(Received for publication, August 8, 1995; and in revised form, October 6, 1995)
From the
The annexin family of proteins is characterized by a conserved
core domain that binds to phospholipids in a
Ca-dependent manner. Each annexin also has a
structurally distinct N-terminal domain that may impart functional
specificity. To search for cellular proteins that interact with the
N-terminal domain of annexin I, we constructed a fusion protein
consisting of glutathione S-transferase fused to amino acids
2-47 of human annexin I (GST-AINT; AINT = annexin I
N-terminal). Extracts from metabolically labeled A431 cells contained a
single protein (M
10,000) that bound to
GST-AINT in a Ca
-dependent manner. A synthetic
peptide corresponding to amino acids 2-18 of annexin I inhibited
the binding of the 10-kDa protein to GST-AINT with half-maximal
inhibition occurring at
15 µM peptide. In cellular
extracts, endogenous annexin I and the 10-kDa protein associated in a
reversible Ca
-dependent manner. Experiments with
other annexins and with N-terminal truncated forms of annexin I
indicated that the 10-kDa protein bound specifically to a site within
the first 12 amino acids of annexin I. The 10-kDa protein was purified
from human placenta by hydrophobic and affinity chromatography. Amino
acid sequence analysis indicated that the 10-kDa protein is the human
homologue of S100C, a recently identified member of the S100 subfamily
of EF-hand Ca
-binding proteins.
Annexins are a family of intracellular proteins that bind in a
reversible Ca-dependent manner to phospholipids
preferentially located on the cytosolic face of plasma membranes (see (1) and (2) for a review). Annexins are structurally
distinct from the EF-hand family of Ca
-binding
proteins. The Ca
and phospholipid binding sites
reside in a core domain that shares approximately 50% amino acid
sequence identity between different annexin gene products(1) .
Each annexin family member also contains an N-terminal domain that is
not structurally related in the different annexins(1) .
Various members of the annexin family have been implicated in a number of different intracellular processes including vesicular trafficking (3) , membrane fusion during exocytosis(4) , signal transduction(1) , and ion channel formation(5, 6, 7) . However, the physiological function has not been established for any of the annexins. The different annexin gene products show cell-specific expression(1) , thereby implying that each annexin performs a different biological function. Since the N-terminal domains are not related, this domain may impart functional specificity.
Extensive
research has focused on the interaction of annexins with phospholipid
bilayers and cell membranes(2) . The hypothesis that annexins
exert their biological effects by interacting with other proteins has
received less attention. If this hypothesis is valid, we speculate that
the annexin-protein interaction would most likely be mediated by their
unique N-terminal domains. In fact, the N-terminal domain of annexins
II and XI are involved in interactions with p11 (8) and
calcyclin(9) , respectively. Calcyclin and p11 belong to the
S100 subgroup of the EF-hand family of Ca-binding
proteins (see (10) for a review). Most S100 proteins are small (M
= 9,000-12,000), although recent
research detected functional S100 domains in profilaggrin (11) and trichohyalin(12) , which are much larger
proteins. S100 proteins have been proposed to play roles in cellular
proliferation, cellular differentiation, and inflammation, but neither
the exact physiological function nor the molecular mechanisms by which
these putative roles are accomplished are known(10) . Since
these proteins appear to lack enzymatic activity, it is widely assumed
that they bind to other cellular proteins and change their activity or
cellular location. Like the annexins, S100 proteins are expressed only
in certain cell types.
Annexins I and II have attracted considerable
interest because of their possible involvement in cell growth and
differentiation. Annexin I is phosphorylated on Tyr-21 in a
Ca-dependent manner by the epidermal growth factor
receptor protein kinase (13, 14) and at an adjacent
site by protein kinase C(15) . Annexin II is phosphorylated at
analogous sites by pp60
(16) and protein
kinase C (17) in response to extracellular effectors. The
N-terminal domain of annexin I is also the site of selective
proteolytic clips (13, 18) and of
transglutaminase-catalyzed dimerization(19) .
