(Received for publication, November 15, 1994; and in revised form, December 21, 1994)
From the
The 14-3-3 protein family has received considerable attention
recently in the literature, because of the finding that and
isoforms interact with and activate Raf. We had previously shown that
these 14-3-3 isoforms also exist as phosphorylated forms in mammalian
and avian brain. The presence of this modification enhances the
activity of 14-3-3 as an inhibitor of protein kinase C nearly 2-fold.
In this report we show by a combination of electrospray mass
spectrometry and protein microsequencing that
and
are in vivo post-translationally modified forms of
and
, respectively, and the site of phosphorylation, serine 185, is in
a consensus sequence motif for proline-directed kinases.
14-3-3 was the name given to a protein family with a particular
migration pattern on two-dimensional DEAE-cellulose chromatography and
starch gel electrophoresis(1) . 14-3-3 proteins all have a pI
of around 4.5 and mass of 30,000 Da on SDS-polyacrylamide gel
electrophoresis but by size exclusion chromatography are dimers of
60,000 Da(2) . There are seven major mammalian brain forms of
14-3-3, named -
after their respective elution
positions on HPLC(
)(3, 4) . Their sequences
have been determined(5) , and recently we showed that two
isoforms are identical in primary structure to
and
isoforms, respectively(6) . The 14-3-3 family is highly
conserved, and individual isoforms are either identical or contain a
few conservative substitutions over a wide range of mammalian species.
Homologues of 14-3-3 proteins have also been found in
plants(7, 8) , insects (9) amphibians(10) , yeast(11, 12) ,
and the nematode Caenorhabditis elegans(13) .
The major roles ascribed to the mammalian 14-3-3 proteins have been as activators of tyrosine and tryptophan hydroxylases(3, 14) , inhibitors or activators of protein kinase C(2, 15, 16) , an involvement with exocytosis(17) , and association with melatonin biosynthesis in the pineal gland(18) . In non-mammalian species, 14-3-3 may play a role in the regulation of gene expression(8) . A yeast homologue of 14-3-3, BMH1(11) , but not plant 14-3-3 (8) affects the growth characteristics when overexpressed in budding yeast. The two 14-3-3 proteins (Rad24 and Rad25 gene products) in fission yeast (Schizosaccharomyces pombe) function in determining the timing of mitosis(12) , suggesting a role in the cell cycle and DNA damage control. A plant 14-3-3 homologue, GF14, binds to the G box promoter element of inducible genes implying a role in regulation of transcription(8) .
The eukaryotic host factor that activates
exoenzyme S from Pseudomonas aeruginosa has recently been
shown to be 14-3-3 (19) . Exoenzyme S ADP-ribosylates Ras
and other GTP-binding proteins. 14-3-3 isoforms associate with polyoma
virus middle T antigen, which in turn associates with other proteins
involved in the regulation of cell proliferation(20) . Middle T
antigen also binds SHC, Grb2, and SOS. The authors suggest a role for
14-3-3 in regulation of Ras and G protein function. The proteins that
interact with middle T antigen include phosphatidylinositol 3-kinase,
which itself modifies Raf activity. A number of groups have recently
shown that 14-3-3
and
associate with and activate Raf
protein kinase both in the cytosol and at the membrane of mammalian
cells and in yeast(21, 22) . Rat brain 14-3-3
activates the Xenopus oocytes' cytosol counterpart of
Raf (REKS or Ras-dependent extracellular signal-regulated kinase kinase
stimulator; (23) ) Expression of 14-3-3 in Xenopus oocytes also leads to activation of Raf (24) . The
principal region of Raf with which 14-3-3 isoforms interact, is
contained in a construct which includes the zinc finger
domain(21) .
Raf plays a key role in the
``mitogen-activated protein kinase cascade'' and is at a
convergent point for different signals, which regulate growth factor
and hormone effects on differentiation and proliferation (reviewed in (25) ). Raf exists as a multiprotein complex, which can be
translocated to the plasma membrane in the presence of Ras-GTP. The
focus of many of the recent publications on 14-3-3 proteins is that the
and
isoforms are part of this complex and have been shown
to activate Raf.
