From the Departments of Molecular Biology and
Genetics, ¶ Neuroscience, and
Ophthalmology and the
** Howard Hughes Medical Institute, Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205
Received for publication, December 26, 2000, and in revised form, March 30, 2001
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ABSTRACT |
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The RdgC/PPEF family of serine/threonine
protein phosphatases is distinguished by the presence of C-terminal
EF-hands and neuron-specific expression, including frequent expression
in primary sensory neurons. Here we report that the sole
Caenorhabditis elegans PPEF (CePPEF) homolog is also highly
expressed in primary sensory neurons and is not found outside the
nervous system. Neurons expressing CePPEF include the ciliary
chemosensory neurons AWB and AWC; and within these neurons, CePPEF is
highly enriched in the sensory cilia. In transgenic C. elegans and in transfected 293 cells, CePPEF is
membrane-associated, and the N terminus of CePPEF is necessary and
sufficient for this membrane association. [3H]Myristate
and [3H]palmitate labeling studies in 293 cells
demonstrated that this association was mediated by myristoylation at
Gly2 and palmitoylation at Cys3. Introducing
the G2A or C3S mutation into CePPEF greatly reduced membrane
association in 293 cells and in transgenic nematodes. A recombinant
C-terminal fragment of CePPEF containing two putative EF-hands bound
between one and two Ca2+ ions/protein, and mutation of
residues presumed to ligand calcium in the two putative EF-hands led to
diminished calcium binding. These results establish the first direct
evidence for fatty acylation and calcium binding of a PPEF family
member and demonstrate a remarkable conservation of sensory neuron
expression among the members of this distinctive family of protein phosphatases.
Phosphorylation and dephosphorylation of serine and threonine
residues are known to be important in several types of sensory neurons.
In vertebrate photoreceptors, where light-dependent
phosphorylation and dephosphorylation have been extensively
characterized, light-induced rhodopsin activation of transducin is
terminated by phosphorylation of multiple serine and threonine residues
at the rhodopsin C terminus and the subsequent binding of arrestin to
phosphorhodopsin (1-3). Recycling of rhodopsin back to the dark state
involves replacement of the all-trans-retinal chromophore
with 11-cis-retinal, release of arrestin, and
dephosphorylation (4, 5). A similar phosphorylation-dephosphorylation cycle occurs in Drosophila photoreceptors (6, 7). In
Drosophila, rhodopsin dephosphorylation appears to be
catalyzed by the protein product of the retinal degeneration C gene
rdgC, and hyperphosphorylation of rhodopsin due to
rdgC mutation produces a rapid light-dependent photoreceptor degeneration of the R1-R6 photoreceptors (8-11).
High throughput sequencing and database homology searches from a
variety of species have revealed several predicted protein sequences
with extensive homology to RdgC (12, 13). Collectively, these
proteins are referred to as PPEFs (for protein
phosphatases with EF-hands) in
recognition of the most distinctive features of their primary sequence:
a serine/threonine protein phosphatase domain and two or more
C-terminal EF-hand calcium-binding motifs. Members of this family
identified to date include the single Drosophila PPEF
(RdgC), two mammalian PPEFs (PPEF-1 and PPEF-2), a puffer-fish PPEF,
and a single Caenorhabditis elegans PPEF
(CePPEF).1 As rdgC
mutants show elevated levels of phosphorylated rhodopsin (10, 14), and
the dephosphorylation of Drosophila rhodopsin in
photoreceptor extracts is stimulated by calcium (14), the C-terminal
EF-hands of RdgC have been postulated to regulate
calcium-dependent rhodopsin dephosphorylation. However,
direct evidence that calcium binds to the EF-hand motifs has not been
reported for any PPEF family member.
Mammalian and Drosophila PPEFs are expressed principally or
exclusively within the nervous system and are highly enriched in a
subset of primary sensory neurons. Drosophila RdgC has been immunolocalized to photoreceptors and the mushroom bodies within the
brain (9); PPEF-1 transcripts have been localized by in situ
hybridization to the inner ear, the dorsal root ganglia, and several
brainstem nuclei in the developing mouse (13); and PPEF-2 has been
localized by in situ hybridization and immunostaining to
photoreceptors and pinealocytes in the adult rodent (12). These
observations suggest that the PPEFs may play a conserved role in
diverse sensory systems.
Aside from Drosophila rhodopsin, the identities of PPEF
substrates have remained elusive. By analogy, it would be plausible to
suppose that mammalian PPEF-2 specifically dephosphorylates phosphorhodopsin, but current evidence argues against this idea. In
particular, protein phosphatase 2A activity has been detected in rod
outer segment extracts and can efficiently dephosphorylate phosphorhodopsin (5, 15-17). Moreover, immunolocalization of one
isoform of PPEF-2 that contains the EF-hand motifs localizes this
protein to photoreceptor inner segments rather than outer segments
(12). (The subcellular localization of a second PPEF-2 isoform that
lacks the C-terminal domain is unknown.) These observations suggest
that, in mammals, protein phosphatase 2A is the rhodopsin phosphatase
and therefore that some aspects of PPEF function may not be conserved
between vertebrate and invertebrate photoreceptors.
