profiling substrate phosphorylation at the phosphopeptide level

Profiling substrate phosphorylation at the phosphopeptide level

Andrea Gatti*

Department of Biochemistry, University of California, Riverside, CA 92521, USA

Received 18 June 2002

Abstract

The identification of substrates is a key aspect in the study of the biological function of protein kinases. The procedure here

described is aimed at profiling substrate phosphorylation at the phosphopeptide level by sequentially involving (i) the assessment of

the in vitro activity of individual protein kinases on a complex mix of immobilized proteins, (ii) the fractionation of the phos-

phopeptides being released upon proteolysis of substrates, and (iii) the final identification of the targeted sequences. In particular,

the protein sample is spotted onto nitrocellulose membrane and then subjected to a solid-phase kinase assay in the presence of

[32P]ATP, prior to solid-phase proteolytic digestion and two-dimensional phosphopeptide mapping. Radiolabeled phosphopeptides

are subsequently isolated and sequenced to identify the substrates being targeted by the examined protein kinase. Using the c-isotypeof p21-activated protein kinase (c-PAK) and its known in vitro substrates, I verified that both the specificity of substrate phos-

phorylation and its efficiency are similar upon solid- and liquid-phase conditions. To demonstrate the feasibility of the overall

experimental system, I then employed a fairly crude cell extract as a source of candidate substrates and successfully identified the

sequence of a putative substrate of c-PAK.� 2003 Elsevier Science (USA). All rights reserved.

Keywords: PAK; Solid-phase kinase assay; Substrate specificity

The large number of protein kinases and the evenlarger spectrum of the respective substrates involved in

cell signaling reflects the high level of complexity of the

overall phenomenon of protein phosphorylation. To

better understand the molecular basis underlying the

specificity of protein phosphorylation, I investigated the

possibility of generating 2-D1 peptide maps to profile

substrate phosphorylation at the phosphopeptide level.

Traditionally, the search for relevant substrates ofprotein kinases has been conducted by comparative 2-D

gel electrophoresis of protein samples frommetabolically

radiolabeled cells, followed by amino acid sequencing of

selected phosphoproteins. New methodologies to iden-

tify substrates of protein kinases by screening cDNAexpression libraries with appropriately chosen molecular

probes such as antibodies and binding proteins have re-

cently become available [1]. Notably, many of these re-

cent applications employ nitrocellulose membrane to

immobilize the expression clones. In some cases, the

screening assay is based on solid-phase phosphorylation

of plaque proteins by exogenously added protein kinases

[2]. The idea of using a solid-phase kinase assay in thepresent studywas also encouraged by the conclusions of a

report of Valtorta et al. [3], according to which SDS–

PAGE-fractionated and nitrocellulose-immobilized pro-

teins are effectively phosphorylated by soluble protein

kinases with a specificity similar to that obtained in a

conventional (i.e., liquid-phase) kinase assay. Note that

in the present study the use of immobilized substrates was

chosen to facilitate the overall flow of operations prior topeptide profiling (e.g., by allowing a direct solid-phase

proteolytic cleavage of substrates).

As a model protein kinase, I employed the c-isotype ofp21-activated protein kinase (c-PAK) [4,5], also known

as PAK-2 [6], a serine/threonine kinase whose activity is

Analytical Biochemistry 312 (2003) 40–47

www.elsevier.com/locate/yabio

ANALYTICAL

BIOCHEMISTRY

* Present address: Via Stringher 27, Rome 00191, Italy. Fax: 39-06-

36300832.

E-mail address: [email protected] Abbreviations used: 2-D, two-dimensional; DEAE, diethylamino-

ethyl cellulose; H4, histone 4; HPLC, high-performance liquid chro-

matography; IMAC, immobilized metal affinity chromatography;

MBP, myelin basic protein; MS, mass spectrometry; PAK, p21-

activated protein kinase; PRS, postribosomal supernatant; TDX1,

thioredoxin peroxidase 1; TFA, trifluoroacetic acid.

