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INTRODUCTION |
Calcium regulates a number of intracellular activities in
stimulated cells by associating with specific motifs in calcium-binding proteins. In some cases the calcium-binding motif is embedded in an
enzyme or structural protein that is directly regulated by calcium.
However, in the case of the important signaling protein calmodulin,
calcium first binds to calmodulin and then the calcium-calmodulin complex binds to and regulates a wide variety of "target" proteins. Copines, proteins of unknown function first described in
Paramecium (1, 2), are expressed from multigene families in
plants, animals, and protozoa. They bind phospholipid membranes through the action of two "C2 domains" in the N-terminal portion that are
activated by calcium. The C-terminal half of the copine molecule is
distantly related to the "A domain" (3) (or "I domain") that
enables the extracellular portion of integrins to bind extracellular matrix proteins. The binding of the integrin A domain to matrix proteins is dependent upon the presence of a bound magnesium or manganese ion through a mechanism that has been termed MIDAS for metal-induced adhesion site (3). Two lines of evidence have led to the
hypothesis that the copine A domain may also be involved in targeted
protein-protein interactions. First, critical residues involved in the
chelation of Mg2+ are conserved in the copine molecule (1),
and second, native copine I binds Mg2+ and Mn2+
(4). It is, however, unknown whether the copine A domain is in fact a
protein-protein interaction motif. We report here, based on in
vivo and in vitro evidence obtained by yeast two-hybrid screening and "pull-down" experiments using immobilized copine partners, that the copine A domain binds to a number of intracellular target proteins. We also demonstrate that full-length copine mediates the Ca2+-dependent association of target
proteins with phospholipids, a phenomenon that could influence the
activities and intracellular localization of the target proteins. The
copines may thus comprise another pathway for calcium signaling to
proteins involved in a wide range of biological activities including
growth control, exocytosis, mitosis, apoptosis, gene transcription, and
cytoskeletal organization.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screen--
A mouse embryo cDNA library (5)
in vector pVP16 was used in a yeast two-hybrid screen based on the
Clontech GAL4 two-hybrid System 3. The A domains
for human copines I, II, and IV (SER260 to the C terminus of copine I;
the homologous portions of copines II and IV) were subcloned from EST
clones obtained from the American Type Culture Collection into the bait
vector pGBKT7 (Clontech). Library and bait
cotransformants of yeast strain AH109 (Clontech) were selected for growth on His
, Ade
medium
and for development of blue color on
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside indicator plates. Approximately 8 × 105 transformants
were screened for interaction with copines I and II, and 1.6 × 106 for copine IV. Library plasmids captured from positive
clones were retransformed into AH109 transformed with the copine A
domain bait vector or with the empty bait vector to identify false
positives that were obtained at a frequency of about 25% of the true positives.
In Vitro Pull-down Assay--
Interacting cDNAs from the
two-hybrid screen were excised from pVP16 with NotI and
subcloned into pGEX4T (Clontech). The
GST1 fusion proteins were
purified by binding to agarose-glutathione beads as previously
described (4) omitting the solubilization in Sarkosyl. The interaction
of copines with MEK1 was studied using a commercial preparation of
murine MEK1-GST bound to agarose beads (Calbiochem 444952). cDNAs
encoding the A domains of copines I and IV, as used in the two-hybrid
screen, were subcloned into pET28a or pET30a (Novagen) and expressed in
Escherichia coli as His-tagged proteins. Purification was
carried out by metal chelation chromatography under denaturing
conditions in 6 M urea using nickel-nitrilotriacetic acid
columns (Novagen 70971-3) according to the manufacturer's protocol.