C. elegans contains a wide variety of sensory neurons
(18-20), and the C. elegans genome is known to contain
several hundred putative G protein-coupled receptors (21). Expression
analysis has shown that these are widely distributed inside and outside the nervous system, with many receptors localizing to primary sensory
neurons, including the three neurons responsible for responding to
volatile odorants: AWA, AWB, and AWC (22-25). In this report, we show
that expression of CePPEF, the sole member of the PPEF family in
C. elegans, is restricted to several neurons in the head and
tail, including the primary sensory neurons AWB and AWC. Within these
sensory neurons, CePPEF is highly enriched in sensory cilia, and this
localization depends on sequences at the extreme N terminus, which
site-directed mutagenesis shows are the sites of both myristoylation
and palmitoylation. Finally, we show that the two consensus EF-hand
motifs within the C terminus of CePPEF bind calcium. These observations
reveal a mechanism for membrane association of the PPEFs; strengthen
the idea that PPEF activity is directly regulated by calcium binding;
and suggest that some substrates of CePPEF are found in the sensory
cilia, possibly including G protein-coupled receptors in the cilia of
AWB and AWC.
cDNA and Genomic Sequences--
Generation of Transgenic C. elegans--
GFP reporter vectors
(60 µg/ml) were mixed with pRF4-Rol6 DNA (100 µg/ml), spun down,
and injected into the gonads of L4 or early adult N2 hermaphrodites.
Injected animals were recovered and designated as F0. F1 animals with
the roller phenotype were isolated and observed for progeny that also
showed the roller phenotype. F2 animals with the roller phenotype were
generated from at least two different F0 injections for each GFP
reporter vector construct analyzed. Adult animals were photographed
after anesthetizing in 1 mM levamisole.
Antibody Preparation and Purification--
Fusion proteins
between the bacteriophage gene 10 protein and the C-terminal 142 amino
acids of CePPEF were expressed in Escherichia coli, purified
by preparative SDS-PAGE, and used for immunization of rabbits. Fusion
proteins between the same CePPEF fragment and maltose-binding protein
(MBP) were also produced in E. coli, purified to apparent
homogeneity on an amylose resin (New England Biolabs Inc.), covalently
cross-linked to Affi-Gel 15 (Bio-Rad), and used to affinity purify the
rabbit polyclonal antibodies.
C. elegans Immunostaining--
Animals were synchronized by
collecting N2 hermaphrodites from a single 10-cm plate, mixing gently
in 10% bleach + 1 M NaOH for 1 min, washing several times
in M9 medium, and then gently rocking for 36 h at room temperature
to allow all embryos to hatch. Approximately 100 L1 larvae were placed
on a Probe-On Plus glass slide (Fisher), coverslipped, and then
freeze-cracked under slight pressure. Animals were fixed in Immunoblotting of C. elegans Lysates--
Three 10-cm plates of
wild-type N2 animals or transgenic animals with the roller phenotype
were resuspended in PBS and washed extensively. Nematodes were lysed in
PBS with 1% Triton X-100; sonicated; and centrifuged at 731 × g at 4 °C to remove cell debris, genomic DNA, and cuticle
fragments. The protein concentration in the supernatants was determined
using the Bradford assay (Bio-Rad), and 20 µg of each lysate were
subjected to SDS-PAGE analysis and electrophoretic transfer.
Immunoblots were visualized with the Supersignal West Pico system
(Pierce) after sequential incubation with the CePPEF C
terminus-specific antibody and a horseradish peroxidase-conjugated goat
anti-rabbit antibody.
293 Subcellular Fractionation--
Twenty-four hours after
transient transfection using the calcium phosphate method, cells were
washed in PBS, dissociated in PBS with 5 mM EDTA, collected
by centrifugation at 418 × g, resuspended in 6 ml of
PBS with protease inhibitors (1 µg/ml each chymostatin, leupeptin, aprotinin, and pepstatin A and 100 µg/ml
phenylmethylsulfonyl fluoride), and disrupted with a Polytron 10/35
homogenizer (Kinematica). Nuclei were removed by centrifugation at
731 × g for 5 min at 4 °C, and a portion of the
post-nuclear supernatant was saved as the lysate fraction. The
remaining sample was centrifuged at 58,450 × g for 30 min at 4 °C. The high speed supernatant was recovered, and the
pellet was resuspended in 4 ml of 1× SDS sample buffer, sonicated for
1 min, and diluted to 6 ml. 6× SDS sample buffer was added to the
lysate and supernatant fractions to a final concentration of 1×, and
20 µl of lysate, pellet, and supernatant fractions were then analyzed
by SDS-PAGE and immunoblotting.
Immunostaining of 293 Cells--
293 cells on gelatin-coated
coverslips were transfected with 0.25 µg each of GFP/pCIS and
CePPEF/pCIS using LipofectAMINE (Life Technologies, Inc.). Twenty-four
hours after transfection, cells were fixed for 10 min in 4%
paraformaldehyde in PBS and then permeabilized in ice-cold methanol for
10 min. Cells were blocked for 1 h in PBS with 5% normal goat
serum and incubated overnight at 4 °C in PBS with 5% normal goat
serum and a 1:100 dilution of affinity-purified CePPEF C
terminus-specific antibody. The following day, CePPEF was visualized by
sequential addition of a biotinylated anti-rabbit antibody and Texas
Red-streptavidin. Images were analyzed by confocal microscopy using a
1-µm slice thickness.
Metabolic Labeling and Immunoprecipitation--
293 cells were
split 1:5 into 12-well plates and transfected 36 h later with 0.5 µg of CePPEF/pCIS using LipofectAMINE. For myristate labeling,
[3H]myristate was added to Dulbecco's modified Eagle's
medium/nutrient mixture F-12 with 10% calf serum at a specific
activity of 500 µCi/ml (26), and cells were incubated in labeling
medium for 5 h. For palmitate labeling, cells were transferred to
serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12
for 12 h and then to the same medium with 1% calf serum for
1 h. [3H]Palmitate was added to Dulbecco's modified
Eagle's medium/nutrient mixture F-12 with 5% calf serum at a specific
activity of 1000 µCi/ml (27), and cells were incubated in labeling
medium for 1 h. After labeling, cells were washed in PBS and then
lysed in radioimmune precipitation assay buffer (10 mM
sodium phosphatase, pH 7.2, 150 mM NaCl, 1% Nonidet P-40,
1% sodium deoxycholate, and 0.1% SDS) with protease inhibitors (see
above). Lysates were added to 25 µl of protein G-Sepharose beads
precoated with anti-Myc monoclonal antibody, rotated at 4 °C for
3 h, and then washed extensively in radioimmune precipitation
assay buffer. Beads were resuspended in 50 µl of 1.5× SDS sample
buffer (without dithiothreitol for palmitate-labeled samples) and
subjected to SDS-PAGE. Gels were fixed, treated with ENHANCE
(PerkinElmer Life Sciences), dried, and analyzed by autoradiography.