0003-2697/02/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

PII: S0003 -2697 (02 )00426-8

mail to: [email protected]

stimulated by binding to small G proteins such as Cdc42and Rac [7]. Although little information on the in vivo

activity of c-PAK is available, proteins such as myelin

basic protein (MBP) and histone 4 (H4) are known to be

phosphorylated by c-PAK in vitro [8,9]. To profile the

liquid- and the solid-phosphorylation of MBP and H4 at

the phosphopeptide level, the respective tryptic digests

were fractionated by 2-D peptide PAGE, a procedure

recently developed to map radiolabeled [10] and fluo-rescent [11] peptides. Note that such a 2-D gel system has

been employed in the analysis of in vitro [12] and in vivo

[13] phosphorylation of c-PAK. By combining manual

and automated amino acid sequencing, the specificities of

phosphorylation of MBP and H4 were demonstrated to

be independent of whether the respective kinase assays

were performed under liquid- or solid-phase conditions.

The use of the solid-phase kinase assay with a fairly crudecell extract and purified c-PAK, followed by 2-D peptide

PAGE and standard peptide purification steps, was

subsequently shown to allow the identification of the

target sequence of a novel substrate of c-PAK.

Methods

Preparation of protein samples

Postribosomal supernatant (PRS) was obtained from

rabbit reticulocytes as previously described [14]. PRS was

diluted 10� with 10mM Tris–HCl, pH 7.4 (buffer A),

prior to being loaded onto a 5-ml diethylaminoethyl cel-

lulose (DEAE; Whatman) column preequilibrated with

the same buffer. After washing the column with 15 vol ofbuffer A, proteins were eluted with 3 vol of buffer A sup-

plemented with 0.5M NaCl. The DEAE eluate was dia-

lyzed at 4 �C against double-distilled water for 24 h prior

to being used.When indicated, purifiedMBP frombovine

brain (Fluka) or proteins from the dialyzed DEAE eluate

were immobilized on nitrocellulose (0.45 lm; Schleicher& Schuell) by spotting a 0.01- to 10-ml aliquot of the

sample onto amembrane square (0.25–100 cm2) placed ona heating block set at 50 �C. Note that the binding ca-

pacity of the employed nitrocellulose is approximately

0.1mg protein/cm2, as indicated by the supplier.

In vitro phosphorylation

Liquid-phase phosphorylation was carried out in a

50-ll reaction mixture containing 20mM Tris–HCl, pH7.4, 10mM MgCl2, 30mM b-mercaptoethanol, and

0.2mM [c-32P]ATP (1000 cpm/pmol), in the presence of

0.1 lg of GST–c-PAK and 0.5 lg of GTP–Cdc42 [15].

The reaction was terminated after 30min incubation at

30 �C by addition of 2� sample buffer (50mM Tris–

HCl, pH 6.8, 2% SDS, 10% glycerol, and 30mM b-mercaptoethanol), prior to SDS–PAGE on 12.5%

polyacrylamide gel and subsequent electrotransfer ontonitrocellulose. Solid-phase phosphorylation was carried

out in 0.05–5ml of the above-described reaction buffer

in the presence of 0.1–10 lg of GST–c-PAK, 0.5–50 lgof GTP–Cdc42 [15], and the additional nitrocellulose-

bound protein sample. The reaction was terminated by

extensive washing of the nitrocellulose square in double-

distilled water. When indicated, preactivation of GST–

c-PAK was carried out in presence of GTP–Cdc42 andunlabeled ATP, prior to solid-phase kinase assay with

[32P]ATP. This step was included to reduce the incor-

poration of radioactivity on GST–c-PAK and enhance

the detection of labeled substrates.