Fractions containing the purified A domains as assessed by SDS-PAGE (6)
were combined and renatured by slowly removing the urea by dialysis
against decreasing concentrations of urea in 50 mM
Tris-HCl, 50 mM KCl, 2 mM MgCl2,
and 1 mM dithiothreitol. Pull-down experiments were carried
out by incubating the GST target domain fusion proteins bound to
agarose-glutathione beads with the His-tagged copine A domains for
2 h on a shaker at room temperature in 130 µl of 140 mM NaCl, 10 mM Hepes-NaOH, pH 7.4, 2 mM MgCl2, 0.1% Triton X-100. MgCl2
was omitted from this buffer and from the dialysis buffer in
experiments aimed to test the Mg2+ sensitivity of the
binding. Because of the variable efficiency of binding of GST-tagged
proteins to agarose-glutathione beads, the amount of beads used for
each protein was varied between 30 and 130 µl to obtain similar
amounts of protein as judged by SDS-PAGE (roughly equivalent to a
10-µg band of bovine serum albumin). Blank beads were used when
necessary to make up the volume of beads to 130 µl. The amount of
copine A domain ranged from 3 to 9 µg (7). After incubation, the
supernatant was withdrawn and the beads were washed three times with 1 ml of the same buffer containing no copine. Separation of beads from
buffer was achieved by sedimentation at 100 × g for 1 min. Finally, proteins were eluted from the beads with electrophoresis
sample buffer and loaded on SDS-PAGE gels. To reveal the presence of
His-tagged proteins, gels were transferred to nitrocellulose membranes
and probed with 1:1,000 commercial mouse monoclonal anti-His antibodies
(Novagen 70796-3). Detection was carried out using 1:10,000 polyclonal goat anti-mouse, peroxidase-labeled antibodies (American Qualex), and a
chemiluminescence kit (Pierce Supersignal). In addition to GST alone as
a control (Fig. 1), two additional negative controls were used: GST
fused to residues 95-221 of the 24-kDa subunit of murine mitochondrial
NADH-ubiquinone oxidoreductase (GenBankTM Q9D6J6), and GST
fused to residues 28-171 of the murine protein of unknown function
MGC19415 (AAH11286). These constructs generated fusion proteins that
are of a size comparable with the interacting proteins but that do not
bind copines.
Protein Phosphatase 5 Activity--
Protein Phosphatase 5 was
expressed and purified as a GST fusion protein as described (4),
omitting solubilization in Sarkosyl, using the plasmid pET GST-PP5 (8)
kindly provided by Dr. Sandra Rossie (Purdue University). The
phosphatase was eluted from the agarose beads by incubation for 1 h with an equal volume of 50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione, 0.1%
-mercaptoethanol. Full-length human copine I was produced in yeast with the expression vector YEpDB60 previously used for the production of recombinant annexins (9) and the protein was isolated by
calcium-dependent binding to lipids (4). This recombinant
protein was found to have calcium-dependent lipid binding
properties indistinguishable from those of native bovine copine I (10).
Phosphatase activity was measured colorimetrically by monitoring
transformation of colorless p-nitrophenol phosphate into
yellow p-nitrophenol (11). Assays were carried out in 200 µl of 50 mM Tris-HCl, 10 mM
p-nitrophenol phosphate, 10 mM
MgCl2, 30 mM arachidonic acid, 0.1%
-mercaptoethanol, 200 µg of egg albumin, 0.5-1.8 µg of protein
phosphatase 5, and 4.5-6.8 µg of copine, full-length or A domain.
Reactions were started by the addition of the substrate, allowed to
proceed for 15 min at room temperature, and stopped with 800 µl of
0.25 M NaOH. Mixtures were centrifuged at 20,000 × g for 1 min and the absorbance of the supernatants measured
at 410 nm. Reactions were linear up to 30 min.
Phospholipid Overlay Assay--
Overlay assays were carried out
as described by Cheever et al. (12) with minor
modifications. Briefly, pieces of nitrocellulose membrane were spotted
with 1-µl (100 pmol) drops of phosphatidylserine dissolved in
1:2:0.8, chloroform:methanol:H2O and allowed to dry for
1 h. Membranes were blocked for 1 h with 3% fatty acid-free bovine serum albumin (Sigma A-8806) in TBST (10 mM
Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) Tween 20). All
subsequent incubations and washes were carried out in TBST containing
0.5% fatty acid-free bovine serum albumin and 3 mM
CaCl2 or 5 mM EGTA. Blocked membranes were then
sequentially incubated for 1 h each with: 0.5 µg/ml full-length
recombinant human copine I produced in yeast, 0.01 µg/ml GST-copine
target fusion protein or GST, 1:2000 commercial rabbit polyclonal
anti-GST antibodies (Sigma G-7781) and 1:10,000 polyclonal goat
anti-rabbit, peroxidase-labeled antibodies (American Qualex). For these
experiments a full-length murine MEK1-GST fusion protein was generated
in E. coli using an expression vector kindly provided by Dr.
Thomas Sturgill of the University of Virginia. After each incubation
membranes were rinsed three times for 30 min with excess buffer.