Calcium Binding Assays--
Mutations in the C terminus of
CePPEF were generated by polymerase chain reaction. MBP fusions with
the C-terminal 142 amino acids of wild-type or mutant CePPEF were
produced in E. coli. Bacteria were lysed in 10 mM imidazole, pH 7.0, 200 mM KCl, and 0.1 mM EDTA and then purified to near homogeneity by amylose
affinity chromatography. Fusion proteins were dialyzed extensively
against 10 mM imidazole, pH 7.0, and 200 mM
KCl, which had been prerun over a Chelex 100 column. Binding assays
were performed at 4 °C in 10 mM imidazole, pH 7.0, 200 mM KCl, and 0.1 µCi/ml 45CaCl2
(Amersham Pharmacia Biotech) with the indicated concentrations of
unlabeled CaCl2. In each binding reaction, the protein
concentration was set to 20% of the Ca2+ concentration.
The binding mixture was incubated at 4 °C for 2 h, and bound
calcium was purified by running 50 µl of the binding mixture over a
Sephadex G-50 spin column prechilled to 4 °C. Columns were
spun at 731 × g for 1 min at 4 °C. The separation
of bound Ca2+ (in the void volume) from free
Ca2+ (retained in the column) occurs within several
seconds. Calcium binding at each Ca2+ concentration was
assayed in triplicate; a control reaction without protein was analyzed
for each Ca2+ concentration and subtracted as background.
Binding data were fit to a second-order polynomial equation.
For analysis of calcium binding under equilibrium conditions, Sephadex
G-50 spin columns were pre-equilibrated with a solution that was
identical to the binding assay contents described above, except for the
absence of protein. Pre-equilibration was accomplished by five cycles
of adding 100 µl of the pre-equilibration solution to the Sephadex
G-50 spin column followed by centrifugation at 731 × g
for 1 min at 22 °C and two cycles of adding 50 µl of the pre-equilibration solution to the Sephadex G-50 spin column followed by
centrifugation at 731 × g for 1 min at 22 °C.
Fusion proteins and binding reactions were prepared as described above
and incubated at 22 °C for 30 min. Binding reactions were then added
to the pre-equilibrated Sephadex G-50 spin column and centrifuged at 731 × g for 1 min at 22 °C. Calcium binding at each
Ca2+ concentration was assayed in nine independent Sephadex
G-50 spin columns; three control reactions without protein were also
analyzed for each Ca2+ concentration and subtracted as
background. Binding data were fit to a two-site Aldair-Klotz model (28)
with the following equation: mol Ca2+/mol protein = 0.675·[m1·10(6 Neuronal Localization of CePPEF in Transgenic C. elegans--
As a
first step in determining the cellular expression pattern of the
CePPEF gene in C. elegans, a transgene construct
was generated that fused 3.0 kb of DNA sequence upstream of the start codon to a GFP-LacZ reporter carrying an NLS (aa-(1)-NLS-GFP-LacZ) (Fig. 1). The large size of the GFP-LacZ
fusion protein facilitates retention in the nucleus. Injection of the
aa-(1)-NLS-GFP-LacZ construct into C. elegans revealed
reporter gene expression in several anterior neurons, including AWB,
AWC, AVA, AVB, AVX, BAG, and URX (Fig.
2F). The ASE neuron showed
inconsistent transgene expression. To assess the subcellular
localization of the CePPEF protein and to determine whether additional
transcriptional regulatory elements exist in intronic sequences, a
transgene was constructed in which a 9.5-kb genomic DNA fragment
extending from 3 kb 5' of the start codon to a point 32 base pairs
upstream of the CePPEF stop codon in exon 7 was fused
in-frame to GFP (FL-GFP) (Fig. 1). This FL-GFP transgene directed GFP
expression to the same set of neurons as the transgene construct
described above (Fig. 2, compare A and B with
F), and expression in a single posterior neuron was also
more clearly observed (Fig. 2E). Within the expressing cells, the FL-GFP fusion protein localized efficiently to several structures beyond the cell soma, including axons that form the ventral
nerve cord (Fig. 2D); dendrites that extend to the anterior tip of the animal (Fig. 2, A and B); and the
cilia of neurons AWB, AWC, and BAG (Fig. 2C).
The transgene constructs described above showed minimal expression in
non-neuronal cells. However, we observed transgene expression in
intestinal, hypodermal, or muscle cells in occasional transgenic lines
in which 3.0-kb genomic DNA fragments extending just past the
CePPEF start codon directed production of short
N-terminal regions of CePPEF fused to GFP (described below). As these
expression patterns occurred sporadically, they likely reflect
transgene effects rather than an expression pattern relevant to the
endogenous CePPEF gene.
Immunolocalization of CePPEF--
As a more direct method for
determining the pattern of CePPEF protein localization,
affinity-purified rabbit antibodies raised against the C-terminal 142 amino acids of CePPEF were used for immunostaining of C. elegans L1 larvae. The anti-CePPEF antibodies recognized a single
polypeptide band of ~80 kDa on immunoblots of proteins from 293 cells
transfected with CePPEF cDNA, and no bands were seen with proteins
from untransfected cells (Fig.