Tryptic digestion and phosphopeptide mapping

Nitrocellulose-bound proteins were digested with10 ng/ll trypsin (DPCC-treated; Sigma) in 50mM am-

monium bicarbonate and 1% (w/v) Zwittergent 3-16

(Fluka) at 30 �C for 24 h. I have previously reported [10]

that under similar conditions of proteolytic digestion the

vast majority (approximately 90%) of the radiolabeled

phosphopeptides of immobilized substrates can be ex-

tracted from the nitrocellulose.

Tryptic digests were subjected to Sep-Pak cartridges(50mg; Waters) prior to 2-D peptide PAGE, as previ-

ously described [10]. The 2-D peptide PAGE consists of

nondenaturing isoelectric focusing in a gel tube as the

first dimension (native IEF) and electrophoresis on a

40% polyacrylamide alkaline slab gel as the second

dimension (40% PAGE). After 2-D peptide PAGE,

radiolabeled phosphopeptides were visualized by auto-

radiography of the dried 40% gel.

Isolation and purification of phosphopeptides

When using a purified protein as substrate of c-PAK,

the respective phosphopeptides were extracted from the

40% gel and directly subjected to a cleanup procedure

through Sep-Pak cartridges prior to manual and auto-

mated amino acid sequencing [10]. In contrast, two addi-tional steps of purification were required when the source

of substrates was a complex mixture of proteins such as

the DEAE eluate of PRS. Prior to the cleanup through a

Sep-Pak cartridge, the sample eluted from the 40% gel

was subjected to an immobilized metal affinity chroma-

tography (IMAC), essentially as described by [16]. No-

tably, the inclusion of the IMAC step involved a minimal

loss of radioactivity (less than 20%), thus confirming theefficiency of such type of chromatography in the isolation

of phosphopeptides from relatively complex protein di-

gests [17]. Briefly, 200 ll of Chelating Sepharose (Phar-

macia) was packed onto a column and washed with

double-distilled water. The column was then activated

with 30mM FeCl3 until metal appeared in the eluate.

After column equilibration with 0.1% trifluoroacetic

A. Gatti / Analytical Biochemistry 312 (2003) 40–47 41

acid (TFA), the sample eluted from the 40% gelwas loaded and the column was washed with 0.1%

TFA (10 vol), prior to elution of phosphopeptides

in 100mM phosphate buffer, pH 10. After IMAC and

a sub-sequent cleanup through a Sep-Pak cartridge,

the isolated phosphopeptide was finally purified by re-

versed-phase high-performance liquid chromatography

(HPLC). The elution from HPLC was monitored by

checking optical density and radioactivity of the fractionsto isolate the radiolabeled phosphopeptide.

Manual and automated amino acid sequencing

Prior to amino acid sequencing, each phosphopeptide

was immobilized on a disk of arylamine membrane (Se-

quelon-AA; Millipore), as described by the supplier. The

disk was routinely cut into two parts; approximately 3/4of the disk was applied to an amino acid sequencer

(Procise 492; Applied Biosystems), while the remaining 1/

4 was subjected to manual Edman degradation as de-

scribed by others [18]. The computer program BLAST

was utilized to match the identified phosphopeptide with

sequences retrieved from the protein databases.

Results

Phosphorylation of known substrates

A comparative analysis of conventional and solid-

phase phosphorylation of an established in vitro sub-

strate of c-PAK (i.e., MBP) was carried out to validate

the choice of using the nitrocellulose-immobilized pro-teins as source of substrates of the purified c-PAK. Note

that to minimize the impact of c-PAK autophosphory-

lation on the total incorporation of radioactivity, a

preincubation of c-PAK with Cdc42 and unlabeled ATP

(preactivation) was routinely carried out prior to the

addition of ½32P]ATP.When performing the liquid-phase kinase assay, a

25-lg aliquot of purified MBP was incubated with pre-activated c-PAK in the presence of [32P]ATP. After in-

cubation, samples were subjected to SDS–PAGE,

followed by protein transfer onto nitrocellulose. Auto-

radiography of the resulting blot membrane allowed

excision of the nitrocellulose band containing the

phosphorylated substrate, prior to solid-phase tryptic

digestion as previously described [10]. When performing

the solid-phase kinase assay, 25 lg of MBP was spottedonto a 0.25-cm2 nitrocellulose square placed on a heat-

ing block set at 50 �C. The nitrocellulose square was thensubjected to a brief washing with double-distilled water

and incubated with preactivated c-PAK in the presence

of [32P]ATP. After incubation, the nitrocellulose square

was extensively washed to remove free radioactivity and

then subjected to solid-phase tryptic digestion [10].

