1 µl of 1 M
MgCl2 and 60 µl of (diluted) enzyme were added. After 30 min, the reaction was stopped and the lipids were extracted. Lipids
were separated by TLC and visualized by exposure to film or
PhosphorImager. In the case of the PI(4,5)P2 assay,
the spots were visualized using EN3Hance spray (PerkinElmer Life
Sciences) and scraped from the plates (Silica Gel 60, Merck) and
radioactivity was measured.
[32P]PI(3,4,5)P3 was prepared by making
vesicles containing 50 µg of PI(4,5)P2 and 100 µg of
phosphatidyl serine and incubation with PI3K (purified from baculovirus
using an expression vector kindly provided by B. Vanhaesebroeck (25))
and 70 µCi [ Identification of Four Inositol 5-Phosphatase in D. discoideum--
The first potential inositol 5-phosphatase in
D. discoideum was identified by performing a PCR with
degenerated primers. The obtained PCR product was used as probe for
cDNA library screening. The gene found using this method was called
Dd5P1 (D. discoideum 5-phosphatase 1) and codes
for a protein of 678 amino acids (see "Appendix B"). To identify
other putative inositol 5-phosphatases in D. discoideum, we
searched the D. discoideum data base for sequences showing
homology to the conserved motifs I and II,
WXGDXN(Y/F)R and P(A/S)W(C/T)DR(I/V)L
respectively, which are characteristic for inositol 5-phosphatases (6).
Using the partial sequence obtained from the data base, complete
sequences were obtained from cDNA library screens. Three putative
inositol 5-phosphatases were identified (see "Appendix B"), coding
for proteins consisting of 1800, 1377, and 787 amino acids,
respectively. A comparison of the sequence obtained from cDNA
clones with the genomic data base sequences revealed the presence of
one intron in each inositol 5-phosphatase gene. The position of the
introns is indicated by a triangle in Fig.
1.
High Diversity in Inositol 5-Phosphatase Domain Composition Present
in D. discoideum--
Alignment of the catalytic domain of the four
D. discoideum inositol 5-phosphatases with other inositol
5-phosphatase catalytic domains shows a high degree of identity between
the amino acid sequence of the four proteins and type II inositol
5-phosphatases (Fig. 2) (see "Appendix
C"). All of the four proteins contain the conserved amino acids
present in motifs I and II that are important for inositol
5-phosphatase activity (26-28). Also, the amino acids strongly
conserved in other regions of the inositol 5-phosphatase domains
are present in all four D. discoideum inositol 5-phosphatases, suggesting that they are active inositol
5-phosphatases.
The four inositol 5-phosphatases differ with respect to their domain
composition (Fig. 1). Dd5P1 shows the least complex composition containing only the inositol 5-phosphatase domain. A BLAST analysis with the catalytic domain shows the highest score (Expect value = 2e
Besides the inositol 5-phosphatase domain, Dd5P2 contains a region
homologous to RCC1 ("Appendix D") (30). The structure of RCC1 has
been solved, revealing a seven-bladed propeller with each blade
comprising by four
The other two inositol 5-phosphatases identified in D. discoideum are homologous to human inositol 5-phosphatases.
Dd5P3 resembles the synaptojanin-like proteins found in both human and
yeast with the highest BLAST score of the catalytic domain with that of
INP52p from fission yeast (37% identity, 59% similarity) (34).
Similar to human Synaptojanin and yeast INP52p, Dd5P3 has a
SacI-like domain including the conserved
RXNCXDCLDRTN motif (35) in front of the inositol
5-phosphatase domain (see "Appendix E"). The SacI domains of Synaptojanin and INP52p have been shown to remove
the phosphate group of PI(4)P, PI(3)P, and at a low
rate, both phosphates of PI(3,5)P2(36). The long
C-terminal part of Dd5P3 does not have any homology with known domains
and consists of poly(Q) and poly(N) repeats.
Dd5P4 is homologous to human OCRL (10) and INPP5b (37, 38), consisting
of a inositol 5-phosphatase domain followed by a RhoGAP domain (see
"Appendix F"). The catalytic domain has the highest BLAST score
with the catalytic domain of human INPP5b (44% identity, 60%
similarity). RhoGAP domains are known to catalyze the GTPase activity
of Rho proteins. The crystal structure of human RhoGAP has been solved
indicating a role for two conserved amino acids,
Arg-85p50RhoGAP and Asn-194p50RhoGAP, in
GAP-activated GTP hydrolysis (39). The role of these amino acids is
supported by mutational analysis (39, 40). Mutational analysis has also shown that the conserved Arg is not predominantly involved in the
binding of Rho proteins (40). In Dd5P4, the Arg and the Asn are
substituted by an Ile and Gln, respectively. This observation may
suggest that Dd5P4 does not exhibit high RhoGAP activity, but may still
bind Rho proteins.
