(Received for publication, December 8, 1995; and in revised form, February 9, 1996)
From the
Neurocalcins belong to a family of neuronal specific EF hand
Ca-binding proteins defined by recoverin. Previously,
we reported the cloning and initial characterization of neurocalcin in Drosophila melanogaster (Teng, D. H.-F., Chen, C.-K., and
Hurley, J. B.(1994) J. Biol. Chem. 269, 31900-31907). We
showed that the Drosophila neurocalcin protein (DrosNCa) is
expressed in neurons and that bacterially expressed recombinant DrosNCa
(rDrosNCa) can be myristoylated. Here, we present two lines of evidence
that DrosNCa is fatty acylated in vivo. First, the mobility of
affinity-purified native DrosNCa on two-dimensional gel electrophoresis
is identical to that of myristoylated rDrosNCa and distinct from that
of nonacylated rDrosNCa. Second, the membrane binding properties of
native DrosNCa are similar to those of myristoylated rDrosNCa; both of
these proteins bind to membranes at 0.2 mM Ca
, whereas nonacylated rDrosNCa always remains
soluble. It has been shown that recoverin inhibits the phosphorylation
of rhodopsin when Ca
is present (Kawamura et
al., 1993) and that a dependent recoverin/rhodopsin kinase
interaction underlies the inhibitory effect of recoverin (Chen et
al., 1995). Given the similarities between recoverin and
neurocalcin, we examined the effect of DrosNCa on rhodopsin
phosphorylation. We find that rDrosNCa is capable of inhibiting bovine
rhodopsin phosphorylation in vitro in a
Ca
-dependent manner. The inhibitory activity of
rDrosNCa is enhanced by myristoylation, and the potency of its effect
is similar to that of recoverin. Two other related EF hand proteins,
guanylate cyclase-activating protein-2 and calmodulin, are only poor
inhibitors in these phosphorylation assays. in vitro inhibition of rhodopsin phosphorylation therefore appears to be an
assayable property of a subset of recoverin-like proteins.
Neurons are highly specialized cells that have evolved elaborate
mechanisms to sense, integrate, and transmit signals. The calcium ion
(Ca) is a universal second messenger that is a key
player in neuronal signaling. Dynamic fluxes in the intracellular
concentration of Ca
during neuronal excitation,
recovery, and adaptation or potentiation, are interpreted by
Ca
-binding proteins that act as transducers; the
binding or release of Ca
causes these proteins to
switch into an ``on'' or ``off '' state in the
regulation of effector activity.
Many Ca-sensing
transducers are members of the EF hand superfamily of
Ca
-binding proteins (Moncrief et al., 1990),
one of the best characterized being calmodulin. In the past few years,
a new and rapidly expanding EF hand subfamily has been defined by
recoverin and its cognates (Dizhoor et al., 1991; Hurley et al., 1993). Electrophysiological analyses on mammalian
recoverin and frog S-modulin reveal that these photoreceptor proteins
prolong photoexcitation in high free Ca
concentrations (Gray-Keller et al., 1993; Kawamura,
1993). Accumulated biochemical evidence suggests that
recoverin/S-modulin delays photorecovery by preventing rhodopsin kinase
from phosphorylating rhodopsin in high Ca
conditions
(Kawamura et al., 1993; Chen et al., 1995; Klenchin et al., 1995). Indeed, Chen and co-workers(1995) have recently
found that recoverin directly binds to rhodopsin kinase at high
Ca
levels. Recoverin is heterogeneously fatty
acylated on its amino terminus (Dizhoor et al., 1992), and
this posttranslational modification enhances the association of
recoverin with membranes in high Ca
conditions
(Dizhoor et al., 1993). However, the presence of a fatty acyl
moiety on recoverin is not necessary for its inhibition of rhodopsin
kinase (Chen et al., 1995). Flaherty et al.(1993)
have shown that the crystallographic structure of recoverin, with
Ca
bound in its EF3 site, is compact and that the
protein is composed of two domains, each having one functional EF hand.