Tryptic digests were subjected to 2-D peptide PAGEto fractionate the radiolabeled phosphopeptides, as

previously described [10]. As shown in Fig. 1, the 2-D

phosphopeptide maps resulting from liquid- (Fig. 1A)

and solid-phase (Fig. 1B) phosphorylation of MBP by

c-PAK are indistinguishable. Manual and automated

sequencing of the isolated phosphopeptides demonstrate

identical specificity and similar efficiency of MBP

phosphorylation (Fig. 2). In particular, microsequencinganalysis of phosphopeptides 1 and 2 resulting from both

liquid- and solid-phase kinase assay showed that Thr-33

and Ser-114 are the major sites of MBP being phos-

phorylated by c-PAK, regardless of the kinase assay

conditions. Incidentally, Thr-33 and Ser-114 have been

described as major phosphorylation sites of MBP fol-

lowing conventional kinase assay with the catalytic do-

main of c-PAK [19].To verify that a similar conclusion could also apply to

other c-PAK substrates, a comparative analysis of solid-

and liquid-phase phosphorylation was also carried out

with H4. As in case of MBP, the specificity of H4

phosphorylation by c-PAK was found to be indepen-

dent of the type of kinase assay (A. Gatti, unpublished

observation). In summary, the overall analysis of the in

vitro phosphorylation of two established substrates of c-PAK validates the choice of using the solid-phase assay

as an alternative to the conventional, liquid-phase

in vitro kinase assay.

Phosphorylation of unknown substrates and identification

of the respective phosphopeptides

The possibility of employing a solid-phase kinaseassay to screen a complex mixture of proteins was next

examined. In particular, the DEAE eluate of PRS

Fig. 1. Comparison between liquid-phase and solid-phase kinase as-

says. A 25-lg aliquot of purified MBP from bovine brain was either

subjected to liquid-phase kinase assay prior to 12.5% SDS–PAGE and

subsequent transfer to nitrocellulose (A) or directly spotted onto a

nitrocellulose square prior to solid-phase kinase assay (B). After

tryptic proteolysis of the nitrocellulose-bound MBP, the same amount

of tryptic digest was loaded onto the 2-D peptide PAGE. The frac-

tionated 32P-labeled phosphopeptides were visualized upon autoradi-

ography of the dried peptide gel. Only the relevant sections of the

autoradiograms with the major phosphopeptides are shown. Similar

results were obtained in two additional experiments.

42 A. Gatti / Analytical Biochemistry 312 (2003) 40–47

obtained from rabbit reticulocytes was chosen as thesource of candidate substrates of c-PAK because such

sample was previously reported to be rich in various

substrates of c-PAK [8]. Initially, a 0.2-ml aliquot

(200 lg of protein) of the DEAE eluate was spotted onto

a 1-cm2 nitrocellulose square prior to a solid-phase ki-

nase assay with a preactivated c-PAK in the presence of

[32P]ATP. Subsequent solid-phase tryptic digestion was

followed by 2-D peptide PAGE, whose autoradiographyrevealed multiple phosphopeptides (Fig. 3C).

