(Received for publication, August 18, 1995; and in revised form, August 29, 1995)
From the
The 14-3-3 protein family plays a role in a wide variety of cell
signaling processes including monoamine synthesis, exocytosis, and cell
cycle regulation, but the structural requirements for the activity of
this protein family are not known. We have previously shown that the
14-3-3 protein binds with and activates phosphorylated tryptophan
hydroxylase (TPH, the rate-limiting enzyme in the biosynthesis of
neurotransmitter serotonin) and proposed that this activity might be
mediated through the COOH-terminal acidic region of the 14-3-3
molecules. In this report we demonstrate, using a series of truncation
mutants of the 14-3-3 isoform expressed in Escherichia
coli, that the COOH-terminal region, especially restricted in
amino acids 171-213, binds indeed with the phosphorylated TPH.
This restricted region, which we termed 14-3-3 box I, is one of the
structural regions whose sequence is highly conserved beyond species,
allowing that the plant 14-3-3 isoform (GF14) could also activate rat
brain TPH. The 14-3-3 box I is the first functional region whose
activity has directly been defined in the 14-3-3 sequence and may
represent a common structural element whereby 14-3-3 interacts with
other target proteins such as Raf-1 kinase. The result is consistent
with the recently published crystal structure of this protein family,
which suggests the importance of the negatively charged groove-like
structure in the ligand binding.
The 14-3-3 protein family consists of acidic, dimeric proteins
with relative molecular masses of 60 kDa distributed widely among
eukaryotic cells (for reviews, see (1) and (2) ).
Numerous biochemical activities have been attributed to this family of
proteins, including calmodulin (CaM) (
)kinase II-dependent
activation of enzymes involved in neurotransmitter synthesis,
Ca
-dependent stimulation of noradrenalin secretion,
and the regulation of the activity of
Ca
-phospholipid-dependent protein kinase
C(2) . Recent findings, particularly in fission yeast, have
shown that the 14-3-3 family is associated with the products of
proto-oncogenes and oncogenes such as Raf-1, Bcr, Bcr-Abl, and
Polyomavirus middle tumor antigen, suggesting that this family of
proteins is also involved in cell transformation and mitogenic
signaling pathways (for a review, see (3) ). We have previously
shown that the 14-3-3 protein activates TPH in concert with
phosphorylation of the hydroxylase by CaM kinase II (4, 5) and demonstrated that this activation results
from the binding of 14-3-3 protein to the phosphorylated
hydroxylase(6) . From these results, together with the
structural features of the 14-3-3 protein(4) , we proposed that
the acidic COOH-terminal region of the 14-3-3 protein might be involved
in the interaction with phosphorylated TPH. However, little evidence
has been provided in this and other systems on the structural
requirements of the 14-3-3 protein for its biological activities.
In this study, we used the TPH system as a model to assess the functional region of the 14-3-3 protein and showed that the acidic COOH-terminal region, especially restricted in residues 171-213, is a primary site for the interaction of 14-3-3 with phosphorylated TPH.
Figure 2:
A, schematic illustration of wild-type and
mutant forms of 14-3-3. For simplicity, only 14-3-3 regions of the
fusion proteins are shown. For wild-type 14-3-3, the acidic
COOH-terminal region is indicated by a hatched box. For
mutants, dotted lines are used to depict 14-3-3 regions, whose
relative NH and COOH termini are denoted by adjacent
numbers. B, purified 14-3-3 fusion proteins (1-2 µg
each) analyzed by SDS-PAGE (Coomassie Blue staining). Since the GST has
a molecular mass of 26 kDa, the expected sizes of the 14-3-3 fusion
proteins, calculated from amino acid sequences, are: 55 kDa
(1-246), 36 kDa (1-77), 47 kDa (1-170), 54 kDa
(1-237), 51 kDa (1-213), 49 kDa (1-190), 36 kDa
(167-213), 32 kDa (167-213), 50 kDa (
171-213).
The molecular mass markers were bovine serum albumin (66.2 kDa),
ovalbumin (43 kDa), and carbonic anhydrase (28.7
kDa).
We used the bacterial expression system that produces
proteins in E. coli as fusions with an affinity tag, GST, to
prepare recombinant proteins, because this system permits affinity
purification of active proteins. Bovine brain 14-3-3 isoform was
expressed in this system, and the GST-fused
protein was purified
by affinity chromatography on cross-linked glutathione resin. The
purified protein showed a single protein band on SDS gel with an
expected molecular mass of
55 kDa (see Fig. 2B, lane 1), and this protein cross-reacted with polyclonal
antibodies to bovine brain 14-3-3 protein (data not shown).