Different Transcription Levels during Development--
To
determine the transcription levels of the D. discoideum
inositol 5-phosphatases, Northern blot analysis was performed. Very low
transcription levels were observed for the four inositol 5-phosphatases
in all stages of the D. discoideum life cycle (Fig. 3). Dd5P1 and Dd5P3
were equally transcribed in all stages with the exception of the even
lower transcription of Dd5P3 in the vegetative stage. The
levels of transcription of Dd5P2 were higher during
aggregation than during growth and multicellular development. The
transcription of Dd5P4 was relatively high during growth, decreased during aggregation, and returned to almost vegetative levels
in the multicellular stages. Furthermore, a smaller transcript of 2.5 kilobases was observed in the multicellular stages.
Activity toward Soluble and Lipid Substrates of D. discoideum
Inositol 5-Phosphatases--
To determine whether the four putative
inositol 5-phosphatases identified in D. discoideum can function as inositol 5-phosphatases, the
catalytic activity and specificity of the inositol 5-phosphatase domains were studied in vitro. The catalytic domains were
expressed as His tag fusion proteins in E. coli and
purified. The protein of the expected size could be detected by Western
blot analysis for Dd5P1-3 (Fig. 4). The
Western blot analysis performed for Dd5P4 showed a band at a higher
position than expected (62 instead of 55 kDa), but the purified protein
did show inositol 5-phosphatase activity. Unfortunately, because of the
very low expression levels obtained for the catalytic domain of Dd5P1,
no activity could be determined for this protein.
The purified catalytic domains of Dd5P2, Dd5P3, and Dd5P4 were
incubated with the phospholipids PI(4,5)P2 and
PI(3,4,5)P3 and the water-soluble inositol phosphates
Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Substrate
degradation was quantified using thin-layer chromatography for the
phospholipids and ion-exchange chromatography for the water-soluble
inositol phosphates. The results are summarized in Fig.
5 demonstrating good degradation of
PI(4,5)P2 by all inositol 5-phosphatases, whereas
PI(3,4,5)P3 is degraded predominantly by Dd5P2 and also by
Dd5P4 but not by Dd5P3. Good degradation of the water-soluble inositol
phosphates Ins(1,4,5)P3 and Ins(1,3,4,5)P4 is
observed for Dd5P2, Dd5P3, and Dd5P4, showing the best degradation of
Ins(1,4,5)P3 by Dd5P3, whereas Ins(1,3,4,5)P4
is best degraded by Dd5P2 (Fig. 5C). Comparing the activity
of the four enzymes for each substrate indicates the relatively
preferred substrates (Table I). The
protein with the highest activity toward Ins(1,4,5)P3 is
the catalytic domain of Dd5P3. This protein, compared with the other
proteins, is poor in dephosphorylation of lipid-soluble substrates.
This substrate specificity is almost opposite to the specificity of the
homologue INP51p, which degrades PI(4,5)P2 but does not
hydrolyze Ins(1,4,5)P3. Dd5P2, in contrast, is a very good
PI(3,4,5)P3- and Ins(1,3,4,5)P4-metabolizing
enzyme.
Gene Inactivation Leads to Defects in Growth and
Development--
Upon starvation, D. discoideum wild-type
cells show chemotaxis toward the cAMP secreted by other
starving cells. The formed aggregate develops into a migrating slug or
fruiting body. The spores of the fruiting body are resistant to severe
conditions and maturate into single amoeba under better conditions. To
get an indication of the function of inositol 5-phosphatases in
D. discoideum, the four identified inositol 5-phosphatase
genes have been knocked out. The effect of gene inactivation on
chemotaxis has been investigated by determining the response of cells
toward different cAMP concentrations using the small-droplet chemotaxis assay. Single gene inactivation of either Dd5P3 or
Dd5P4 or double gene inactivation of Dd5P2 and
Dd5P3 (Dd5P2/3
The single disruption of Dd5P4 did affect growth and
development. The growth rate in axenic medium was significantly lower for Dd5P4 cells compared with wild-type AX3 cells (Fig.