To test the
hypothesis that annexin I interacts with other cellular proteins via
its N-terminal domain, we searched for proteins in cellular extracts
that would bind to a fusion protein containing amino acids 2-47
of human annexin I. We detected Ca-dependent binding
of a 10-kDa protein to the fusion protein and to intact annexin I.
Amino acid sequence analysis identified the protein as
S100C(20, 21, 22) , a recently identified
member of the S100 family of proteins.
Figure 1:
Ca-dependent binding
of a 10-kDa protein to GST-AINT. Lysates from
S-labeled
A431 cells were incubated with GST or GST-AINT in the presence of
Ca
. Glutathione-agarose was added and the beads,
along with any associated proteins, were pelleted and washed by
repeated centrifugation. The proteins bound to GST (lanes a, c, and e) or GST-AINT (lanes b, d,
and f) were either analyzed directly (lanes a and b) or eluted with EGTA and then analyzed (lanes c and d). The proteins remaining associated with GST (lane
e) or GST-AINT (lane f) after extraction with EGTA also
were analyzed. Samples were analyzed by SDS-PAGE and were visualized by
autoradiography. Methods are described under ``Experimental
Procedures.'' The arrow indicates the position of the
10-kDa protein that bound to GST-AINT.
Figure 2:
Inhibition of 10-kDa protein binding to
GST-AINT by an annexin I N-terminal peptide (AI 2-18). Panel
A, lysates from S-labeled A431 cells were incubated
with GST-AINT in the presence of Ca
and either no
peptide (lane a), 100 µM AI 2-18 (lane
b), or 100 µM of a control peptide (CPEP, lane
c). The GST-AINT bound proteins were eluted with EGTA, analyzed by
SDS-PAGE, and visualized by autoradiography. Methods are described
under ``Experimental Procedures.'' The arrow indicates the position of the 10-kDa protein. Panel B,
the reactions were performed, as described in panel A, with
increasing concentrations of AI 2-18 peptide in the binding
assay. The samples were analyzed by SDS-PAGE, and the 10-kDa protein
was visualized by autoradiography. The intensity of the band at 10-kDa
was analyzed by densitometry, and the results are expressed as a
percent of the intensity observed in the absence of peptide.
Experimental points shown are the average of duplicate
reactions.
Figure 3:
An endogenous annexin I10-kDa
protein complex binds to phospholipid vesicles. Lysates from
S-labeled A431 cells were incubated with phospholipid
vesicles in the presence of Ca
. The vesicles and any
associated proteins were pelleted by centrifugation, washed in the
presence of Ca
, and then incubated with either no
peptide (lanes a and b), 100 µM AI
2-18 (lanes c and d), or 100 µM CPEP (lanes e and f). The vesicles were removed
by centrifugation, and the supernatants containing protein eluted by
the peptide were saved for analysis (lanes a, c, and e). The proteins that remained associated with the vesicle
pellet after the peptide incubation also were analyzed (lanes
b, d, and f). Methods are described under
``Experimental Procedures.'' The samples were analyzed by
SDS-PAGE, and the proteins were visualized by
autoradiography.
To
confirm that the 10-kDa protein associated with endogenous annexin I, a
polyclonal antiserum was used to immunoprecipitate annexin I from S-labeled lysates of A431 cells. In the presence of
Ca
, a 10-kDa protein co-immunoprecipitated with
antibodies to annexin I (Fig. 4, lane c), but not with
a rabbit preimmune serum control (Fig. 4, lane a). In
the presence of EGTA, no 10-kDa protein was detected associated with
immunoprecipitated annexin I (Fig. 4, lane b).
Figure 4:
Co-immunoprecipitation of a 10-kDa protein
with annexin I. Rabbit preimmune control antiserum (lane a) or
antiserum against annexin I (lanes b and c) were used
to immunoprecipitate S-labeled proteins from extracts of
A431 cells (see ``Experimental Procedures''). The incubations
were in buffers containing either Ca
(lanes a and c) or EGTA (lane b). The immunoprecipitated
proteins were resolved by SDS-PAGE and visualized by autoradiography.
