©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
14-3-3 and Are the Phosphorylated Forms of Raf-activating 14-3-3 and
IN VIVO STOICHIOMETRIC PHOSPHORYLATION IN BRAIN AT A Ser-Pro-Glu-Lys MOTIF (*)

(Received for publication, November 15, 1994; and in revised form, December 21, 1994)

Alastair Aitken (§) Steve Howell David Jones Joel Madrazo (¶) Yasmina Patel

From the Laboratory of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 14-3-3 protein family has received considerable attention recently in the literature, because of the finding that beta 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 alpha and are in vivo post-translationally modified forms of beta and , respectively, and the site of phosphorylation, serine 185, is in a consensus sequence motif for proline-directed kinases.


INTRODUCTION

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 alpha- after their respective elution positions on HPLC(^1)(3, 4) . Their sequences have been determined(5) , and recently we showed that two isoforms are identical in primary structure to beta 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 beta 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 beta and isoforms are part of this complex and have been shown to activate Raf.

We have previously established that alpha and are the post-translationally modified forms of beta and , respectively(26) . Electrospray mass spectrometry (ESMS) revealed that each pair of sheep and chicken alpha/beta 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 alpha and isoforms. It should be noted that approximately 40% beta 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 alpha and beta 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 alpha 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 alpha and isoforms was identified.


MATERIALS AND METHODS

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.

Purification of 14-3-3

14-3-3 was isolated from sheep and chicken brain by a combination of anion-exchange and hydrophobic interaction chromatography, according to the methods in (2) and (27) . The concentration of the purified proteins was estimated by amino acid analysis (on a Beckman 121MB or a Beckman 6300 analyzer with post column ninhydrin detection), and by the Bio-Rad protein assay (according to manufacturer's instructions).

Reverse Phase Separation of 14-3-3 Isoforms

Pure 14-3-3 was separated into individual isomers on reverse-phase HPLC, using Aquapore RP300 (C(8), 4.6 times 100 mm (Refs. 3, 4, and 27), and Bakerbond WP-C(4) (4.6 times 250 mm and 10 times 250 mm) wide pore columns. The separations employed shallow water/acetonitrile gradients containing trifluoroacetic acid. The isoforms eluted between approximately 50-56% (v/v) acetonitrile.

Electrospray Mass Spectrometry

ESMS was performed on a Fisons VG Platform instrument. On-line trapping was used to purify and desalt proteins before introduction to the ESMS source(26) , which resulted in a very large increase (down to fmol/µl level) in sensitivity(32) . This comprised a Polymer Laboratories (UK) poly(styrene/divinylbenzene) PLRP-S, 8-µm particle, 300-Å pore size, 0.7-mm microbore column (slurry-packed in house). The sample was loaded on this trapping column in a low concentration of organic modifier, washed free of interfering salts, etc., with acetonitrile/water/acetic acid 15:84:1 (v/v/v) at a flow rate of 2-500 µl min. Proteins were eluted with acetonitrile/water/acetic acid 50:49:1 (v/v/v) at a flow rate of 10 µl min by switching a Rheodyne valve to put this column on-line with the source.

Ethanethiol Derivatization

This was carried out as described in (33) with the modification of the use of acetonitrile instead of the normal solvents. This minimized subsequent manipulations. Extended reaction time (18 h at 50 °C) was used, since the beta-elimination step of derivatization of a phosphoserine adjacent to a proline residue is slow.

Isoform Digestion and Peptide Purification and Sequencing

Sheep brain 14-3-3 alpha and isoforms that had been separated on the wide pore reverse phase HPLC columns were digested with cyanogen bromide and with trypsin as described previously(2, 34) . All resulting peptides were screened by ESMS to search for the modified site (which would have a mass 80 Da higher than predicted by its amino acid composition). The tryptic peptide containing the site of phosphorylation was subdigested with Glu-C proteinase (4 µg) overnight in 0.1 M ammonium bicarbonate, pH 7.8, to ensure that cleavage at the Glu-Ile bond was as complete as possible. The unmodified beta and isoforms and recombinant isoform were also digested separately as controls. Peptides were fractionated on HPLC using a water/acetonitrile gradient (0-50% in 0.1% (v/v) trifluoroacetic acid) on a Vydac reverse-phase C(18) column (25 times 0.46 cm). Peaks (A) were collected, concentrated and the peptides sequenced on Applied Biosystems 470A gas-phase and 477A pulsed liquid-phase protein sequencers with Applied Biosystems 120A on-line phenylthiohydantoin (PTH)-amino acid analyzers (34) . Data collection and analysis were performed with an Applied Biosystems 900A module calibrated with 25 pmol of PTH-amino acid standards.

Alkali-labile Phosphate Analysis

The presence of alkali-labile phosphate (protein-linked O-phosphoester) was tested by incubation with 1 M NaOH at 37 °C for 16 h. The phosphate liberated from sheep brain 14-3-3 was measured as follows. The solution was neutralized with trichloracetic acid. Centrifugation removed unhydrolyzed protein, and the phosphate was measured by the color formed with molybdate-ascorbate reagent against a standard curve of phosphate solutions tested at the same time(35) .


RESULTS AND DISCUSSION

We have previously established by protein sequencing that the alpha and beta (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 alpha/beta 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 beta 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(2)-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 alpha 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, DeltaSer, 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 alpha and isoforms. Accurate ESMS data was obtained for all CNBr peptides from all regions of alpha 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 alpha and isoforms were also examined retrospectively from previous studies.


Figure 1: Electrospray mass spectrometry of phosphopeptide T2 from alpha and 14-3-3. This figure shows the ESMS spectrum of the phosphopeptide T2 from tryptic digestion of alpha and 14-3-3 (upperpanel). The unmodified peptide from a parallel tryptic digestion of beta 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 alpha 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 alpha 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 alpha 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 alpha and isoforms (as well as beta 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. Delta-Ser indicates that only the DTT adduct of dehydro-Ser was detected at this position, due to the sequencing chemistry, which results in beta-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 alpha and forms of 14-3-3 and from tryptic (and Glu-C subdigestion) of 14-3-3 containing a mixture of both alpha and forms.



The focus of much of the recent excitement about 14-3-3 proteins is that the beta 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 (alpha and ) are in fact in vivo phosphorylated forms of beta 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 (alpha 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. (^2)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 beta 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.


FOOTNOTES

*
This work was funded by the Medical Research Council (United Kingdom) and by a grant from the Wellcome Trust (to J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-81-959-3666; Fax: 44-81-906-4477.

Permanent address: CIGB, Ave. 31, Cubanacan, P. O. Box 6162, Habana, Cuba.

(^1)
The abbreviations used are: HPLC, high performance liquid chromatography; ESMS, electrospray mass spectrometry; DTT, dithiothreitol; PTH, phenylthiohydantoin derivative of an amino acid.

(^2)
D. Schoenwasser, P. J. Parker, and A. Aitken, unpublished data.


ACKNOWLEDGEMENTS

We thank H. Aitken for human 14-3-3 placenta protein.


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