7). Whereas the doubling time of
wild-type cells is ~15 h in shaking culture, the doubling time of
Dd5P4-deficient cells is 41 h. The growth rate on bacterial lawns
was affected as well for Dd5P4 Overexpression of Full-length Dd5P4 but Not the Separate Domains
Rescues Dd5P4 The implicated role of phosphoinositide molecules in signal
localization has lead to an increased interest in metabolizing enzymes
such as inositol 5-phosphatases. The amount of data on mammalian
inositol 5-phosphatases has expanded rapidly over the last few years,
showing important functions for inositol 5-phosphatases in several
processes (6).
Human inositol 5-phosphatases are divided in two groups. Type I
inositol 5-phosphatases only convert the water-soluble substrates Ins(1,4,5)P3 and Ins(1,3,4,5)P4. They do not
convert lipid substrates. Type II inositol 5-phosphatases do convert
phosphoinositides (e.g. PI(4,5)P2 and
PI(3,4,5)P3), and in most cases, they also convert water-soluble substrates. The insertions present in Type I enzymes may
prevent the enzymes from binding to the membrane surface, which may
explain the differences in substrate specificity between Types I and II
inositol 5-phosphatases (28). The insertions present in Type I inositol
5-phosphatases are not present in either one of the D. discoideum inositol 5-phosphatases. As expected by this
sequence analysis, the D. discoideum 5-phosphatases Dd5P2, Dd5P3, and Dd5P4 catalyze the dephosphorylation of both water-soluble and lipid substrates, which classify them as Type II inositol 5-phosphatases. The D. discoideum genome has been sequenced
to near completion, making it unlikely that more inositol
5-phosphatases containing the motifs WXGDXN(Y/F)R
and P(A/S)W(C/T)DR(I/V)L are present. Therefore, probably no Type I and
only four Type II inositol 5-phosphatases are present in D. discoideum.
Type II inositol 5-phosphatases can be divided in three subgroups on
the basis of domain composition. SHIP1 represents the group of SH2
domain containing inositol 5-phosphatases Synaptojanin, the
SacI domain containing enzymes, and OCRL, the RhoGAP domain containing inositol 5-phosphatases (Fig. 1). Mammals contain all three
groups of proteins. Six non-mammalian organisms containing putative
inositol 5-phosphatases have been sequenced completely so far. The
metazoa C. elegans and D. melanogaster lack
inositol 5-phosphatases containing a SH2 domain, but both contain one
Synaptojanin-like protein ((41) and Q9W296, respectively) and one
OCRL-like protein (O17590 and O46049, respectively). The diversity in
the plant Arabidopsis thaliana, the yeast strains
Saccharomyces cerevisiae and Schizosaccharomyces
pombe, and the microsporidia Encephalitozoon cuniculi
is even less. The inositol 5-phosphatases of A. thaliana contain only WD40 repeats as additional domains (e.g. Q9SKB7
and O80560), whereas in yeast only Synaptojanin-like proteins are present (28, 34). The one inositol 5-phosphatase of
E. cuniculi (CAD25856) only contains the inositol
5-phosphatase catalytic domain. In prokaryotes, no inositol
5-phosphatases have been identified.
The four inositol 5-phosphatases in D. discoideum show a
high diversity in domain composition compared with the evolutionary position of this organism between plants and yeast. A homologue of OCRL
(Dd5P4) and a homologue of Synaptojanin (Dd5P3) were identified. Also,
a unique combination of a RCC1-like domain in front of the inositol
5-phosphatase domain was identified (Dd5P2). This combination of
domains has not been found in any other protein reported or present in
the GenBankTM so far.
Knock-out strains with one or a combination of two inactive genes for
Dd5P1, Dd5P2, and Dd5P3 show no defects in growth and development,
suggesting a redundancy among these proteins. Chemotaxis toward cAMP is
similar or slightly improved in all of the inositol 5-phosphatase
knock-out cell lines compared with wild type. Recently, it has been
shown that the 3-phosphatase PTEN is the major
PI(3,4,5)P3/PI(3,4)P2-degrading enzyme. It has
been suggested that inositol 5-phosphatases add an addition layer of
regulation of these molecules, fine-tuning the chemotactic signal (42).
Our results support the role of the group of inositol 5-phosphatases as
a minor negative regulator of chemotaxis in D. discoideum.
It will be interesting to study the effects of either overexpression or
inactivation of the inositol 5-phosphatases in a PTEN-null background.