3A). Immunoblots of wild-type
C. elegans lysates showed two bands, one at 80 kDa, which
corresponds closely to the predicted molecular mass of the full-length
protein, and a fainter band of slightly lower molecular mass, which may
correspond to either a CePPEF degradation product or a heretofore
unrecognized splice variant (Fig. 3B). Immunoblots of
lysates from FL-GFP transgenic animals showed a higher molecular mass
band at ~120 kDa derived from the CePPEF-GFP fusion protein in
addition to the endogenous CePPEF band. These data strongly imply that
the anti-CePPEF antibodies specifically recognize CePPEF in C. elegans lysates. Immunostaining of wild-type L1 larvae with the
anti-CePPEF antibodies demonstrated CePPEF in several cell bodies in
the region of the nerve ring, in dendritic processes leading from these
cells to the anterior end of the animal, and in sensory cilia at the
extreme anterior end of the animal (Fig. 3C). This
expression pattern closely resembles that observed with the GFP
transgenic animals, indicating that the CePPEF-GFP transgenes
accurately reproduce the endogenous CePPEF expression pattern.
The CePPEF N Terminus Is Necessary and Sufficient for Localization
to Axons, Dendrites, and Cilia--
The extreme N terminus of CePPEF
contains the myristoylation consensus sequence
(MGXXX(S/T) ... ) as well as the consensus sequence for
N-terminal palmitoylation (MGC ... ) (26, 27), suggesting that
CePPEF may be membrane-associated via an N-terminal lipid modification.
A membrane association of this type could play an important role in
localizing CePPEF to axons, dendrites, or cilia. As an initial step in
assessing this possibility, we generated transgenic lines in which 1, 5, 8, 11, or 14 N-terminal amino acids from CePPEF were fused to an
NLS-tagged GFP (aa-(1)-NLS-GFP, aa-(1-5)-NLS-GFP, etc.) (Fig. 1). The
NLS-GFP reporter showed little nuclear retention, presumably due to its
small size. The aa-(1)-NLS-GFP and aa-(1-5)-NLS-GFP transgenic lines
showed localization primarily in the nucleus and cell body and weak or
no localization in dendrites and cilia (Fig.
4, A and B). In
contrast, the aa-(1-11)-NLS-GFP and aa-(1-14)-NLS-GFP transgenic
lines showed GFP localization primarily in axons, dendrites, and cilia,
but not in the cell soma (Fig. 4, D and E). The
aa-(1-8)-NLS-GFP fusion showed an intermediate pattern, with
localization in axons, dendrites and cilia as well as in the cell body
and nucleus (Fig. 4C).
To test whether the myristoylation and/or palmitoylation consensus
sites are required for localization outside of the cell soma,
site-directed mutants were constructed that disrupted either one or
both fatty acylation sites. In one set of experiments, Gly2
was substituted with alanine in the context of aa-(1-14)-NLS-GFP or
FL-GFP to simultaneously disrupt the myristoylation and palmitoylation consensus sequences. In both cases, the G2A mutant proteins localized to the cytoplasm and nucleus, suggesting that myristoylation, palmitoylation, or both modifications together confer membrane attachment and concomitant localization to axons, dendrites, and cilia
(Fig. 4, compare F with E and H with
G). To further refine this analysis, Cys3 was
substituted with serine in the context of FL-GFP to eliminate only the
palmitoylation site. This mutant protein showed low levels of axonal,
dendritic, and ciliary staining (Fig. 4, compare I with
G), suggesting that palmitoylation in particular is critical for membrane attachment.
Role of the CePPEF N Terminus in Membrane Association and Fatty
Acylation in 293 Cells--
As an independent test of the ability of
the CePPEF N terminus to confer membrane association, we examined the
properties of CePPEF and its various N-terminal mutants in 293 cells.
293 cells transfected with the wild-type CePPEF cDNA or with a
CePPEF cDNA carrying the G2A or C3S mutation were analyzed by both
subcellular fractionation and confocal microscopy. Subcellular
fractionation showed that all of the wild-type protein, but little of
the G2A or C3S mutant protein, was associated with the membrane
fraction following high speed centrifugation (Fig.
5). Attempts to perform an analogous
experiments using C. elegans failed because the tough C. elegans cuticle protects the animal from rupture by
methods compatible with subcellular fractionation (data not shown).
Confocal microscopy of transfected 293 cells immunostained with
anti-CePPEF antibodies revealed wild-type CePPEF at the plasma
membrane, whereas the G2A and C3S mutants were found in the cytoplasm
(Fig. 6). As observed in transgenic
animals, the N-terminal 14 amino acids of CePPEF were sufficient to
confer plasma membrane association when joined to GFP, and mutation of
Gly2 or Cys3 resulted in cytosolic localization
(Fig. 7).
To define the presumptive N-terminal lipid modifications of CePPEF,
wild-type and mutant CePPEFs tagged with triple-Myc epitopes at their C
termini were expressed in 293 cells, metabolically labeled with
[3H]myristic acid or [3H]palmitic acid, and
immunoprecipitated. The wild-type protein incorporated both myristate
and palmitate. By contrast, the G2A mutant, which affected both the
myristoylation and palmitoylation consensus sites, incorporated neither
label, and the C3S mutant, which affected only the palmitoylation
consensus site, incorporated myristate, but not palmitate (Fig.
8A). Both the wild-type and mutant proteins accumulated to comparable levels as judged by immunoblotting (Fig. 8B). The results of these experiments,
as well as those described above, are summarized in Table
I.
The CePPEF C Terminus Binds between 1 and 2 mol of Calcium/mol of
Protein--
The PPEF family of protein phosphatases are so named
because they contain two consensus EF-hand motifs near their C termini; but to date, there is no direct evidence that any PPEF family member
binds calcium. Moreover, the loose nature of the EF-hand consensus has
led to the suggestion that there may be as many as five EF-hand motifs
within a single PPEF sequence (9). To directly assess the
calcium-binding capacity of the CePPEF C terminus, a C-terminal segment
of 142 amino acids containing the two CePPEF consensus EF-hand motifs
was expressed as a C-terminal fusion to MBP in E. coli and
purified by amylose affinity chromatography. Additionally, we
constructed mutants EF1' and EF2', which would be predicted to impair
calcium binding to the consensus EF-hands starting at amino acids 642 and 682, respectively, and the corresponding double mutant, EF1'+2'
(Fig. 9A).