We have previously established that and
are the post-translationally modified forms of
and
,
respectively(26) . Electrospray mass spectrometry (ESMS)
revealed that each pair of sheep and chicken
/
and
/
brain isoforms were different in mass by 80
Da(27) . This was suggestive of phosphorylation (or sulfation
on tyrosine). In a variety of other tissues(28) , we found no
evidence for
and
isoforms. It should be noted that
approximately 40%
14-3-3 is expressed in mammalian species using
an alternative initiator methionine codon 6 nucleotides upstream of the
major initiation site (26) . With Thr (residue mass 101 Da) as
the second amino acid, this initiator Met is removed as predicted (29) and
and
forms with masses approximately 100 Da
higher are detected, which provides additional evidence that they arise
from the same gene product. The presence of
0.2 mol/mol
alkali-labile phosphate was observed after two separate prolonged
incubations with 1 M NaOH (in retrospect, the identification
of the site of phosphorylation on a serine residue adjacent to proline
is consistent with the observed relatively high alkali stability; (30) ). Together
and
14-3-3 constitute 19% of total
brain isoforms(27) . The nature of the modification was also
analyzed by incubation of mixed brain 14-3-3 with a range of
phosphatases and sulfatases. However, in no case was significant loss
of the modified form observed. This may also have been due, in
retrospect, to the poor phosphatase specificity for the site adjacent
to proline. 14-3-3 isoforms separated in trifluoroacetic
acid/acetonitrile (pH
2) on reverse phase HPLC were not restored
to their native state, when simply neutralized. A method was developed
to renature 14-3-3 isoforms to the native state judged by two criteria:
circular dichroism (which measures secondary structure) and dimeric
structure(31) . We subsequently showed that the phospho-form
(
) interacted more strongly with kinase C than the unmodified
isoform (28) . This was measured by the level of
inhibition of protein kinase C when assayed in the absence of
diacylglycerol or phorbol ester (which will overcome this inhibition; (2) and (31) ).
In the present study, the site of
phosphorylation on 14-3-3 and
isoforms was identified.
All reagents were analytical grade, from BDH, Sigma, or Boehringer Mannheim. Protein assay reagent was obtained from Bio-Rad. HPLC and fast protein liquid chromatography solvents and water were obtained from Romil.
We have previously established by protein sequencing that the
and
(as well as the
and
) isoforms of mammalian
and avian brain 14-3-3 are identical in primary structure but differ
only in a post-translational modification(6, 26) . In
the present study, we have identified the site of phosphorylation in
/
and
/
14-3-3 isoforms in mammalian brain by ESMS
with on-line trapping(26, 32) . Since the site of
phosphorylation was endogenous to the 14-3-3 isoforms in protein
purified from brain, there was no
P radioactivity present.
No attempt was made, in the present study, to incubate brain homogenate
protein with [
P]phosphate. This could have led
to the labeling of these isoforms by the endogenous kinase and so
facilitate identification of the site. There is, however, a great
danger of trace labeling by another (or the same kinase) on another
site. This study therefore has led undoubtedly to the identification of
an intrinsic high stoichiometry site of phosphorylation.
The site of
phosphorylation was identified on the CNBr, tryptic, and Glu-C peptides
(CN6, T2, and Glu-C2). Except for T2 (Fig. 1), ESMS data from
the tryptic peptides which are included within CN6 revealed no masses
that were 80 Da higher than predicted from their sequences. In most
cases, the unphosphorylated CNBr, tryptic, and Glu-C peptides (from
unmodified and
forms) were also analyzed by ESMS and by
sequencing after reaction with ethanethiol, as controls. Yields of
PTH-amino acids from sequencing of CN6 were lower than normal, due to
partial cyclization of the NH
-terminal glutamine residue to
form pyroglutamic acid (29) . Both this peptide and T2 were
poorly soluble in HPLC buffers and low recovery from the reverse phase
columns was the norm. Lyophilization was avoided if at all possible.