The three-dimensional structure of recoverin also reveals the presence
of a hydrophobic crevice composed of aromatic and aliphatic amino acid
residues distributed in the N-terminal half of the protein that may be
involved in interactions with a target factor(s). Interestingly, Polans et al.(1991) have found that recoverin is an antigen in the
autoimmune response of people inflicted with cancer-associated
retinopathy, but its role in this degenerative disease is unclear.
Numerous vertebrate cognates of recoverin have been identified
including visinin (Yamagata et al., 1990), multiple isoforms
of neurocalcin (Hidaka and Okazaki, 1993; Terasawa et al.,
1992), hippocalcin (Kobayashi et al., 1992), and neuronal
visinin-related proteins like Vilip (Lenz et al., 1992) and
NVPs (Kajimoto et al., 1993). Protein sequence comparisons
indicate that recoverin, S-modulin, and visinin define a distinct
subclass that is expressed in photoreceptor cells. Another subclass of
these neuronally expressed Ca-binding proteins is
comprised of bovine neurocalcin, rat hippocalcin, rat NVPs, and chicken
Vilip. The evolutionary diversity of these EF hand recoverin-like
proteins is further exemplified by the recent discoveries of yet
another subclass defined by GCAP-1 (Palczewski et al., 1994)
and GCAP-2 (Dizhoor et al., 1995), two mammalian photoreceptor
proteins that activate membrane guanylate cyclase when the free
Ca
levels are low.
Two invertebrate homologues of
recoverin have been reported to date: Drosophila frequenin and
neurocalcin. Pongs et al.(1994) identified frequenin from
studies on the Shaker-like V7 mutants of D. melanogaster. They
proposed that the overexpression of frequenin in V7 mutants causes the
augmented facilitation of neurotransmitter release at neuromuscular
junctions. In addition, Pongs et al. reported that recombinant
frequenin is capable of activating bovine rod outer segment membrane
guanylate cyclase in a Ca-dependent manner in
vitro, but the in vivo target of frequenin has not been
established. In a search for homologues of recoverin in D.
melanogaster, we discovered Drosophila neurocalcin (nca), a gene coding for a protein that is 88% identical
to the bovine neurocalcin
isoform on the primary sequence level
(Teng et al., 1994). Initial characterization revealed that
the Drosophila neurocalcin protein (DrosNCa) is expressed in
neurons and that bacterially expressed recombinant DrosNCa could be
myristoylated by N-acyl transferase. Here, we present
evidences that native DrosNCa is fatty acylated in vivo and
that this modification significantly enhances its
Ca
-dependent association with Drosophila membranes. In addition, we have found that at high Ca
concentrations in reconstituted assays, both recombinant
myristoylated and nonacylated DrosNCa are capable of inhibiting the
phosphorylation of bovine rhodopsin by rhodopsin kinase, but the
recombinant DrosNCa proteins neither stimulate nor inhibit mammalian
photoreceptor membrane guanylate cyclase in high or low Ca
conditions.
Figure 1:
Analyses of
myristoylated and nonacylated rDrosNCa by gel electrophoresis. A, native gel analysis. 2.5 µg of purified non-rDrosNCa (n) or myr-rDrosNCa (m), in the presence of 20 mM -mercaptoethanol and either 3 mM EGTA or 1 mM CaCl
, was resolved on a 10% polyacrylamide gel. B, SDS-polyacrylamide gel electrophoresis in reducing
conditions. 1.5 µg of non-rDrosNCa or myr-rDrosNCa, boiled in SDS
sample buffer containing 200 mM
-ME and either 3 mM EGTA or 1 mM CaCl
, was resolved on a 15%
SDS-polyacrylamide gel. Molecular weight (MW) standards are
shown, and their corresponding sizes are indicated in kDa. C,
SDS-PAGE in nonreducing conditions. 25 µg of recombinant protein
was analyzed as described in B using SDS sample buffer without
-ME. All of the protein bands were visualized by staining with
Coomassie. Using liquid chromatography-coupled electrospray mass
spectrometry, the extent of myristoylation of the purified recombinant
protein preparations was determined to be greater than
90%.