Inactivation of Dd5P4 resulted in defects in growth and
development. The reduced growth rate, either in axenic culture or grown
on bacterial plates, suggests a role for Dd5P4 in endocytosis. Development is also affected as the cells form multiple-tipped aggregates. The relative high transcription levels of Dd5P4 at vegetative and multicellular stages support the role of Dd5P4 in growth
and development. Knock-out strains of PI3K1 + 2 are defective in
chemotaxis, growth, and development (15, 16). The cells grow slowly on
bacterial lawns and in axenic medium and form multiple-tipped
aggregates resembling the phenotype of Dd5P4. No enzyme activity
measurements have been reported for the PI3K in D. discoideum, but they have been suggested to catalyze the formation
of PI(3)P, PI(3,4)P2, and PI(3,4,5)P3
on basis of sequence homology. Our results support a role for
PI(3,4,5)P3 and/or PI(3,4)P2 in growth and
development; Dd5P4 regulates the levels of these phospholipids by
degradation of PI(3,4,5)P3 and production of
PI(3,4)P2. On the other hand, the effect of inactivation of
Dd5P4 could also be assigned to its action on either
PI(4,5)P2 or Ins(1,4,5)P3, two signaling
molecules also implicated in endocytosis (20, 21). In addition, the
RhoGAP domain could be responsible for the function of Dd5P4 in
endocytosis. Rho proteins have been shown to play a role in both
development and growth. The defects in growth and development of
Dd5P4
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP
using the random primer method (High Prime, Roche Molecular Biochemicals). Blots were incubated overnight at 65 °C in
hybridization solution (0.5 M
NaH2PO4/Na2HPO4, pH
7.0, 7% SDS, 0.2 mg/ml bovine serum albumin). Stringency of the final
washes was 0.1 M
NaH2PO4/Na2HPO4, pH
7.0, 1% SDS at 65 °C. Bands were visualized using a PhosphorImager.
-D-galactopyranoside at a cell density of A600 nm = 0.4-0.6
in 2× YT (16 g/liter Trypton peptone, 10 g/liter yeast extract, 0.09 M NaCl). After 3-5 h of induction, 0.5-3.0 liters of cell
culture were harvested, resuspended in binding buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole), lysed with lysozyme (0.5 g/liter) and
phenylmethylsulfonyl fluoride (1 mM), and ultrasonificated.
Soluble proteins were purified from supernatant by the binding of the
His tag to nickel-nitrilotriacetic acid and elution of the protein
(binding buffer with 60, 100, 80, and 60 mM imidazole for
Dd5P1, Dd5P2, Dd5P3, and Dd5P4, respectively).
cells, the obtained full-length cDNA
clone was inserted into the BglII site of pMB74. To clone
the inositol 5-phosphatase catalytic domain, the domain was excised
from the pRSETB vector (see above) with BamHI and
BglII and cloned into the BglII site of pAH2. The RhoGAP domain was obtained by PCR using primer OE4GAPs1 (see
"Appendix A") and the universal T7 primer with cDNA as
template. The PCR product was digested with
BamHI/XhoI and cloned into the
BamHI/XhoI site of pRSETB. The insert was
sequenced and cloned into the BglII site of pAH2.
-mercaptoethanol, 1 mg/ml bovine serum albumin), containing either
100 µM
Ins(1,4,5)P3/[3H]Ins(1,4,5)P3 or
10 µM
Ins(1,3,4,5)P4/[3H]Ins(1,3,4,5)P4.
After 15 min of incubation the reaction was stopped and the products
were separated on Dowex columns.
-32P]ATP (3000 Ci/mmol) in reaction
buffer (50 mM Tris-HCl, pH 7.4, 1.5 mM
dithiothreitol, 100 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 100 µM ATP). After 30 min, the reaction was stopped and lipids were extracted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Domain composition of the D. discoideum inositol 5-phosphatases Dd5P1-4 and the
human inositol 5-phosphatases Synaptojanin (Syj),
OCRL, and SHIP1. The black region represents the
inositol 5-phosphatase catalytic domain. RCC1, SacI, RhoGAP
(GAP), and SH2-like domains are shown in gray.
Inverted triangles indicate the position of the introns,
whereas asterisks indicate the place of gene
disruption.
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Fig. 2.
Amino acid sequence alignment of inositol
5-phosphatases. Amino acids conserved between the D. discoideum inositol 5-phosphatases Dd5P1, Dd5P2, Dd5P3, Dd5P4,
human OCRL (AAA59964), SHIP2 (O15357), yeast SPsynaptojanin
(SPsyj, 1I9Y_A), and INP52p (NP_014293) are shown in
black (100% identity) or gray (80-100%
identity). Asterisks in the top row indicate
amino acids interacting with a metal ion, and open
circles indicate amino acids directly interacting with inositol
phosphates.