Each CePPEF C-terminal fusion protein was assayed for calcium binding
based on its ability to bind and retain 45Ca2+
during rapid passage through a small Sephadex G-50 spin column at
4 °C. This method has been used previously to measure the binding of
radioligands to solubilized
In a second set of experiments designed to examine Ca2+
binding to the MBP-wild-type CePPEF C-terminal fusion protein under equilibrium conditions, a method first described by Hummel and Dreyer
(32), the Sephadex G-50 spin columns were extensively pre-equilibrated
with a protein-free solution that was otherwise identical to the
solution used for the binding reaction. In these experiments, the
maximal observed binding for the wild-type C-terminal fusion protein
was 1.4 mol of Ca2+/mol of protein (Fig. 9C).
Fitting these data to a two-ligand binding site dissociation curve
yielded macroscopic dissociation constants of 36 and 1.0 µM, affinities typical for EF-hands (28).
This work represents the first characterization of CePPEF, the
sole member of the PPEF family of serine/threonine protein phosphatases
encoded within the C. elegans genome. The results presented
above show that (a) CePPEF gene expression is
confined to a small subset of neurons, including primary sensory
neurons; (b) the CePPEF protein is highly enriched in
sensory cilia and, to a lesser extent, in axons and dendrites;
(c) there are adjacent N-terminal myristoylation and
palmitoylation sites in CePPEF that play an important role in correct
membrane and subcellular localization; and (d) the CePPEF C
terminus contains two EF-hand motifs that can bind calcium. We discuss
below each of these findings in the context of PPEF function.
EF-hands at the C Terminus of CePPEF Bind Calcium--
We have
measured the stoichiometry and affinity of the C terminus of CePPEF for
calcium using centrifugation through a small gel filtration column to
effect a rapid separation of free and bound calcium. This assay has
been used previously for the analysis of ligand-receptor and
enzyme-substrate interactions (29-32) and may also be generally useful
for the analysis of calcium binding to proteins that are difficult to
assay by equilibrium dialysis due to protein adsorption to the dialysis
membrane or protein denaturation or degradation during the extended
incubation required for equilibrium dialysis. Moreover, for proteins
with multiple calcium-binding sites, the analysis of binding
stoichiometry is likely to be more straightforward with this gel
filtration binding assay compared with assays that monitor intrinsic
tryptophan fluorescence or competition with fluorescent calcium chelators.
Using this calcium binding assay, we found that the CePPEF C terminus
bound between 1 and 2 mol of calcium/mol of protein, with dissociation
constants in the low to mid micromolar range. Mutation of 2 amino acids
predicted to coordinate calcium in each of the two consensus EF-hand
motifs demonstrated that most of the calcium binding activity is
referable to these EF-hand sequences. Mutations in the second EF-hand
had a relatively greater effect on calcium binding than mutations in
the first EF-hand, possibly due to the additional mutation of the
glycine critical for permitting a sharp bend in the calcium-binding
loop of the second EF-hand (see Fig. 9A) (33). Whether the
reduction in maximal calcium binding observed in these EF-hand mutants
reflects lower equilibrium calcium affinities and/or increased rates of
calcium dissociation is an open question. Finally, we noted that the
Ca2+-binding affinity of the C-terminal fusion proteins
tested here may differ from that of the full-length protein under
in vivo conditions as a result of interactions with the more
N-terminal PPEF domains or with other proteins.
If calcium binding to the C terminus of PPEF proteins is a general
mechanism for directly regulating phosphatase activity as suggested by
the calcium stimulation of rhodopsin dephosphorylation in
Drosophila photoreceptor extracts (14), it will be of
interest to determine the concentration dependence of this regulation, whether one or both calcium ions must bind to shift the enzyme between
inactive and active states, and whether calcium binding activates the
catalytic activity of some PPEFs and inhibits others. In rough
agreement with this model, Huang and Honkanen (34) have reported
that recombinant PPEF-1 exhibits modest calcium-stimulated phosphatase
activity on the pseudosubstrate p-nitrophenyl phosphate with
a half-maximal concentration for calcium stimulation of ~500 µM. We note, however, that this concentration of
half-maximal activation is significantly higher than the typical range
of EF-hand calcium-binding affinities (28) and therefore may reflect a calcium effect independent of the EF-hands.
An additional mode of regulation by calcium deserves consideration:
calcium-dependent membrane association, as seen, for
example, in the "myristoyl-switch" mechanism of the
recoverin family of EF-hand proteins (35-37). To test this hypothesis,
native PPEF-2 from bovine retina and recombinant CePPEF from
transfected 293 cells were analyzed by immunoblotting after
homogenization in PBS with 1 mM CaCl2 or 1 mM EGTA, followed by separation of membrane and cytosol
fractions via centrifugation. The presence or absence of
Ca2+ did not alter the membrane versus cytosolic
distribution of either protein (data not shown). Although it remains
possible that our choice of in vitro conditions inhibited or
masked a calcium-regulated membrane association, these data are most
consistent with a model in which calcium binding by the C-terminal
EF-hands serves simply to regulate phosphatase activity, as seen in the
regulation of the catalytic Function of N-terminal Myristoylation and Palmitoylation--
At
its N terminus, CePPEF contains myristoylation
(MGXXXS ... ) and palmitoylation (MGC ... )
consensus sites (26, 27); and in transfected 293 cells, the wild-type
protein incorporates [3H]myristate and
[3H]palmitate and is efficiently localized to the plasma
membrane. Mutation of Cys3 in the palmitoylation consensus
sequence still allows for myristoylation, but largely eliminates
membrane association both in 293 cells and in transgenic nematodes,
suggesting that palmitoylation is critical for membrane attachment.