When myr-rDrosNCa and non-rDrosNCa were subjected to SDS-PAGE
without -mercaptoethanol, the formation of dimers was detected (Fig. 1C). Under these nonreducing conditions, multiple
forms of monomeric (arrows) and dimeric (arrowhead)
DrosNCa were observed, and their mobilities differed from those of the
-ME-treated proteins (Fig. 1B). Considering that
three cysteine residues are present in DrosNCa, these results suggest
that rDrosNCa can form intra- and/or intermolecular disulfide bonds.
Curiously, the distribution of the monomeric and dimeric species for
myr- and non-rDrosNCa were different, suggesting that lipid
modification could influence the formation of the disulfide linkages.
Figure 2:
Tryptophan fluorescence. Fluorescence
emission spectra of 1 µM of myr-rDrosNCa (A) and
non-rDrosNCa (B), excited at 292 nm in buffers containing
either 1 mM HEDTA (solid line) or 0.1 mM CaCl (dashed line), are
shown.
Figure 3: Purification of native Drosophila neurocalcin. A, native DrosNCa was purified from a soluble extract of Canton-S adult heads by affinity chromatography using an AP6515 anti-DrosNCa antibody column. Fractions obtained from this purification scheme were subjected to SDS-PAGE on a 15% gel that was stained with Coomassie Blue. Lane 1, molecular mass standards (sizes indicated are in kDa); lane 2, 40 µg of protein from the Canton-S adult head extract; lane 3, approximately 1 µg of protein recovered in the low pH elution. B, Western blot. The following fractions were separated on a 15% SDS-polyacrylamide gel, transferred onto nitrocellulose, and probed with 0.2 nM AP6094. Lane 1, 40 µg of protein from the soluble Canton-S head extract; lane 2, 40 µg of protein from the flow-through fraction of the AP6515 column; lane 3, 4 µl of the low pH eluate. Based on densitometric scans and comparison to signals of rDrosNCa standards, it was estimated that there was originally 12.5 µg of DrosNCa in the 38 mg of protein in the Drosophila head extract, and approximately 1.7 µg of DrosNCa was recovered in the low pH elution. This corresponds to a yield of 14% from the antibody column.
Two-dimensional gel electrophoresis was
performed to determine if native DrosNCa behaved like myr-rDrosNCa or
non-rDrosNCa. As shown in Fig. 4A, myr-rDrosNCa
resolved at a more acidic pH relative to non-rDrosNCa in the IEF
dimension, and myr-rDrosNCa migrated slightly faster than non-rDrosNCa
in the SDS-PAGE dimension as previously observed in Fig. 1. We
found that in IEF gels having different gradients of ampholytes,
myr-rDrosNCa consistently resolved at a more acidic pH relative to the
5.2 pI, 45-kDa standard (arrow), whereas non-rDrosNCa settled at a more
basic pH. This result is consistent with the fact that a fatty acyl
modification on DrosNCa results in the loss of one positive charge in
the -amine group of the N-terminal glycine residue. The apparent
pI values of both rDrosNCa proteins are close to the calculated pI of
5.1 for the deduced nca polypeptide product.
Figure 4: Two-dimensional gel electrophoresis. A, a mixture of 1.5 µg of non-rDrosNCa (n), 1.5 µg of myr-rDrosNCa (m), and 8 µg of two-dimensional molecular mass standards was first resolved in an IEF gel and then separated on a 15% SDS-polyacrylamide gel; the proteins were stained with Coomassie Blue. B, two-dimensional Western blot of 40 µg of total protein from a soluble fraction of Canton-S adult heads homogenized in 0.5% SDS and 5 mM EGTA. C, two-dimensional immunoblot of a mixture of 5 ng of non-rDrosNCa, 5 ng of purified native DrosNCa, and 8 µg of two-dimensional molecular mass standards. D, a mixture of 5 ng of myr-rDrosNCa, 5 ng or purified native DrosNCa and 8 µg of two-dimensional standards. The blots shown in B-D were initially stained with Ponceau-S to visualize the proteins, and subsequently probed with AP6094 primary antibody and a goat anti-rabbit HRP-conjugated secondary antibody and detected by using chemiluminescence. All of the two-dimensional gels were resolved in the presence of 2.5 mM EGTA.