Finally, membranes were washed with TBST containing 3 mM
CaCl2 or 5 mM EGTA and detection was carried
out using a commercial chemiluminescence kit (Pierce Supersignal) supplemented with 3 mM CaCl2 or 5 mM EGTA. All steps were carried out at room temperature.
The GST and GST fusion proteins were prepared by the method described
for protein phosphatase 5 above. For competition experiments incubation
with full-length copine I was carried out in the presence of variable
concentrations of copine I A domain (see Fig. 2) prepared as described above.
Derivation and Application of a Consensus Sequence for the
Copine-binding Coiled-coil Domain--
The amino acid sequences
encoded by the interacting cDNAs obtained in the two-hybrid screen
for the following proteins were aligned by the University of Wisconsin
Genetics Computer Group PRETTY program (13): octamer-binding protein,
radixin, bicaudal D, CDC42-binding kinase, and WTAP. The Blosum62
scoring matrix was used with a gap creation penalty of 8 and a gap
extension penalty of 2. The program was used to generate a consensus
sequence based on a parameter setting of 3 of 5 similar (nonidentical) matches. The consensus sequence obtained (see "Results" and
"Discussion") was used to probe Release 72 of the Protein
Information Resource of the National Biomedical Research Foundation
with the FASTA program (14) with the Blosum50 scoring matrix, gap
creation penalty of 12, and gap extension penalty of 2. The 20 closest matches included several of the proteins used to derive the consensus sequence (radixin, the CDC42-binding kinase, and octamer-binding protein), some known and unknown sequences homologous to these proteins, and three entries for the mitogen-activated protein kinase
kinase, MEK1.
General Methods--
SDS-polyacrylamide gels were run according
to Laemmli (6). Protein concentrations were determined according to
Bradford (7).
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RESULTS AND DISCUSSION |
Identification of Copine Targets by Yeast Two-hybrid
Screening--
To search for possible targets for copines we used the
isolated A domains of three of the seven human copines (copines I, II,
and IV) as baits in a yeast two-hybrid assay to screen a mouse embryo
cDNA library. Unique classes of interacting cDNAs, summarized in Table I, were isolated for each of the
three copines.
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Table I
Copine targets
COP, copine "I", "II", or "IV" A domain used to identify
the target cDNA in the initial two-hybrid screen. ID, GenBankTM
accession number for the target cDNA. If the mouse sequence as
obtained in the screen is not present in GenBankTM, the accession
number of the closest human homolog is given. NAME (SYNONYM), common
name of the target protein (synonyms given in parentheses). ISOLATES,
number of times overlapping clones were obtained for the same cDNA
in the two-hybrid screen. RESIDUES, amino acid residues encoded by the
cloned target cDNAs. If more than one clone was obtained, the
minimum overlapping sequence is represented. DOMAIN, recognized
structural domains encoded by the partial target cDNA. COILS,
graphic output of the COILS program giving the probability that the
target protein domain will form a coiled-coil. Probability from 0 to 1 is plotted as a function of residue number (as given in the column
"RESIDUES"). Dashed horizontal line represents probability of 0.5. TWO-HYBRID INTERACTION, level of interaction seen in the two-hybrid
assay with the A domains of copines I, II, or IV. Range from " "
to "++++" depending on growth rate and supression of the pink color
of ade2 mutants on adenine-deficient medium.
IN VITRO INTERACTION, ability of target protein domain fused
to GST to pull down the A domains of copines I or IV, ranging from
" " to
"++".
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Characteristic of the library used for the screen, the isolated
cDNAs are generally less than full-length (average size about 400 to 500 base pairs), of a size likely to encode only domains of the
target proteins. Each copine was found to select from the library
certain target cDNAs that dominated the screen. Screening with
copine I repeatedly identified protein phosphatase 5 (8 of 10 isolates), copine II the NEDD8-conjugating enzyme UBC12 (4 of 10 isolates), and copine IV the single stranded RNA-binding protein,
octamer-binding protein (18 of 38 isolates). In addition, a complement
of other specific targets for each copine was also observed as detailed
in Table I. Interestingly, copine I was found to be a target of copine
IV. Although it was previously demonstrated that copine I exhibits
calcium-dependent self-association in vitro (4),
interactions between different copines have not been previously
recognized. This result may indicate that copines can function as
oligomers, or that copines can regulate the activities of one another.