These findings are consistent with previous cell fractionation
experiments that found that all palmitoylated, but only some
myristoylated, proteins are membrane-associated (40, 41). They are also
reminiscent of observations on the membrane association of Ras
proteins, in which C-terminal farnesylation is insufficient to confer
membrane association unless combined with palmitoylation at a second
site (42).
Many classes of signaling molecules, including G protein
Another reason for the dual acylation of CePPEF may be to allow the
myristoyl group to mediate a function other than membrane attachment.
Indeed, fatty acyl groups such as the palmitate groups in
G Ciliary Localization of CePPEF--
The patterns of
immunolocalization for CePPEF and of GFP fluorescence for the various
CePPEF-GFP fusion proteins, including those carrying only 14 N-terminal
amino acids from CePPEF, show the greatest concentration of signal
within the sensory cilia, suggesting that the N terminus of CePPEF
carries signals both for membrane targeting and for localizing CePPEF
to cilia. These observations are reminiscent of previous work on the
neuronal protein GAP-43, which is also palmitoylated at the N terminus. Fusion proteins consisting of the N-terminal 10 amino acids of GAP-43
and choline acetyltransferase localize to growth cones and filopodia
when expressed in PC12 cells (58).
The association of CePPEF with membranes and its further enrichment
within cilia could confer any of several advantageous properties.
First, it could promote more efficient encounters with integral
membrane, membrane-associated, or cilium-specific protein substrates;
second, it could facilitate calcium regulation by localizing CePPEF to
the juxtamembrane zone where the amplitude of calcium transients is
greatest; and third, it could allow regulation of CePPEF by
membrane-associated regulatory proteins such as kinases.
Localization of PPEFs to Sensory Neurons in Diverse
Organisms--
The localization of CePPEF described here extends to a
new species the pattern of nervous system-specific expression
seen for the PPEF family. Like other PPEFs, CePPEF is expressed within a subset of primary sensory neurons; and like RdgC, it is localized to
the subcellular compartment in which sensory transduction occurs. This
localization reinforces the impression that the PPEF family displays a
remarkable degree of tissue-specificity relative to other
serine/threonine protein phosphatases. One particularly interesting
domain of CePPEF expression is the cilia of the neurons AWB and AWC, as
several proteins that are critical for odorant detection and/or
adaptation and that are known to be involved in G protein signaling are
also strongly concentrated there (24, 25, 59-63). This suggests that
CePPEF may also participate in the G protein signaling pathway
mediating odorant detection and/or adaptation, possibly by
dephosphorylating G protein-coupled receptors much like rdgC
dephosphorylates Drosophila rhodopsin. A behavioral analysis
of CePPEF mutants utilizing volatile odorants recognized by the neurons
AWB and AWC would be an important step in addressing these hypotheses.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage clones
encompassing the F23H11.8 (CePPEF) locus were
isolated from a C. elegans genomic DNA library and used to
prepare transgene constructs. The full-length green fluorescent protein (FL-GFP) transgene was generated by cloning the genomic region from the
EcoRI site 3.0 kb 5' of the initiator ATG codon to the StuI site 32 base pairs 5' of the stop codon near the end of
exon 7 into pPD95.81. pPD96.04 and pPD95.67 were used to generate the aa-(1)-NLS-GFP-LacZ and aa-(1-14)-NLS-GFP constructs,
respectively, each of which also carries the same 3.0 kb of 5'-sequence
extending to the EcoRI site. The G2A and C3S mutants of
FL-GFP were generated by polymerase chain reaction, followed by
cassette replacement of a BclI-AvrII fragment and
sequencing. The full-length CePPEF cDNA was obtained by reverse
transcription-polymerase chain reaction using random-primed cDNA
from N2 hermaphrodites. Following subcloning, the cDNA sequence was
confirmed to rule out spurious mutations. The CePPEF cDNA was
modified by polymerase chain reaction to insert an optimal Kozak
sequence 5' of the initiator methionine codon and transferred to pCIS,
a mammalian expression vector that utilizes the cytomegalovirus
promoter. Subsequent mutagenesis of the N-terminal region was achieved
by replacing the first 14 codons with synthetic double-stranded DNA
segments containing the appropriate mutations.
20 °C
methanol for 30 min, followed by 15 min in
20 °C acetone.
Freeze-cracked animals were blocked for several hours in Tris-buffered
saline with 0.1% Triton X-100 and 5% normal goat serum, followed by
primary antibody addition at a dilution of 1:100, overnight incubation
at 4 °C, and subsequent incubation with a Cy3-conjugated anti-rabbit antibody.
m0) + 2·m1·m2· 10(12
2·m0)]/[1 + m1·10(6
m0) + m1·m2·10(12
2·m0)] where m0
represents the pCa of the binding mixture and
m1 and m2 represent the
first and second macroscopic binding constants, respectively.
Adjustment of the numerator by a factor of 0.675 is designed to set
maximal binding to the binding observed at 1 mM
Ca2+ and accounts for the lowered maximal binding due to an
estimated 80-90% purity of the fusion protein sample and retention of
10-20% of the fusion protein on the Sephadex G-50 spin column.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
C. elegans transgene constructs
for the analysis of CePPEF gene expression and protein
localization. The intron-exon structure of the CePPEF
gene was derived from a comparison between the complete CePPEF cDNA
sequence (12) and the nucleotide sequence of the corresponding
genomic segment. All constructs contain 3 kb of 5'-sequence between the
indicated EcoRI site and the initiator methionine codon.