To verify that the detected phosphopeptides derived

from the cleavage of substrates, a parallel solid-phase

kinase assay was carried out in the absence of DEAE

eluate (Fig. 3B). The resulting 2-D profile of tryptic digest

resulting from such incubation of preactivated c-PAKwith control nitrocellulose (i.e., not loaded with proteins)

showed few barely detectable spots, presumably dueto the autophosphorylation of nitrocellulose-bound

c-PAK. To verify that the assigned phosphopeptides

shown in Fig. 3C resulted from the kinase activity of

c-PAK rather than from the activity of kinases present in

the DEAE eluate, a parallel sample of DEAE eluate

of PRS was subjected to a solid-phase kinase assay in

the absence of exogenously added c-PAK (Fig. 3A).

After 2-D peptide PAGE of the resulting tryptic digest,

no radiolabeled peptides were detected within the routine

exposure time, thus ruling out the relevance of nonspe-

cific incorporation of radiolabel under the describedconditions.

Next, I tested the possibility of scaling up this

experimental approach to identify the target sequences

of unknown substrates of c-PAK. To this aim, the

Fig. 2. Manual and automated sequencing of phosphopeptides 1 and 2 resulting from liquid- and soild-phase phosphorylation of MBP. After 2-D

peptide PAGE, the radiolabeled phosphopeptides were isolated and bound to Sequelon-AA disks, prior to manual and automated sequencing. The

cycle at which 32P was released by manual Edman degradation and the amino acid sequence identified by automated sequencing are indicated,

together with the relevant sequence of MBP. X designates amino acid residues that could not be unambiguously identified by automated sequencing.

The underlined residues are those found to be phosphorylated.

Fig. 3. Solid-phase phosphorylation of a complex mix of proteins. A

200-lg aliquot of proteins from DEAE eluate of PRS from rabbit

reticulocytes was directly spotted onto a 1-cm2 nitrocellulose square

prior to solid-phase kinase assay in the presence of preactivated c-PAK(C). Parallel assays were also carried out either without the exoge-

nously added c-PAK (A) or without the DEAE eluate (B). Results

shown are representative of two independent experiments.


procedure consisted of the eight steps depicted in Fig. 4:(1) preparation of crude partially purified cell extract,

(2) immobilization of such cell extract onto nitrocellu-

lose membrane, (3) solid-phase phosphorylation by pu-

rified and activated c-PAK in the presence of [32P]ATP,

(4) solid-phase tryptic digestion, (5) cleanup and con-

centration of peptides by using a Sep-Pak cartridge, (6)

peptide fractionation on 2-D peptide PAGE, (7) ex-

traction and purification of individual phosphopeptidesby sequential IMAC/Sep-Pak/HPLC chromatographies,

and (8) manual and automated amino acid sequencingof the HPLC-purified phosphopeptides.

A 10-ml aliquot (10mg of proteins) of the DEAE el-

uate of PRS from rabbit reticulocytes was the starting

material used in the above operations. A major 32P-la-

beled peptide, corresponding to the phosphopeptide a of

Fig. 3C, was extracted from the second dimensional gel of

the preparative 2-D peptide PAGE (data not shown) and

isolated via the described means of peptide purificationprior to automated andmanual Edman degradation. The

Fig. 4. Schematics of the overall procedure. At the end of the operations here outlined, phosphopeptide a was identified as the labeled sequence of

c-PAK-phosphorylated TDX-1 (see Fig. 5).


automated analysis revealed the amino acid sequence ofphosphopeptide a, which exhibits a phosphorylatable

residue in the position of the single phosphorylation site

detected by manual sequencing (Fig. 5). Underscoring

the high purity of the HPLC-purified peptide, the auto-

mated sequencing indicated that the noise due to the

contaminating amino acid residues represented less than

15% of the signal of the identified residues.