54) with the catalytic domain of human SHIP2 (30%
identity, 44% similarity) (29). This score is clearly higher than
scores for non-SHIP-like proteins with the highest score for inositol
polyphosphate 5-phosphatase II of Mus musculus (Expect
value = 7e
37). The homology with SHIP2 does not
expand to the N-terminal part of the protein, because the SH2 domain
present in SHIP2 is not present in Dd5P1.
-sheets (31). RCC1 is known to bind Ran, a
small G-protein present at high concentrations in the nucleus. The
binding of RCC1 to Ran leads to an increased rate of exchange of GDP
for GTP, thus acting as a guanine exchange factor for Ran (32).
The highest BLAST score of the catalytic domain of Dd5P2 is shown with
the catalytic domain of mouse inositol polyphosphate 5-phosphatase
b (INPP5b), a RhoGAP domain containing protein (41%
identity, 59% similarity) (33). The long C terminus of Dd5P2 (~1000
amino acids) does not represent a known domain structure. Three poly(N)
stretches and a poly(D/E) stretch are present in this C-terminal part
of the protein. These kind of stretches are not unusual in D. discoideum proteins.
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Fig. 3.
Northern blot analysis of Dd5P1-4.
A, blots containing RNA isolated from vegetative cells were
probed with part of the catalytic domain of Dd5P1-4. Equal amounts of
RNA were loaded for the four inositol 5-phosphatases. Sizes (in
kilobases) of the transcripts are shown on the right.
B, Northern blot analysis of RNA isolated from vegetative
cells (0), cells starved for 3 (3) or 6 h
(6), tight aggregates (ti), slugs
(sl), or culminants (cu). Equal amounts of RNA
were loaded for all stages.
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Fig. 4.
Western blot analysis of the purified
inositol 5-phosphatase catalytic domains used for activity
measurements. The catalytic domains were expressed in E. coli, purified by binding to nickel-nitrilotriacetic acid, and
eluted with 3 ml of imidazole-containing binding buffer. The proteins
were visualized using Coomassie Brilliant Blue staining (A)
or using a His-tag antibody followed by chemiluminescence
(B). The amount of elute loaded onto the gel was 10 µl of
each sample (A) or 7.5, 1.5, 1.5, 0.05, and 7.5 µl for
Dd5P1, Dd5P2, Dd5P3, Dd5P4, and empty pRSETB vector, respectively
(B). Sizes of standards (in kilodaltons) are shown on the
left. The arrows indicate a band at the expected
size for Dd5P1.
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Fig. 5.
Activity of the catalytic domains of inositol
5-phosphatase Dd5P1-4. A, representative TLC analysis
of the reaction toward PI(3,4,5)P3 using 60 µl of elute
for Dd5P3 and Dd5P4. Results for Dd5P1 were similar to results obtained
for the negative control (empty pRSETB vector) ( ). Results obtained
for Dd5P2 were similar to results obtained for Dd5P4.
PI(3,4,5)P3 and PI(4,5)P2 were used as
standards. B, representative TLC analysis of the reaction
toward PI(4,5)P2 using 60 µl of elute for Dd5P2 and Dd5P3
and 30 µl of elute for Dd5P4. Results for Dd5P1 were similar to
results obtained for the negative control (
). C, activity
of Dd5P1-4 is expressed in either nanomoles of
Ins(1,4,5)P3 or piccolos of Ins(1,3,4,5)P4 per
min per microgram of protein or in percentage of degraded
PI(4,5)P2 or PI(3,4,5)P3 per min per microgram
of protein.
Substrate specificity of Dd5P1-4 towards different substrates
) did not
negatively affect chemotaxis, whereas single disruption of
Dd5P1 or Dd5P2 slightly improved chemotaxis (Fig.
6). Also, the double disruption of either
Dd5P1 and Dd5P2
(Dd5P1/2
) or Dd5P1 and
Dd5P3 (Dd5P1/3
) resulted
in slightly improved chemotaxis. Growth and development of the cells
were also studied. The single gene inactivation of Dd5P1,
Dd5P2, or Dd5P3 did not result in any observable
difference in growth rate in axenic medium or on bacterial lawns (data
not shown). The single disruptants Dd5P1
,
Dd5P2
, and Dd5P3
were
deposited on non-nutrient agar plates or grown on bacterial lawns to
study the development of the cells. Aggregation of the cells proceeded
at a rate comparable to wild-type cells. Slug formation was also normal
as well as the formation of fruiting bodies. The spores that were
formed were viable (data not shown). Also,
Dd5P1/2
,
Dd5P1/3
, or
Dd5P2/3
cells showed no defect in
growth or development.