Exons 1-7 are denoted by boxes. In the aa-(1)-NLS-GFP-LacZ
construct, the CePPEF 5'-sequences are joined at the
initiator methionine codon to DNA sequences encoding a fusion protein
consisting of SV40 NLS-GFP-LacZ. LacZ was included in this fusion
protein to minimize diffusion out of the nucleus. The FL-GFP construct
contains the entire CePPEF coding region, except for the
last 10 amino acids, fused in-frame to GFP at the indicated
StuI site. In the aa-(1-14)-NLS-GFP construct, the first 14 codons of CePPEF are fused in frame to DNA sequences encoding an SV40
NLS-GFP reporter at the indicated SalI site. Constructs
analogous to aa-(1-14)-NLS-GFP but with <14 CePPEF N-terminal amino
acids were also constructed. For enhanced expression in C. elegans, the coding regions of GFP and lacZ
contain 3 and 11 artificial introns, respectively, which are not
shown.
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Fig. 2.
Localization of CePPEF
promoter activity and CePPEF-GFP fusion proteins using transgenic
C. elegans. In each panel, anterior is to
the right. A and B, analysis of FL-GFP transgenic
C. elegans shows the CePPEF-GFP fusion protein localizing to
anterior sensory cilia (arrows), to the cell bodies of
several anterior neurons surrounding the nerve ring, and to a subset of
axons extending posteriorly and dendrites extending anteriorly.
C, at high magnification, the extreme anterior end of an
FL-GFP transgenic animal demonstrates intense staining in the cilia of
BAG, AWB, and AWC (arrow). D and E,
the FL-GFP transgene is also expressed in the ventral nerve cord and in
a single tail neuron (arrowhead), respectively.
F, aa-(1)-NLS-GFP-LacZ transgenic animals show GFP
fluorescence in the nuclei of a subset of neurons near the nerve ring.
Arrows indicate the location of the sensory cilia. The
bars correspond to 25 µm.
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Fig. 3.
Immunostaining with CePPEF-specific
antibodies confirms the gene expression pattern and protein
localization of CePPEF inferred from transgenic animals. Rabbit
polyclonal antibodies were generated against the C-terminal 142 amino
acids of CePPEF and affinity-purified. A, immunoblotting of
293 cells. Lane 1, untransfected cells; lane 2,
CePPEF transfected cells. The bars represent
(from top to bottom) the 97-, 66-, and 46-kDa markers. B,
immunoblotting of C. elegans lysates prepared by sonication.
Twenty micrograms of total protein were loaded in each lane. Lane
1, wild-type (N2 strain) C. elegans; lane 2,
transgenic C. elegans expressing FL-GFP; lane 3,
transgenic C. elegans expressing the G2A mutant of FL-GFP.
The bars represent (from top to bottom) the 220-, 97-, and
66-kDa markers. C, anti-CePPEF immunofluorescent staining of
wild-type C. elegans. Anterior is to the right, and the
position of the sensory cilia is indicated by the arrow.
Autofluorescence from gut granules is seen more caudally and is
indicated by the arrowhead. The bar corresponds
to 25 µm.
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Fig. 4.
The N terminus of CePPEF is necessary and
sufficient for localizing transgenic GFP fusion protein to axons,
dendrites, and cilia of AWB, AWC, and BAG. In each panel, anterior
is to the right, and the position of the sensory cilia is indicated by
an arrow. The bars correspond to 25 µm.
aa-(1)-NLS-GFP (A) and aa-(1-5)-NLS-GFP (B) show
minimal dendritic and ciliary localization; aa-(1-8)-NLS-GFP
(C) show an intermediate level of dendritic and ciliary
localization; and aa-(1-11)-NLS-GFP (D) and
aa-(1-14)-NLS-GFP (E) shows strong dendritic and ciliary
localization. The transgenic line in which Gly2 was mutated
to alanine (G2A) in the context of aa-(1-14)-NLS-GFP shows loss of
axonal, dendritic, and ciliary localization (F) (compare
with E). Also shown is transgenic C. elegans
carrying wild-type FL-GFP (G), FL-GFP carrying the G2A
mutation (H), or an FL-GFP transgene in which cysteine at
position 3 was mutated to serine (C3S) (I). G2A-FL-GFP
animals show a dramatic loss of axonal, dendritic, and ciliary
localization. C3S-FL-GFP animals show diminished axonal, dendritic, and
ciliary localization, with no evidence of the increased ciliary
concentration of CePPEF-GFP characteristic of the wild-type FL-GFP
transgenic lines (compare with G and Fig.
2B).
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Fig. 5.
CePPEF expressed in 293 cells is associated
with the membrane fraction. 293 cells were transiently transfected
with wild-type CePPEF cDNA (WT) or with the
corresponding G2A or C3S mutants. Twenty-four hours after transfection,
the cells were harvested, and a post-nuclear lysate (L) was
centrifuged at 58,450 × g for 30 min to yield
supernatant (S) and pellet (P) fractions. Equal
fractions of each was subjected to SDS-PAGE and immunoblotting using
affinity-purified antibodies directed against the CePPEF C
terminus.
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Fig. 6.
Essential role of Gly2 and
Cys3 in membrane localization of CePPEF in transiently
transfected 293 cells. 293 cells were cotransfected with GFP and
either wild-type (WT) or mutant CePPEF cDNA. Transfected
cells were fixed, permeabilized, and immunostained using the
affinity-purified CePPEF C terminus-specific antibody, a biotinylated
secondary antibody, and Texas Red-streptavidin. Wild-type CePPEF
localize efficiently to the plasma membrane, but each of the CePPEF
point mutants localize to the cytosol. Fluorescent images were obtained
by confocal microscopy at a 1-µm thickness.
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Fig. 7.