Previously, we had reported that AP6094 weakly cross-reacted to rat hippocalcin. This observation raised the possibility that these antibodies would detect homologues or different isoforms of DrosNCa in fly preparations. We therefore performed Western analysis on proteins extracted from Drosophila adult heads that had been separated on a two-dimensional gel. As shown in Fig. 4B, only one spot was recognized by AP6094 in the immunoblot. Under these two-dimensional gel conditions, rat hippocalcin was clearly separated from myr-rDrosNCa and non-rDrosNCa (data not shown). Thus, these data suggest that AP6094 specifically recognizes a single antigen in Drosophila adult heads, that being native DrosNCa.
To ascertain if the gel mobility of purified native DrosNCa was similar to either myr-rDrosNCa or non-rDrosNCa, mixtures of equivalent quantities of native and recombinant DrosNCa were separated on two-dimensional gels, transblotted, and probed with AP6094. Whereas non-rDrosNCa and the native protein resolved as two distinct spots (Fig. 4C), the immunoblot signal for myr-rDrosNCa and the native protein comigrated (Fig. 4D). From these results, we infer that DrosNCa is fatty acylated in vivo.
Figure 5:
Ca-dependent
translocation of DrosNCa. A, subcellular fractionation of
native DrosNCa. Wild type Drosophila Canton-S heads were
homogenized in 25 mM Tris (pH 7.5), 10 mM NaCl, 2
mM MgCl
, 5 mM
-ME, 0.2 mM PMSF containing either 2.5 mM EGTA or 0.2 mM CaCl
. The homogenates were centrifuged, and the
resulting pellet (P) and supernatant (S) fractions
were analyzed on immunoblots using AP6094 anti-DrosNCa antibodies.
Densitometric scans indicated that 70% of DrosNCa was present in the
supernatant fraction of the EGTA sample. B, membrane
association of myr- or non-rDrosNCa. Two hundred nanograms of
recombinant protein was reconstituted with washed Canton-S head
membranes in membrane binding buffer solution containing either 2
mM EGTA or 0.2 mM CaCl
and centrifuged,
and the pellet (P) and supernatant (S) fractions were
examined by Western analyses using AP6094 antibodies. As a control, 200
ng of myr-rDrosNCa in solution containing 0.2 mM CaCl2 without Drosophila head membranes remained soluble under these
conditions (myr-control). To ensure that the immunoblot
signals observed in the samples were solely from recombinant DrosNCa,
the absence of detectable native DrosNCa in the washed Canton-S head
membranes (M) was ascertained.
The
involvement of the fatty acyl moiety of DrosNCa in translocation was
investigated by comparing the membrane binding abilities of
myr-rDrosNCa and non-rDrosNCa. Briefly, recombinant protein was
reconstituted with washed Drosophila head membranes and
centrifuged. The resulting pellet and supernatant fractions were
examined by Western analysis (Fig. 5B). In the presence
of 0.2 mM CaCl, most of the myr-rDrosNCa was
located in the pellet, whereas it was almost completely soluble in the
presence of EGTA. In contrast, non-rDrosNCa was always found in the
supernatant in the presence or absence of Ca
. These
results show that the association of recombinant DrosNCa to the
particulate fraction is dependent on its fatty acyl modification.
Figure 6:
Ca-dependent inhibition
of the phosphorylation of bovine rhodopsin. Phosphorylation assays were
performed as described under ``Experimental Procedures.'' The
final concentrations of ATP, recombinant bovine RK, and urea-stripped
bovine rod outer segment membranes in the reactions were 500
µM, 200 nM, and 20 µM, respectively.
The reaction contents were separated on a 12% polyacrylamide gel and
autoradiographed, and the rhodopsin bands were cut and counted.