Immunoprecipitated annexin I (apparent M
=
35,000) and the associated 10-kDa protein are indicated by the arrows on the right.
Figure 5:
A cellular 10-kDa protein binds to annexin
I. Lysates from S-labeled A431 cells were incubated in the
presence of phospholipid vesicles, Ca
, and the
indicated purified annexin protein (25 µg/ml). The vesicles and any
associated proteins were pelleted by centrifugation, washed in the
presence of Ca
, and the bound proteins were eluted
with EGTA (see ``Experimental Procedures''). Aliquots of the
eluted proteins were analyzed by SDS-PAGE, the gel was stained with
Coomassie Blue, and
S-labeled proteins were visualized by
autoradiography. The Coomassie Blue-stained bands confirmed that the
indicated annexins bound to the vesicles (data not shown). Additions: lane a, none; lane b, annexin I; lane c,
des-1-12-annexin I; lane d, des-1-26-annexin I; lane e, annexin V; lane f, annexin
XII.
Figure 6: Purification of GST-AINT-binding proteins from human placenta. The proteins that bound to GST-AINT were purified from extracts of human placenta, and aliquots from the purification steps were analyzed by SDS-PAGE and Coomassie Blue staining (see ``Experimental Procedures''). Lane a, proteins eluted from the phenyl-Sepharose column with EGTA. Lane b, proteins eluted from the GST-AINT affinity column with EGTA. Lanes c and d, HPLC reverse phase chromatography was used to separate the 10-kDa (lane c) and 6-kDa (lane d) proteins.
Edman degradation of the 10- and 6-kDa proteins resulted in no sequential release of phenylthiohydantoin amino acids, implying the 10- and 6-kDa proteins have blocked N termini. To obtain sequence information from the 10- and 6-kDa proteins, samples were digested with CNBr and the resulting peptide fragments were fractionated by HPLC chromatography. The two major peak fractions from the HPLC column were chosen for Edman degradation sequencing. The sequence XXELAAFT was obtained for the 10-kDa protein, and the sequence NTELAAFTKN was obtained for the 6-kDa protein (Fig. 7). Due to the exact match between these sequences, the 6-kDa protein is proposed to be a proteolytic fragment of the 10-kDa protein. Since the 6-kDa protein appeared to have a blocked N terminus, the proteolytic clip probably occurred toward the carboxyl side of the sequenced peptide. When the partial sequence of the 10- and 6-kDa proteins were compared to the protein sequence data base, an exact match was found to a member of the S100 protein family called calgizzarin from chicken (21) and rabbit(22) . The same protein from pig, termed S100C(20) , had only a single amino acid difference from the 10- and 6-kDa partial sequence. The amino acid sequence of human S100C (calgizzarin) has not been reported. However, the partial sequence and functional properties of the 10-kDa protein that binds to annexin I leave little doubt that it is human S100C (calgizzarin). Since the 6-kDa protein retains the ability to interact with GST-AINT, it appears that the annexin I binding site on S100C is located in the N-terminal half of the protein (see Fig. 7).
Figure 7: Partial amino acid sequence of the 10-kDa and 6-kDa GST-AINT-binding proteins. Peptide fragments of the HPLC-purified 10-kDa and 6-kDa proteins from placenta were generated by CNBr digestion, purified by HPLC, and subjected to Edman degradation sequencing. The same sequence was obtained for both peptides (except the first two and last cycles from the 10-kDa-derived peptide were not interpretable). The peptide sequence obtained is compared to the sequence of S100C/calgizzarin from rabbit (22) chicken (21) and pig (20) . Features of porcine, rabbit, and chicken proteins are diagrammed with the numbers corresponding to the amino acids in porcine S100C. The site of the proposed C-terminal proteolytic clip that generates the 6-kDa protein is indicated by the arrow.