When the cDNAs of targets interacting with each copine were tested
in the two-hybrid assay using the other two copine A domains as bait,
it was found that some targets would interact with only a single copine
(e.g. UBC12, octamer-binding protein, and CDC42-binding
kinase) whereas others were less specific (e.g. Myc-binding
protein, radixin, and E2-230K ubiquitin-conjugating enzyme).
Binding of Copines to Target Proteins in Vitro--
The
interactions between the copine A domains and their targets were
further analyzed in an in vitro pull-down assay (Fig. 1). The cDNAs isolated in the
two-hybrid screen were subcloned into a GST fusion expression vector to
generate immobilized protein fragments to test for binding to
recombinant A domains. The copine A domains were expressed in E. coli as His-tagged fusion proteins that facilitated
isolation of the proteins and detection in the pull-down assay with
anti-His tag antibodies. However, the copine II A domain could not be
obtained in soluble form and so was not examined for in
vitro binding of target proteins. Two yeast two-hybrid clones were
excluded from this study (and from Table I) because one of them failed
to produce a fusion protein in E. coli and the other one
produced a protein that did not bind copine in vitro. All
other clones obtained in the two-hybrid screen produced fusion proteins
that bound to the copine I or IV A domains and are listed in Table I.
Many of the target proteins bound to both copine A domains, although
often with a differential affinity. Sometimes this reflected
specificity that was also apparent in the yeast two-hybrid assay (Table
I). However, in many cases the two-hybrid assay was more discriminating
than the in vitro pull-down assay.

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Fig. 1.
Binding of copine A domains to representative
copine targets in vitro. Copine target proteins
fused to GST and bound to glutathione-agarose beads were incubated with
His-tagged copine I and IV A domains. Beads were subsequently washed
and eluted with SDS-PAGE sample buffer and the extent of interaction
assessed by probing Western blots of the eluted material with anti-His
tag antibodies. Panel A, Ponceau S staining of Western blot
of extracts from beads with bound GST alone (GST), GST-UBC12
ubiquitin-conjugating enzyme (UBC), or GST- collagen
(COL) after incubation with the copine I A (I) or
IV A domains (IV). In each case the major band is the GST
target domain fusion construct. STD, molecular weight
standards, masses given in kDa. Panel B, immunostaining of
the membrane in panel A for copine A domains with an
anti-His tag antibody detected by chemiluminescence. Copine A domains
are bound to UBC12 and collagen, but not to GST alone. Panel
C, immunostaining of MEK1-GST saturated beads for bound copine.
The differential binding of copines I and IV to MEK1 is scored as ++
and +, respectively, in Table I. Incubation of the MEK1-GST saturated
beads with 10 mM glutathione blocked the association of the
copine A domains with the beads (not shown).
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To test the possibility that the interaction between the copine A
domain and the target proteins may occur through the MIDAS mechanism,
the sensitivity of the binding to the removal of Mg2+ was
tested in the in vitro pull-down assay. The binding of all of the proteins listed in Table I to the copine I A domain (or copine
IV A domain when this was the preferred partner) was tested in the
absence of Mg2+ as described under "Experimental
Procedures." In the case of collagen, interaction with the copine I A
domain was completely abolished by the absence of Mg2+,
whereas the rest of the target interactions were insensitive to
Mg2+ removal. This suggests that the MIDAS mechanism may
not be essential for most of the interactions we have observed. In the
case of collagen the in vitro interaction may be
mechanistically similar to that between the integrin A domain and
collagen, for which there is a crystallographic model (15). Because
collagen is the only extracellular copine target that was identified,
the mechanism of binding in this case may be different from the
interactions with intracellular proteins. Although the biological
significance of such an interaction is unclear because copines are
believed to be exclusively intracellular proteins, the interaction may be relevant during developmental or pathological events that involve cell lysis.