This resulted in greatly improved amounts of these peptides. There was
no indication of 80 Da higher mass CNBr and tryptic peptides elsewhere
in peptides derived from other regions of
and
14-3-3. The
presence of phosphotyrosine was eliminated by lack of cross reaction
with specific anti-phospho-Tyr antisera (data not shown). Additionally,
as further proof, the expected levels of PTH-Tyr were seen at cycles 11
and 12 and cycles 18 and 19 of T2 and CN6, respectively. This suggests
the site is one of two serines in these peptides. The precise location
of the phosphate group at cycle 17 of peptide T2 (Ser
in
the protein) was verified by derivatization with ethanethiol, which
converts phosphoserine (but no other phosphoamino acid) to S-ethyl cysteine(34) . This was followed by automated
peptide sequencing. Normal levels of PTH-Ser and the DTT adduct of
dehydroserine,
Ser, were seen at cycle 8 of peptide T2 (data not
shown). Subdigestion of T2 with Glu-C proteinase for an extended time
resulted in cleavage of the Glu-Ile bond (anticipated to be slow) in
excellent yield. As expected the COOH-terminal Glu-Lys bond was not
cleaved, since Glu-C is not an exopeptidase. There is a large amount of
additional evidence for absence of phosphorylation on any other residue
in the entire sequence of both isoforms which can only be summarized
here. This includes the direct identification of the PTH-derivatives of
serine, threonine, and tyrosine (not just the DTT adduct of the dehydro
derivatives of the first two) on all the alternative sites during
separate sequence analysis of CNBr peptides derived from HPLC-purified
and
isoforms. Accurate ESMS data was obtained for all CNBr
peptides from all regions of
and
isoforms except for CN4,
which is also very hydrophobic. However, none of the tryptic peptides
from this region that are included within CN4 showed additional mass.
Peptide sequence data from all regions of
and
isoforms were
also examined retrospectively from previous studies.
Figure 1:
Electrospray mass
spectrometry of phosphopeptide T2 from and
14-3-3. This
figure shows the ESMS spectrum of the phosphopeptide T2 from tryptic
digestion of
and
14-3-3 (upperpanel).
The unmodified peptide from a parallel tryptic digestion of
and
14-3-3 (which elutes approximately 1 min later from the HPLC
column, data not shown) is reproduced in lowerpanel). A2 and A3 are the doubly and
triply charged forms of the peptides. The calculated masses of
phosphorylated and unmodified T2 are 2397.7 and 2317.7 Da,
respectively. The phosphopeptide T2 also contained some of the peptide
from the same region of the
isoform, which is 6 residues longer
since there is no equivalent lysine residue (
14-3-3 is
incompletely separated from
after HPLC of the intact isoforms;
Refs. 3, 4, and 29). The m/z of the triply charged form of
this peptide is 986.3. Similarly, the peak at m/z 778.1 (B3) is the triply charged form of the
peptide (intact
and
14-3-3 isoforms are also incompletely
separated after HPLC; Refs. 3, 4, and 27).
Our findings
are summarized as follows. (a) Brain 14-3-3 proteins contained
the expected amount of alkali-labile phosphate. This indicated the
presence of phosphoserine or phosphothreonine. Phosphotyrosine is
alkali-stable. (b) The absence of any phosphate anywhere in
the proteins except in CN6, T2 and Glu-C2 was conclusive. (c)
Phosphotyrosine was ruled out by specific antisera and the presence of
PTH-tyrosine in the phosphorylated forms of peptides CN6 and T2 (as
well as its presence at normal levels at the position of all other
tyrosines in all other peptides sequenced). (d) The first
serine residue in CN6 (from separate sequencing analyses of and
isoforms) and T2 was detected with the normal levels of PTH-Ser,
in contrast to the second serine (Ser
). (e) S-Ethyl-Cys was detected only at
Ser
. (f) ESMS on the intact HPLC-separated
isoforms never gave any indication of more than one site of
phosphorylation, at least on the same polypeptide chain. (g)
ESMS data on subdigest peptide Glu-C2 which contains only one
phosphorylatable residue, accurately locate the modification. Peptide
Glu-C1 was not modified.
The evidence for Ser as the
only site of phosphorylation on 14-3-3
and
isoforms is
therefore conclusive. The evidence for this site of phosphorylation on
the CNBr, tryptic, and Glu-C peptides (CN6, Glu-C2, and T2) is
summarized in Fig. 2.