We detected a single protein in extracts of cultured A431
cells that underwent reversible Ca-dependent binding
to a fusion protein containing the N-terminal domain of annexin I (Fig. 1). We purified this protein from placenta and obtained a
partial amino acid sequence, which identified it as the human form of
an S100 protein known as porcine S100C (20) and also as chicken (21) or rabbit (22) calgizzarin (Fig. 7).
Partial amino acid sequence analysis of the 10-kDa protein purified
from A431 cells (data not shown) confirmed that the placental and A431
proteins were the same protein. A previous study using labeled S100C
and a gel overlay technique detected a number of bands in extracts from
porcine heart including a 36-kDa band that may have been annexin
I(26) . We speculate that this interaction occurs in intact
cells, since the association of endogenous annexin I and S100C in
extracts of A431 cells could be detected by immunoprecipitation (Fig. 4) and by an assay that exploited
Ca
-dependent interaction of annexin I with
phospholipid vesicles (Fig. 3).
Based on the limited information that is available for S100C, there is a good correlation between the tissue-specific expression of S100C and annexin I. Both proteins are highly expressed in lung and kidney but show minimal expression in brain and liver(20, 27, 28) . We also detected a 10-kDa protein in extracts from diploid human fibroblasts (data not shown) that interacted with GST-AINT in a manner indistinguishable from the S100C protein detected in A431 cells.
Previous studies detected high affinity specific interactions
between two other members of the annexin and S100 families; annexin
II-p11 (8) and annexin XI-calcyclin(29) . Annexin I is
structurally more similar to annexin II than other annexins, and p11 is
structurally more similar to S100C than other S100 proteins. Another
similarity between annexins I and II is that they both are
phosphorylated by tyrosine kinases (the epidermal growth factor
receptor (14) and pp60(16) ,
respectively) and by protein kinase C(15, 17) .
Because of these structural and functional similarities, it is
particularly interesting to compare and contrast the annexin I-S100C
interaction with the annexin II-p11 interaction. Annexin II and p11
form a heterotetramer (2:2 molar ratio) and the site of interaction is
an amphipathic helix formed by the first 14 amino acids of annexin
II(8) . In parallel with the annexin II-p11 interaction, our
studies localized the S100C binding site to the first 12 amino acids of
annexin I (Fig. 5). Chou-Fasman analysis of the first 19
residues of annexin I suggest a high probability for
-helical
formation with residues Met-3, Val-4, Phe-7, Leu-8, Ala-11, and Ile-14
aligning onto one side of a theoretical amphipathic helix (Fig. 8). In comparison, residues Val-4, Ile-7, Leu-8, and
Leu-11 of the N-terminal domain of annexin II are similarly aligned on
one side of an amphipathic helix (Fig. 8), and these hydrophobic
residues are critical for p11 binding (8, 30) . Based
on similarities with annexin II, we speculate that the hypothetical
annexin I hydrophobic face is the binding site for S100C. The first 14
amino acids of annexin I are highly conserved from humans to sponges,
thereby indicating that this putative S100C binding site on annexin I
plays a conserved and critical role in its biological
function(1) . Since previous studies established that cellular
annexin I is sensitive to partial proteolysis at both Trp-12 (13, 18) and Lys-26(13) , it will be of
interest to determine whether the cellular interaction between annexin
I and S100C is regulated by proteolysis.
Figure 8: Helical wheel presentations N-terminal 19 residues of annexins I and II. The single-letter amino acid code for the N-terminal regions of annexins I and II are shown. Hydrophobic amino acids aligned on one side of the helix have been circled.
Although the site of interaction with S100 proteins is in the N-terminal domains of both annexins I and II, the sites of interaction with annexins on S100C and p11 appear to be in different regions of the proteins. Site-directed mutagenesis localized the annexin II binding site to hydrophobic residues in the C-terminal portion of p11(31, 32) . In contrast, the annexin I binding site of S100C appears to be in the N-terminal half of the protein as evidenced by the ability of the 6-kDa protein, which is missing the carboxyl portion of the protein, to interact with GST-AINT ( Fig. 6and Fig. 7).