Identification of a Coiled-coil Copine-binding
Motif--
Examination of the sequences and inferred structural
features of the target domains revealed that a majority (14 of 21)
included sequences predicted to form
helical coiled-coils (Table
I). This is a much higher occurrence of coiled-coils than would be expected to occur randomly. Computer analyses of fully sequenced genomes indicates that 5-9% of the open reading frames are predicted to encode proteins that contain coiled-coils (16), and that only 2-3%
of residues in all proteins are in regions that are predicted to form
coiled-coils (17). In contrast, 34.7% of all the residues encoded by
the interacting target cDNAs obtained in the two-hybrid screen
described here are predicted to be in coiled-coils. Although the actual
structures of most of the target proteins reported here are unknown,
the N-terminal tetratricopeptide repeat domain of protein phosphatase
5, which is the binding site for copine I (see Table I), is known to
form a series of antiparallel
helices (18). Use of the COILS
program (19) to analyze the other targets revealed that many are
predicted to have regions of coiled-coil structure with short
interruptions that could represent hairpin turns (Table I). Therefore,
the copines may favor interaction with antiparallel coiled-coils, a
motif that is also the basis of the interaction of the small
GTP-binding protein Rho with its target proteins (20). Although the
coiled-coils in the copine targets may be intramolecular, it is also
possible that the target proteins formed dimers in the two-hybrid assay
and in the in vitro pull-down assay. However, the two-hybrid
assay as employed here would not have been able to reveal potential
targets that are formed by the association of different protein
subunits to create coiled-coils, as occurs in G-protein
-
dimers
and in soluble NSF attachment protein receptor (SNARE)
complexes. More direct methods must be employed to determine whether
copines may interact with such heterologous coiled-coil motifs.
Recognition of a motif that is bound by copine was found to have
predictive value for identifying copine targets that were not
identified in the two-hybrid screen. As detailed under "Experimental Procedures," a template motif was determined by aligning the
predicted coiled-coil motifs within the copine target fragments of
octamer-binding protein, radixin, CDC42-binding kinase, bicaudal D, and
WTAP and then using the PRETTY program (13) to generate a consensus
sequence (E. . . . . R..R.L.E..EQ.RK.LELR..KQR ... EL.QLD.E.E).
Probing the National Biomedical Research Foundation Protein Information
Resource data base with this sequence in the FASTA program (14)
yielded among the 20 best matches several of the proteins used to
generate the consensus sequence as well as MEK1. The portion of MEK1
identified by this alignment was residues 29-71 in the N-terminal
domain. This portion of MEK1 is strongly predicted by the COILS program (19) to form a coiled-coil. A full-length GST fusion construct of MEK1
was bound by copine I and IV A domains in the pull-down assay (Fig. 1),
verifying the predicted interaction.
Seven proteins that are not known to form coiled-coils also interacted
with copine (Table I). Some of these, such as ALG2 and
-actin, do
have significant
-helical content in the identified interaction
region. Possibly the character of these helices may allow presentation
of a structural motif similar to that involved in the binding of
coiled-coils to copines. However, further structural and deletion
analyses are needed to define these interaction sites more precisely.
Recruitment of Target Proteins to Lipids--
The binding of a
full-length copine to a target protein might have the important
consequence that the target protein would be localized to a membrane
surface in a calcium-regulated fashion by the action of the C2 domains
of copine. To test this possibility the interaction of full-length
copine I with several of the target proteins was examined in detail
using GST fusion proteins of the target cDNAs as well as GST fusion
proteins of full-length protein phosphatase 5 and full-length MEK1. The
full-length copine I was found to recruit these proteins to immobilized
phosphatidylserine in a calcium-dependent manner (Fig.
2), suggesting that copines may indeed be
able to localize their targets to membrane surfaces in the cell in
response to calcium fluxes. This activity was independent of whether or
not the target protein contained a coiled-coil motif. The recruitment
of collagen to the lipid substrate required the presence of
Mg2+ (Fig. 2), reflecting the requirement for
Mg2+ that was seen in the pull-down assay with the copine A
domain. In general, copines bind to mixed lipid systems that contain an acidic phospholipid such as phosphatidylserine, phosphatidylinositol, or phosphatidic acid (4). Copines may therefore recruit target proteins
to the cytoplasmic side of the plasma membrane, as well as to a number
of intracellular membrane systems that contain acidic lipids. However,
recruitment to the plasma membrane could be of particular significance
for protein phosphatase 5 because it has been suggested that one of its
substrates may be the plasma membrane atrial natriuretic peptide
receptor (21). In addition, the mitogen-activated protein kinase
signaling pathway might be enhanced by recruitment of MEK1 to the
complex of proteins involved in signaling from growth factor receptors
at the plasma membrane.

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Fig. 2.