The N terminus of CePPEF is sufficient to
direct membrane localization of a GFP fusion protein in transiently
transfected 293 cells. A fusion protein consisting of the
N-terminal 14 amino acids of CePPEF fused to GFP shows efficient plasma
membrane localization (WT). Two single mutations (G2A and
C3S) and one double mutation (G2A/C3S) in the CePPEF portion of
aa-(1-14)-GFP cause the fusion protein to localize primarily in the
cytosol. Fluorescent images were obtained by confocal microscopy at a
1-µm thickness.
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Fig. 8.
CePPEF is both myristoylated and
palmitoylated in transiently transfected 293 cells. 293 cells
transfected with cDNA encoding wild-type (WT) or mutant
CePPEF carrying a C-terminal triple-Myc tag were metabolically labeled
with either [3H]myristic acid or
[3H]palmitic acid. Cell lysates were immunoprecipitated
with anti-Myc antibodies and subjected to SDS-PAGE for analysis by
either autoradiography (A) or immunoblotting with CePPEF C
terminus-specific antibodies (B).
Summary of wild-type and mutant CePPEF localization and fatty acid
incorporation
indicates low
but detectable levels of membrane association.
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Fig. 9.
The CePPEF C terminus binds between one and
two Ca2+ ions through two consensus EF-hands.
A, the EF-hand consensus sequence (28, 29) (J and
O represent hydrophobic and oxygen-containing amino acid
side chains, respectively); putative EF-hand sequences at the C termini
of RdgC, the human PPEFs (hPPEF), and CePPEF; and mutations introduced
into the first EF-hand (EF1'), the second EF-hand (EF2'), and both
EF-hands (EF1'+2') of CePPEF in the context of a MBP fusion to the
C-terminal 142 amino acids of CePPEF. Dashes indicate the
identical amino acid as the wild-type CePPEF (WT). EF1'
contains mutations in two predicted Ca2+-liganding
residues, and EF2' contains mutations in two predicted
Ca2+-liganding residues and a conserved glycine residue.
B, each fusion protein was expressed in E. coli,
purified to near homogeneity on an amylose resin, and analyzed for
45Ca2+ binding by rapid passage through a gel
filtration column in calcium-free buffer. Continuous lines
are best fitting second-order polynomials. C, the wild-type
CePPEF C-terminal fusion protein was also analyzed after rapid passage
through a gel filtration column pre-equilibrated in a solution
containing the identical concentration and specific activity of
Ca2+ as the binding mixture. The continuous line
represents fitting of the data to the Aldair-Klotz model for two
binding sites. First and second macroscopic binding constants are
given.
-adrenergic (29) and
-aminobutyric acid (30) receptors and of
32PO
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of calcineurin by the
EF-hand-containing
-subunit (38, 39).
-subunits,
non-receptor tyrosine kinases, and G protein regulators, have been
reported to carry either palmitate or myristate at their N termini
(43-47). However, only a few of these molecules are both myristoylated
and palmitoylated. Examples of such doubly modified proteins are
G
i, G
o, and Fyn (46-48). The functional
consequence of having both modifications is unclear, as palmitoylation
alone suffices for efficient membrane association (40, 41, 49). One
possibility is that, in C. elegans, palmitoylation of CePPEF may be heterogeneous such that one subset of CePPEF proteins is palmitoylated and strongly membrane-associated, and a second subset of
CePPEF proteins carries only a myristate group and can therefore shuttle between the membrane and cytosol. A variation on this theme is
suggested by the observation that palmitoylation is a dynamic and
regulated process (50-53). For example, activating G
s
by exposing cells to the
-adrenergic agonist isoproterenol or to
cholera toxin accelerates G
s depalmitoylation (54, 55), whereas activating platelets with thrombin leads to rapid
palmitoylation of several proteins (50). Our observation that CePPEF
immunoreactivity in wild-type nematodes and GFP fluorescence in
full-length CePPEF-GFP (FL-GFP) transgenic animals encompass both the
cell soma and membrane-rich subcellular compartments such as dendrites
and cilia is consistent with the possibility that there may be
populations of CePPEF proteins that are differentially targeted.
s and GAP-43 have been shown to activate or inhibit
protein-protein interactions (56, 57). It is of interest that other
PPEF family members differ with respect to the two fatty acylation
consensus sites within their N-terminal sequences:
Drosophila RdgC lacks both palmitoylation and myristoylation
consensus sites; human PPEF-1 has both sites; and human PPEF-2 has only
the myristoylation site. Thus, the degree of membrane association or
other characteristics referable to fatty acylation are likely to differ
among PPEF family members.
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ACKNOWLEDGEMENTS |
---|
We thank Andrew Fire for the C. elegans-GFP reporter constructs and advice on their use; Geraldine Seydoux for critical advice on C. elegans-related techniques; Cornelia Bargmann for identifying GFP-expressing neurons in our transgenic worm lines; John Williams and Phil Smallwood for assistance in cloning the CePPEF genomic locus and preparing CePPEF-specific antibodies; and Nupur Thekdi, Amir Rattner, Hui Sun, and Geraldine Seydoux for critical comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by the Howard Hughes Medical Institute.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.
§ Trainee of the Visual Neurosciences Training Program and the Medical Scientist Training Program.
To whom correspondence and reprint requests should be
addressed: 805 PCTB, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Tel.: 410-955-4679; Fax: 410-614-0827; E-mail: jnathans@jhmi.edu.
Published, JBC Papers in Press, April 18, 2001, DOI 10.1074/jbc.M011712200
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ABBREVIATIONS |
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The abbreviations used are: CePPEF, C. elegans protein phosphatase with EF-hands; FL-GFP, full-length green fluorescent protein; kb, kilobase pair(s); aa, amino acid(s); NLS, nuclear localization signal; PAGE, polyacrylamide gel electrophoresis; MBP, maltose-binding protein; PBS, phosphate-buffered saline.
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