Database searching revealed the high degree of ho-mology between the identified amino acid residues of

phosphopeptide a and a stretch of sequence of human

thioredoxin peroxidase 1 (TDX1), a protein with per-

oxidase activity expressed in red blood cells in a variety

of isoforms. Given that the sequence of the rabbit iso-

form of TDX1 is not available in the current databases,

the exact degree of homology between the phospho-

peptide a and the corresponding sequence of rabbitTDX1 could not be calculated. However, the identity of

9 of the first 11 amino acid residues provides a signifi-

cant level of confidence. Hence, rabbit TDX1 is likely to

represent an in vitro substrate of c-PAK. The presence

of a tryptic site (Arg-110) in TDX1 in a position im-

mediately upstream of the identified sequence further

supports this conclusion. The two sites that could not be

unequivocally identified by automated sequencing con-sist of the radiolabeled residue and the glutamic acid

residue, both of which are routinely underdetected bythis type of analysis [10].

Discussion

To better understand the molecular basis of the ex-

traordinary specificity underlying cell signaling, it is

important to investigate how the activation of a par-ticular signaling pathway can selectively recruit multiple

effectors. Due to the difficulty of deciphering the com-

plex network of interactions between the protein kinases

and their respective substrates, an experimental system

that facilitates the profiling of substrate phosphoryla-

tion is in high demand. The use of antibodies raised

against phosphorylation sites has recently emerged as an

elective tool in the analysis of the in vivo activity ofprotein kinases. In fact, phosphorylation site-specific

antibodies against specific amino acid sequences allowed

the identification of many substrates. However, the po-

tential use of such antibodies is somewhat limited to

those instances in which critical information on the

nature of the phosphorylation sites is available.

On the contrary, the method presented here does not

depend on any preliminary knowledge, thereby allowingan unbiased search for substrates. With respect to this,

another method based on the unbiased screening of un-

known proteins as potential substrates of individually

examined protein kinases has been recently developed

[20]. Different from the procedure here described, in such

study the target sequences are fractionated and purified

prior to, rather than after, their proteolytic cleavage.

Considering that most recently developed approaches toprotein identification are based on enzymatic fragmen-

tation of the examined proteins [21], the possibility of

profiling substrate phosphorylation at the phosphopep-

tide level—rather than at the phosphoprotein level—is of

clear interest. With respect to this, note that here I opted

for the use of trypsin as protease because trypsin effi-

ciently cleaves the majority of proteins at many sites,

thereby leading to the generation of conveniently sizedpeptides (in relation to the following peptide fraction-

ation/sequencing). However, proteases other than trypsin

(e.g., V8 and Lys-C endoproteases) may be employed to

tailor the size of the resulting peptides to meet specific

requirements.

The serine/threonine kinase PAK has been impli-

cated in a number of biochemical scenarios, involving

cell proliferation, growth arrest [22–24], and responseto stress and apoptotic conditions [25–28]. In the

course of the study of how multisite autophospho-

rylation of the ubiquitous c-PAK correlates to its

activation, a 2-D peptide PAGE system was recently

developed to fractionate the phosphopeptides resulting

from solid-phase digestion of radiolabeled proteins [10,

12]. The high degree of resolution and reproducibility

Fig. 5. Microsequencing of phosphopeptide a. A 10-mg aliquot (10ml)

of proteins from DEAE eluate of PRS was spotted onto a 100-cm2

nitrocellulose square and subjected to solid-phase kinase assay in the

presence of preactivated c-PAK. After tryptic digestion, peptides wereconcentrated by using a Sep-Pak cartridge and fractionated by 2-D

peptide PAGE. Phosphopeptide a was isolated and purified by se-

quential IMAC, Sep-Pak, and a reversed-phase HPLC, prior to

manual and automated sequencing. The cycle at which 32P was re-

leased by manual Edman degradation and the amino acid sequence

identified by automated sequencing are indicated, together with the

sequence of human TDX1 retrieved from a Blast search as the best

matching protein. X designates amino acid residues that could not be

unambiguously identified by automated sequencing. Results shown are

representative of two independent experiments.


by which 2-D profiles of protein digests are generatedby this experimental system, combined with the possi-

bility of identifying the fractionated phosphopeptides,

prompted me to use this peptide mapping system to

profile the kinase activity of c-PAK on unknown

substrates.