Figure 2:
Summary of mass spectrometry and
sequencing analysis for the identification of the site of
phosphorylation. The complete amino acid sequences of sheep brain
and
14-3-3 are shown in single-letter code. The
numbering is different from that in alignments that include all the
mammalian 14-3-3 isoforms since the spaces normally required to
accommodate other isoforms have been removed(5) . The positions
of all CNBr peptides are indicated by the bars above the sequences.
Tryptic peptides that are included within CN6 are indicated below the
sequences. The amino-terminal part of CN6 is expanded below and is
shown in three-letter code for emphasis. The
and
isoforms (as well as
and
) differ only in an Asn-Ser change
within CN6, but this is COOH-terminal to the region of interest. The
experimentally derived masses of all the phosphopeptides (and, for
emphasis, that of Glu-C1, which is not modified (calculated mass 1535.7
Da)) are shown. These are in all cases within 1 Da of the theoretical
mass for a peptide containing a phosphoserine. The calculated mass of
unmodified Glu-C2 is 799.9 Da. All the other phosphorylatable amino
acids (Ser, Thr, and Tyr) within this region are indicated by italics, although these have been eliminated by a number of
other criteria (see text). The relevant results from automated
microsequencing on the phosphopeptides and the S-ethyl-Cys
modified forms are summarized by the half-arrows.
-Ser indicates that only the DTT adduct of dehydro-Ser
was detected at this position, due to the sequencing chemistry, which
results in
-elimination of phosphoserine. The normal ratio of
PTH-Ser/DTT adduct of dehydro-Ser and that of the analogous derivatives
of Thr were detected at the other positions. Normal levels of PTH-Tyr
were also recovered. Due to the alkaline conditions required for the
conversion to S-ethyl-Cys, the Asn (third residue of Glu-C2)
was deamidated and identified as PTH-Asp. The data summarized in this
figure were principally obtained from sequence and ESMS analysis of
separate CNBr digests of
and
forms of 14-3-3 and from
tryptic (and Glu-C subdigestion) of 14-3-3 containing a mixture of both
and
forms.
The focus of much of the recent
excitement about 14-3-3 proteins is that the and
isoforms
are part of the GTP-Ras/Raf complex and have been shown to activate
Raf(25) . Is it simply a coincidence that two of the abundant
mammalian brain isoforms (
and
) are in fact in vivo phosphorylated forms of
and
, respectively? The fact
that these two isoforms are specifically modified gives the
identification of the kinase responsible for this phosphorylation and
the study of a potential regulatory function in the 14-3-3/Raf
interaction a high priority. It cannot yet be ruled out that the 14-3-3
isoforms that have been observed to interact with Raf are in fact the
phosphoforms (
and
). c-Raf, which was specifically
immunoprecipitated from 3T3 cells, did not phosphorylate either
HPLC-purified and renatured 14-3-3 or brain and placenta 14-3-3 isoform
mixture. (
)Significant levels of the phosphoforms are absent
from the latter(28) . There remains, of course, the possibility
that this Raf kinase was already complexed with 14-3-3 and the
phosphorylation may be non-catalytic. The site of phosphorylation in
brain 14-3-3 at a Ser-Pro-Xaa-Lys motif is reminiscent of the
specificity of cyclin-dependent kinases(36) . This SPEK motif
is unique to the
and
isoforms. The ability of this and
other kinases to phosphorylate specific isoforms in vitro is
now being tested. The brain homologue of the Raf family of kinases,
B-Raf, may be a candidate. The human T-cell 14-3-3 isoform (
),
called Bap-1 in that study, associates with and is phosphorylated on
serine by Bcr kinase(37) .
Our structural studies on this family of dimeric proteins are being extended to three dimensional x-ray crystallography, which will indicate more clearly the sites of interaction with kinases and mechanism of inhibition. In brain, the levels of the phosphorylated 14-3-3 forms approach 1:1 with the unmodified isoform. There is the possibility, therefore, that these proteins are phosphorylated to their maximum extent of one phosphate per dimer. The tertiary structure of 14-3-3 will reveal the relative location of this phosphate group on each subunit and should reveal if the site is able to be occupied by more than one phosphate in the dimer.