Light-dependent phosphorylation was determined by substraction of the
-light control to the +light sample. For normalization, the
total number of phosphates incorporated into rhodopsin in the RK
control (-rDrosNCa) was taken to be 100%, and rhodopsin
phosphorylation in the presence of the test proteins was calculated
relative to the control. A, autoradiograph showing the
Ca
-dependent inhibition of bovine rhodopsin
phosphorylation by non- and myr-rDrosNCa. The phosphorylation reactions
were performed in the presence of 1 mM EGTA (EGTA) or 0.1
mM CaCl
(Ca
) with or without 5
µM of non- or myr-rDrosNCa. The RK band corresponds to
autophosphorylated RK. B, the effect of increasing the
concentration of myristoylated (closed shapes) and nonacylated
(open shapes) rDrosNCa (squares) or rBovRv (circles)
on rhodopsin phosphorylation. Calmodulin (open diamonds) and
myristoylated recombinant GCAP-2 (closed triangles) were used
as controls. The final concentration of Ca
in these
reactions was 0.1 mM. The data are the mean of at least three
independent determinations, and error bars represent the
standard deviation.
In this paper, we present two lines of evidence indicating
that Drosophila neurocalcin is a Ca-binding
protein that is fatty acylated in vivo. First, on
two-dimensional gels, native DrosNCa comigrates with myristoylated
rDrosNCa and is distinctly separated from nonacylated rDrosNCa. Second,
the membrane binding properties of native DrosNCa are similar to those
of myr-rDrosNCa; both proteins bind to membranes in high Ca
conditions and become more soluble when free Ca
levels decrease. This study therefore provides the first evidence
of the existence of protein acylation in Drosophila.
Definitive elucidation of the type of lipid modification(s) on DrosNCa in vivo will have to be obtained by mass spectrometry
analyses.
The Ca-dependent membrane association
ability of rDrosNCa is greatly enhanced by fatty acylation. It is
likely that DrosNCa translocation occurs by the
Ca
-myristoyl switch mechanism that has been proposed
for recoverin (Zozulya and Stryer, 1992; Dizhoor et al., 1993;
Hughes et al., 1995; Tanaka et al., 1995). In this
model, the fatty acyl moiety of the Ca
-free form of
the protein resides in a hydrophobic pocket composed of aromatic and
aliphatic amino acid residues distributed in the N-terminal half of the
protein. The binding of Ca
to recoverin induces the
extrusion of its lipid moiety and N-terminal
-helix into solution,
thereby unmasking the hydrophobic pocket; this conformation favors
interactions with membranes, cytoskeleton, and/or a target factor(s).
The fluorescence of rDrosNCa in the presence and absence of
Ca
is consistent with the
Ca
-myristoyl switch model. First, the variation of
fluorescence of myr-rDrosNCa seems to be mainly due to a movement of
its myristoyl group, since no major fluorescence change occurs upon
Ca
-binding to non-acylated rDrosNCa. Second, the
fluorescence of myr-rDrosNCa in its Ca
-bound form is
very similar to that of the protein lacking the myristoyl modification.
Taken together, these observations suggest that the observed
fluorescence change of myr-rDrosNCa upon Ca
binding
is due to the movement of its fatty acyl moiety from a protein
environment into solution.