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Fig. 6.
Small droplet chemotaxis assay.
Chemotaxis toward different concentrations of cAMP (10 9,
10
8, 10
7, and 10
6
M cAMP) was determined for wild-type AX3 and knock-out cell
lines using the small droplet chemotaxis assay. Asterisks,
significant differences from wild-type according to the Student's
t test (p < 0.001) using data of all four
cAMP concentrations; NS, no significant difference from wild
type.
cells.
Amoeba were deposited on a bacterial lawn and incubated at 22 °C.
Approximately 90% wild-type amoeba formed visible plaques within 4 days, whereas <1% Dd5P4
cells formed visible
plaques after 9 days. The development of the Dd5P4 cells
when grown on bacterial lawns was affected as well. The
inactivation of Dd5P4 lead to the formation of
multiple-tipped aggregates (Fig. 8).
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Fig. 7.
Growth rate of wild type and mutant
strains. Doubling times were 15 h for wild-type AX3, 41 h for Dd5P4 , 15 h for
Dd5P4
/Dd5P4, 47 h for
Dd5P4
/cat5P4, and 48 h for
Dd5P4
/GAP5P4.
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Fig. 8.
Development of wild-type AX3,
Dd5P4 , and
Dd5P4
/Dd5P4 on K. aerogens. Pictures were taken of tipped aggregates
using ×40 magnification.
Cells--
The overexpression of the
full-length Dd5P4 restored the defects of
Dd5P4
cells in growth and development,
confirming that the disruption of Dd5P4 was indeed
responsible for the observed defects. Growth rate in axenic medium was
comparable to the growth rate of wild-type cells (AX3) with a doubling
time of 15 h (Fig. 7). When grown on bacterial plates, ~90% of
the amoeba formed visible plaques within 4 days, and the
multiple-tipped phenotype observed for the
Dd5P4
cells could not be observed anymore
(Fig. 8). Overexpression of the inositol 5-phosphatase catalytic domain
or the RhoGAP domain of Dd5P4 in the Dd5P4 knock-out strains
(Dd5P4
/cat5P4 and
Dd5P4
/GAP5P4, respectively) did not restore
any of the defects. As can be seen in Fig. 7, growth rate in axenic
medium was not enhanced by overexpression of either one of the domains.
Also, growth rate and development on bacterial lawns were not restored
(data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells can be rescued by overexpression
of the full-length protein. These defects can not be restored by
transfection of D. discoideum cells with an expression
vector containing either the inositol 5-phosphatase catalytic domain or
the RhoGAP domain. Although we can not exclude that the domains are not
properly expressed or folded, the fact that the inositol 5-phosphatase
catalytic domain expressed in E. coli is catalytically
active would suggest that inositol 5-phosphatase activity is not
sufficient to restore the defects of
Dd5P4
cells. It is possible that the RhoGAP and
inositol 5-phosphatase catalytic domain act together to perform its
function in growth. The binding of a Rho protein to the RhoGAP domain
could affect the inositol 5-phosphatase activity. This would
lead to a direct interaction between the Rho and phosphoinositide
pathways. It would be interesting to see if and which Rho protein binds
to the GAP domain of Dd5P4.
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ACKNOWLEDGEMENTS |
---|
We are indebted to all of the teams involved in the Dictyostelium sequencing projects. We thank L. Drayer and H. Otsuka for their contribution in cloning and analyzing Dd5P1.
![]() |
FOOTNOTES |
---|
* This work was supported by The Netherlands Organization of Scientific Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains appendices A-F.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY184992, AY184993, AY184994, and AY184995.
¶ To whom correspondence should be addressed. Tel.: 31-503634172; Fax: 31-503634165; E-mail: P.J.M.van.Haastert@chem.rug.nl.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M208396200
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ABBREVIATIONS |
---|
The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PI, phosphatidylinositol; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; Ins(1, 3,4,5)P4, inositol 1,4,5-tetrakisphosphate; SH2, Src homology 2; SHIP, SH2 domain-containing inositol 5'-phosphatase; INPP, inositol polyphosphate 5-phosphatase; PH, pleckstrin homology; GAP, GTPase-activating protein.
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