Before
truncation of the recombinant protein, we examined, using TPH system,
whether the GST-fused protein produced in E. coli indeed shares similar properties with bovine brain
isoform (Fig. 1). The fused
protein added to the TPH assay
mixture stimulated the activity of TPH about 2-fold more than the level
of TPH measured in the absence of the fused protein (Fig. 1A). This effect was dose-dependent, and the
concentration of the
protein necessary for half-maximal
activation (V
/2) of TPH was about 20
nM. These values were almost equal to the published values
with bovine brain 14-3-3
(4, 5) . In addition,
no stimulation of TPH activity was observed, as analyzed in the absence
of CaM kinase II or in the presence of GST alone (Fig. 1A, dotted lines), confirming the
previous data(4, 5) that this activation of TPH needs
both phosphorylation of TPH and the 14-3-3 protein.
Figure 1:
Characterization of the recombinant
14-3-3 protein. A, CaM kinase II-dependent activation of TPH
by the GST-fused 14-3-3 protein. The activity of TPH was assayed with
the indicated amounts of the fused 14-3-3 protein () as described
under ``Experimental Procedures.'' Control experiments were
performed under the same conditions without CaM kinase II (
) or
with GST alone (
). The results are the means of triplicate
determinations and are expressed as a percentage of the activity in the
absence of the fused 14-3-3 protein. B, association of the
GST-fused 14-3-3 protein with the phosphorylated TPH. TPH (
1
µg) were incubated under nonphosphorylating (lanes 1 and 3) or phosphorylating (lanes 2 and 4)
conditions in the presence of GST (2 µg, lanes 1 and 2) or GST-fused
protein (2 µg, lanes 3 and 4), and glutathione-agarose beads were added to the mixture.
Bound TPH with the fused protein immobilized on the beads was assayed
by its enzymatic activity using the beads or by Western blot with TPH
antibody (inset). The cross-reacting band was visualized by
horseradish peroxidase-conjugated goat antibodies against rabbit IgG
and ECL reagent (Amersham Corp.). The arrowhead indicates the
position of TPH. C, formation of a complex between the
recombinant 14-3-3 protein and the phosphorylated TPH in the brainstem
extract. The rat brainstem extract (500 µg) was incubated under
nonphosphorylating (lanes 1 and 3) or phosphorylating (lanes 2 and 4) conditions in the presence of GST (5
µg, lanes 1 and 2) or GST-fused
protein (5
µg, lanes 3 and 4) and was then analyzed as in B. ND, not detected.
We next examined
whether the fused protein can interact with TPH in a
phosphorylation-dependent manner. TPH was incubated with the fused
protein under its phosphorylating or nonphosphorylating conditions (see
``Experimental Procedures''), and glutathione-agarose beads
that bind with the fused protein were added to the mixtures. Bound TPH
with the fused
protein immobilized on the beads was then assayed
by its enzymatic activity and by Western blot with a TPH antibody. As
illustrated in Fig. 1B (lanes 3 and 4), TPH bound with the fused
protein only under its
phosphorylating condition. Incorporation of phosphate to TPH under this
condition was confirmed using [
-
P]ATP (data
not shown, see (6) ), suggesting that, like the bovine brain
protein(6) , the recombinant protein binds with TPH in a
phosphorylation-dependent manner.
We also performed similar
experiments using crude brainstem extract supplemented with the
recombinant protein (Fig. 1C). As shown in Fig. 1C, lane 4, TPH present in the brainstem
extract also bound to the added
protein, indicating that this
binding occurs in the crude extract. Again, no interaction of TPH to
the
protein was detected under nonphosphorylating conditions (lane 3). In both experiments, GST alone did not bind to TPH (Fig. 1, B and C, lanes 1 and 2). All these properties of the expressed fusion protein were
same as the reported characteristics of brain 14-3-3 protein.
Furthermore, like the bovine brain protein(9, 10) ,
the expressed protein activated protein kinase C. (
)Thus, we
concluded that the expression system used in this study can produce the
protein that is suitable for analysis of the functional region of the
14-3-3 protein.
A series of truncation mutants were made (Fig. 2A), and the expressed proteins were purified
with the same procedure described above. As judged by SDS-PAGE (Fig. 2B), the mutants were almost pure with a major
band of the expected sizes. The truncated GST-fused proteins were then
examined in a similar way to that described in the legend of Fig. 1B for their ability to bind with phosphorylated
TPH (see also the legend to Fig. 3). This analysis confirmed the
importance of the COOH-terminal acidic region of protein
(residues 170-246) for the interaction with TPH, because the
deletion mutants lacking this region (mutants 1-78 and
1-170, Fig. 2A) were found no longer bound with
TPH (Fig. 3A, lanes 2 and 3, compare
with lane 1). In addition, deletions of up to 33 COOH-terminal
residues (1-213) almost retained the activity of full-length
protein (1-246), but further deletion of 23 residues
(1-190) reduced the activity (Fig. 3A, lanes
4-6). These deletions suggest that residues 171-213
located in the COOH-terminal acidic region of the
protein may be
necessary for the interaction with the phosphorylated TPH.