In this paper, we report that DrosNCa is capable of
inhibiting the phosphorylation of bleached rhodopsin in reconstituted
assays in a Ca-dependent manner. We show that its
potency is similar to that of recoverin and that its inhibitory
activity, like that of recoverin, is enhanced by myristoylation. Our
results on BovRv are in agreement with the findings of Chen et
al.(1995) but in contrast to Calvert et al.(1995), who
report that myristoylated and nonacylated BovRv have similar inhibitory
potencies. It seems plausible that differences between our experimental
procedures and those of Calvert et al. may account for the
discrepancies in results. Three experimental differences stand out in
particular: (i) in Calvert et al., 100% of the rhodopsin was
bleached during the experiment, whereas we bleached only 0.05% of the
rhodopsin; (ii) we used recombinant rhodopsin kinase that is produced
using the baculovirus-Sf9 expression system, whereas Calvert et al. extracted the enzyme from bovine photoreceptors; and (iii), the
methods of purifying rhodopsin kinase differed, e.g. the
rhodopsin kinase preparations of Calvert et al. included
transducin, arrestin, and 0.4% Tween 20, whereas our protein
preparations were highly purified and did not contain detergent. We
have found that detergent can interfere with the
Ca
-dependent association of rhodopsin kinase and
recoverin. (
)
Although our experiments do not explore the
mechanism by which DrosNCa inhibits rhodopsin phosphorylation by
rhodopsin kinase, the similarity between the inhibitory effects of
DrosNCa and recoverin suggests that these two proteins act through the
same mechanism(s). It has been shown that recoverin and rhodopsin
kinase associate in a Ca-dependent manner and that
this interaction is required for the inhibitory effect of recoverin on
rhodopsin phosphorylation (Chen et al., 1995). One might
therefore postulate that a direct interaction between DrosNCa and
rhodopsin kinase is necessary for the inhibition of rhodopsin
phosphorylation by DrosNCa. Further investigations are required to test
this hypothesis. Ca
-dependent inhibition of rhodopsin
phosphorylation has also been observed using another recoverin cognate,
hippocalcin,
but other recoverin-related proteins like
GCAP-2 or the more distant calmodulin are only poor inhibitors in these
phosphorylation assays. As such, the capacity of in vitro inhibition of rhodopsin phosphorylation appears to be a newly
discovered property of a subset of recoverin-like proteins including
recoverin, neurocalcin, and hippocalcin. This is further supported by
recent evidence that neuronal recoverin-like proteins of the neuronal
calcium sensor family inhibit in vitro phosphorylation of
rhodopsin (De Castro et al., 1995).
Rhodopsin kinase
belongs to a subfamily of serine/threonine kinases that desensitize
activated seven-transmembrane receptors involved in signal
transduction. Cognates of rhodopsin kinase include mammalian ARK1,
ARK2, and G protein-coupled receptor kinases 4-6, as well as Drosophila G protein-coupled receptor kinases 1-4
(Cassill et al., 1991; Inglese et al., 1993). Given
that recoverin-like proteins colocalize with homologues of rhodopsin
kinase in neuronal tissues and that several cognates of recoverin
inhibit rhodopsin phosphorylation in a Ca
-sensitive
manner, it is tempting to speculate that members of these two protein
subfamilies interact to confer Ca
-dependent
attenuation to seven-transmembrane receptor quenching events. However,
at least two issues raise questions about the ability of recoverin-like
proteins to control receptor activity in vivo. First,
recoverin and its cognates appear to be exclusively expressed in
neuronal tissues, whereas GRKs and seven-transmembrane receptors are
present in non-neuronal cells as well. Second, the documented data on
the inhibitory effects of recoverin and its cognates are based on in vitro studies using mammalian rhodopsin kinase. If the
recoverin and rhodopsin kinase subfamilies interact as proposed, one
should be able to demonstrate that recoverin-like proteins also inhibit
ARK1,
ARK2, and/or G protein-coupled receptor kinases
4-6. So far, Chen et al.(1995) have found that recoverin
cannot inhibit
ARK1 in reconstituted assays.
Neuronal signaling
is attenuated by mechanisms involving the universal second messenger
Ca. Recoverin and its homologues probably function as
Ca
-sensing regulators in neuronal processes, but
their precise mode(s) of action remains unresolved. It is plausible
that each recoverin-like protein differentially controls a receptor
kinase. Alternatively, it is conceivable that these EF hand proteins,
like calmodulin, may interact with multiple targets. Indeed, multiple
proteins have been reported to associate with recoverin and neurocalcin
in a Ca
-dependent manner (Chen et al., 1995;
Okazaki et al., 1995). Further studies are therefore required
to elucidate the physiological roles of this diverse class of
Ca
-binding proteins.