Most S100
proteins that have been studied undergo a conformational change when
they bind Ca that results in the exposure of a
hydrophobic domain, which often interacts with other
proteins(10) . Consistent with these studies, we found that
S100C interacts in a Ca
-dependent manner with
endogenous annexin I ( Fig. 3and Fig. 4) and GST-AINT (Fig. 1). Since the GST-AINT construct lacks the annexin I
Ca
binding sites, we speculate that the
Ca
binding site required for this interaction resides
on S100C. Previous studies with purified S100C have shown that it binds
Ca
in vitro(21) . In contrast, the
annexin II and p11 interaction is
Ca
-independent(33) . Amino acid substitutions
and deletions in the two p11 EF-hand motifs prevent p11 from binding
Ca
(33, 34) , and these changes have
been proposed to lock p11 into an exposed conformation even in the
absence of Ca
.
Like the interaction of annexin I
with S100C, the interaction of annexin XI with calcyclin is dependent
on Ca(29) and occurs in the N-terminal half
of calcyclin (35) and within the N-terminal domain of annexin
XI(9) . Unlike annexin II and possibly annexin I, Chou-Fasman
analysis of the binding site on annexin XI indicates that it is
unlikely to adopt a helical conformation(9) .
Based on
length, the N-terminal domains of the annexin family in mammals can be
divided into three categories. Annexins VII and XI are long (170
residues); annexins I, II, and XIIIb are intermediate (
45
residues); and the others are short (
15 residues). Since the three
annexins that have been shown to interact with S100 proteins are in the
long and intermediate category, it is tempting to speculate that the
annexins with short N-terminal domains do not undergo this interaction.
Indeed, we did not detect cellular proteins that associated with the
short N-terminal domain of annexin V (Fig. 5). Furthermore, it
will be interesting to determine whether annexin VII, which has a long
N-terminal domain, will interact with an S100 protein.
The N-terminal domains of the annexins are structurally unrelated, and there is no evidence that they evolved from a common precursor. Thus, it would appear that interaction with S100 proteins evolved independently in the three different annexins. This implies that these annexin-S100 complexes serve an important cellular function.
In
summary, this study shows the interaction with S100C may be involved in
the cellular function of annexin I. Because little is known concerning
the physiological function of either the annexins or the S100 family of
proteins, it is difficult to speculate on the physiological function of
their interaction. Previous studies of annexin II showed that
interaction with p11 affected the cellular localization of annexin II.
The heterotetramer of annexin II and p11 is primarily associated with
cytoskeletal (36, 37) and Triton-insoluble
complexes(38) , whereas annexin II monomers are primarily
cytoplasmic (36) . Annexin XI localized to nuclear structures
even though it lacks an obvious nuclear localization sequence, and this
localization is speculated to be mediated by calcyclin(39) .
Based on these parallels, it is possible that the interaction between
annexin I and S100C may target this complex to particular regions of
the cell in a Ca-dependent manner. Alternatively, the
binding of S100C to the N-terminal domain of annexin I may modulate the
activity of the core domain. Parallel studies of annexin II showed that
the half-maximal Ca
concentration required for
membrane binding and aggregation is significantly higher for the
annexin II monomer than for the annexin II-p11
heterotetramer(40, 41) . Although the properties of
the annexin I-S100C complex have not been characterized, previous
studies showed that post-translational modifications to the N-terminal
domain of annexin I modulated the Ca
and phospholipid
binding properties of the core domain(14, 42) . The
N-terminal domain also is known to modulate annexin I-induced
phospholipid vesicle aggregation, even though this activity does not
directly reside in the N-terminal
domain(43, 44, 45) . The elucidation of a
definitive role for S100C in the targeting or regulation of annexin I
requires more information concerning the biological function of annexin
I.