Figure 3:
Analysis of the site of protein
responsible for the binding with phosphorylated TPH. A, effect
of truncation forms of
protein on TPH binding. TPH (
1
µg) was phosphorylated by CaM kinase II for 20 min in the presence
of the indicated proteins (25 pmol each) and then analyzed for its
binding as in the experiment shown in Fig. 1, B and C (inset). B, effects of mutants
167-246, 167-213, and
171-213 on TPH binding.
The experimental conditions were same as in A except that 50
pmol of each mutant protein was used. Arrows indicate the
position of TPH.
To test
this assumption, deletions were made to produce three additional
mutants, the mutant 167-246 (carrying the complete COOH-terminal
region), the mutant 167-213 (carrying the 171-213 region),
and the mutant lacking the 171-213 region (171-213, Fig. 2A), and these mutants were analyzed in terms of
TPH binding. This analysis revealed that the two mutants, 167-246
and 167-213, bound to TPH to a similar extent (Fig. 3B, lanes 1 and 2), but the
mutant
171-213 did not (lane 3). These results
proved the above assumption and provided direct evidence that the
structural region consisting of residues 171-213, which we termed
14-3-3 box I, is a primary site for the interaction of the
protein to the phosphorylated TPH. We also observed that all of the
mutants which lacked the box I and failed to bind phosphorylated TPH,
such as the mutants 1-170 and
171-213, had no
activities toward TPH even with the excess amount over TPH. This
suggests that the box I structure is also essential for the activity of
the
protein. Our results, however, cannot exclude the
possibility that an additional region(s) to the box I participates in
the interaction with TPH because the truncation of the box I might
induce conformational changes in another part of the molecule and
thereby prevent the mutants lacking the box I from the interaction with
the hydroxylase.
The amino acid sequence of the 14-3-3 box I is one
of the highly conservative sequences extended among the members of the
14-3-3 family. Fig. 4displays the alignment of corresponding
sequences of the 14-3-3 box I from bovine brain 14-3-3 isoforms with a
known sequence as well as the plant and yeast counterparts, GF14 and
BMH1, respectively. The sequence similarity suggests that the 14-3-3
box I represents a common structural element, which may be involved in
the association of these 14-3-3 isoforms to TPH. Consistent with this
assumption, the previous data have shown that the rat brain TPH could
be activated by all these bovine isoforms as well as by the plant GF14 (5, 11) , although at present whether the yeast BMH1
protein activates TPH is not known. We also note that the 14-3-3 box I
includes Ser, which has been reported to be the site of
phosphorylation in the
and
14-3-3 isoforms by
proline-directed protein kinase(12) .
Figure 4:
Alignment of the amino acid sequences
(single-letter notation) of 14-3-3 box is of bovine brain isoforms, Arabidopsis GF14, and yeast BMH1. The sequences of bovine
,
,
, and
were from (5) and (9) , and that of
was from our recent study (T. Isobe and
T. Ichimura, unpublished data). The amino acid sequences of Arabidopsis GF14 and yeast BMH1 were from (11) and (17) , respectively. Amino acid sequences are numbered from the
N terminus of each isoform. Amino acids identical in all these isoforms
are shown in reverse type.
Recently, the crystal
structures of the homodimeric proteins of the 14-3-3 (13) and
(14) isoforms have been reported. These
proteins have a similar tertiary fold consisting of a bundle of nine
anti-parallel
-helices of each monomer, and the dimers form a
large negatively charged channel or groove. Both reports have suggested
that this groove may represent the ligand binding surface of the 14-3-3
molecules. In the tertiary structure, the 14-3-3 box I represents
helices 7 and 8 and a part of the linker between helices 8 and 9, which
are located near the edge of the groove. In this viewpoint, our results
are consistent with the proposal from the crystal structure studies and
further emphasize the important role of the COOH-terminal structure
including helices 7 and 8 in the ligand/14-3-3 protein interaction.
Increasing evidence suggests that the phosphorylation may be a
common mechanism that regulates the binding of 14-3-3 to its target
molecules. For example, the interaction of 14-3-3 with Raf-1 and Bcr
protein kinases was ultimately prevented by the treatment of these
kinases with protein phosphatase(15) . It has also been shown
that the 14-3-3 reduces the dephosphorylation of Raf-1 with
protein phosphatases through the interaction with Raf-1 and blocks the
dephosphorylation-induced enzymatic inactivation of Raf-1(16) .
The facts that the mode of interaction between these protein kinases
and 14-3-3 is similar to the TPH/14-3-3 interaction described here and
that the 14-3-3 box I is a highly conserved structure from yeast to
mammals (Fig. 4) imply that the box I structure may serve as a
common binding site for many target proteins including these protein
kinases, but this awaits further investigation.