Calcium-dependent recruitment of
copine-binding proteins to immobilized lipids by full-length copine
I. Phosphatidylserine spotted onto a nitrocellulose membrane was
incubated with full-length copine I (COP I FL) in the
presence of calcium followed by incubation with copine-binding proteins
expressed as GST fusion proteins. Binding was detected by
chemiluminescence using anti-GST antibodies and peroxidase-labeled
secondary antibodies. Panel A shows the results obtained
with five representative copine-binding proteins as indicated in the
header. As shown in row 1, all proteins were recruited to
immobilized lipids. In the case of -collagen (CO),
recruitment required the addition of 2 mM Mg2+
to all incubations and washes (CO+Mg). Rows 2 and
3 show that no recruitment was observed with the GST portion
of the molecule or in the absence of calcium. Rows 4 and
5 show that the recruitment depends on the presence of COP I
FL and is not promoted by the copine I A domain (COP I A).
Rows 6-8 show the phospholipid- and
calcium-dependent binding of copine to nitrocellulose
membranes. In these cases, incubation with copine-binding proteins was
omitted and copine was detected using anti-copine I antibodies (4).
Copine binding occurs only in the presence of phospholipid and calcium
(row 8). Panel B shows that the recruitment of
protein phosphatase 5 (PP5) by COP I FL and calcium is
inhibited by the presence of increasing concentrations of COP I A
domain. Labels on the right indicate the COP I A/COP I FL
concentration ratio. For both panels A and B
exposure time was the same for all spots located in each column.
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The copine I A domain by itself was unable to promote the association
of the target proteins with lipids (Fig. 2), demonstrating that this
property of copine depends upon the calcium- and lipid-binding C2
domains. Interestingly, the copine A domain was able to prevent, in a
dose-dependent manner, full-length copine I from recruiting target proteins to lipids (Fig. 2). It is likely that the A domain is
acting in these experiments in a dominant-negative fashion by competing
for the copine-binding site on the target protein and thus preventing
the full-length copine from attaching the target to lipids through the
C2 domains. The A domain construct might therefore prove to be a useful
dominant-negative probe for testing the functional significance of
target relocalization because of copine in cellular systems.
Effects on Target Protein Enzymatic Activity--
The binding of a
copine to a target protein might have a direct effect on enzymatic or
other functional properties of the target, independent of the effect on
target localization to membranes. In the case of protein phosphatase 5, the tetratricopeptide repeat domain, which is the binding site for
copine (see Table I), is known to regulate the activity of the
catalytic domain (22). In addition, mutagenesis of the coiled-coil
region in the N terminus of MEK1, where we speculate copine may bind,
is also known to influence the catalytic activity of MEK1 (23). To
determine whether copine may influence the activity of protein
phosphatase 5, the activity of the phosphatase against the model
substrate para-nitrophenyl phosphate was tested in the
presence or absence of the copine I or IV A domains or full-length
copine I. These protein constructs were all found to activate the
phosphatase by 30-40% (Table II).
Although this is a modest activation, it is possible that more
significant activation would be seen with specific protein substrates
(which are generally unknown for this phosphatase) or in the presence
of different lipid activators. As these experiments were done in the
absence of calcium, the result also demonstrates that the copine C2
domains do not have to be activated by calcium for copine to bind a
target protein. The importance of the C2 domains might therefore be
limited to their ability to attach the copine-target complex to
membranes. The possibility that the activity of MEK1 is also regulated
by copines will be important to examine.
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Table II
Activation of protein phosphatase 5 by copine
Basal phosphatase activity (100%) was 2.7 nmol/mg-min. Assay performed
in the presence of 0.5 µg of phosphatase, 6.0 µg of copine I, 4.5 µg of copine 1 A domain, 6.8 µg of copine IV A domain. Copine
preparations alone exhibited no phosphatase activity; boiling for 15 min eliminated the ability of the copine preparations to activate the
phosphatase.
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CONCLUSION |
Multiple copines are expressed in a given organism (1, 2), thus
multiple independent and branching pathways for calcium signaling may
exist within cells based on this family of proteins. Because of their
ability to associate with membranes the copines may be particularly
important for regulating signaling processes on membrane surfaces. With
the exception of collagen, all of the copine target domains identified
here are known or predicted to be on intracellular proteins or on the
intracellular portions of membrane proteins. Therefore, these targets
should be accessible to copines in vivo. In many cases the
targets are signaling molecules themselves, including calcium-binding
proteins, kinases, a phosphatase, ubiquitin (or NEDD8)-conjugating
enzymes, and transcription modulators. Mutation of a copine in
Arabidopsis has been shown to lead to alterations of growth
patterns and apoptotic responses to stress (24, 25). Possibly this
reflects alterations in calcium signaling networks based on
copine-target interactions that underlie these processes.