The idea of using a solid-phase kinase assay to profile

substrate phosphorylation was based on the seminal

report of Valtorta et al. [3], according to which thepatterns of protein phosphorylation resulting from the

use of nitrocellulose-immobilized substrates are similar

to those obtained with the conventional liquid-phase

kinase assay [3]. In the present study, the respective sites

of phosphorylation of MBP (Figs. 1 and 2) and H4 (data

not shown) were found to be equally targeted upon

solid- and liquid-phase kinase assay, thus ruling out the

possibility of an artifactual phosphorylation of nitro-cellulose-immobilized substrates.

Notably, the solid-phase kinase assay may circum-

vent several problems routinely associated with con-

ventional in vitro kinase assays. In liquid-phase kinase

assay, both intramolecular and intermolecular interac-

tions may mask relevant domains of potential sub-

strates. This may be the case for enolase, whose in vitro

phosphorylation by src takes place exclusively underacidic conditions [29]. Compared to soluble proteins,

immobilized proteins are more readily screened by

in vitro kinase assay. In fact, nitrocellulose-bound pro-

teins can be more concentrated per unit of reaction

volume and the same [32P]ATP-containing incubation

buffer can be used with multiple batches of nitrocellu-

lose-bound proteins. Compared to proteins in solution,

immobilized proteins are more effectively digested byendoproteases such as trypsin.

Note that in the present study the protein sample was

directly spotted onto nitrocellulose, rather than being

run on SDS–PAGE and blotted onto the membrane as

originally described by Valtorta et al. [3]. In addition to

allowing larger amounts of candidate substrates to be

screened, direct spotting of proteins onto nitrocellulose

avoids the typical problems of electroblotting such asthe limited recovery of high-MW proteins.

A systematic effort to optimize the overall procedure

for preparative purposes lay outside the scope of this

study. When selecting for the final step of peptide

identification, it is reasonable to take into account the

recent progress in the sensitivity and capability of mass

spectrometry (MS) technologies. Approximately 1 pmol

of a given phosphopeptide is required for automatedamino acid sequencing, while a sensitivity extending into

the low-femtomole range is becoming common for

MS-based technologies [30]. Regardless of whether the

final analysis is automated Edman degradation or a

MS-based technology, the sequencing of the isolated

phosphopeptides together with the finding of phosphory-

latable residues in the position of sites of phosphorylation

(being assessed by manual sequencing) is likely to besufficiently informative for the identification of the

respective substrates.

In the case of phosphopeptide a, a database search

revealed high homology with an antioxidant enzyme,

namely TDX1. Note that the presumed site of TDX1

phosphorylation is fully consistent with the recognition

determinant [(K/R)RX(S/T)] of c-PAK phosphorylation

previously described [9]. TDX1, originally termed nat-ural killer enhancing factor-B, belongs to a highly con-

served and widely expressed family of antioxidants

known to be present in red blood cells [31]. TDXs rep-

resent the enzymes that link the reduction of H2O2 to

thioredoxin, thereby protecting cells from apoptosis in-

duced by serum deprivation and etoposide [32]. Inter-

estingly, by using a modified protocol of the present

procedure, a peptide from an additional protein withperoxidase activity (i.e., catalase) was also identified as

in vitro target sequence of c-PAK (A. Gatti, unpub-

lished observation). Taking into consideration that c-PAK has been recently found to be activated by stimuli

leading to cytostatic and apoptotic conditions [33,34], a

role for c-PAK in the regulation of cellular redox can be

anticipated.

A number of substrates of protein kinases have beenidentified upon in vitro screening of cDNA expression

libraries and subsequent DNA sequencing of positive

clones [1]. Such strategy is based on the use of nitro-

cellulose to immobilize the expressed proteins to be

screened with appropriately chosen molecular probes

(e.g., antibodies, purified kinases, binding proteins, etc.).

Compared to the use of cDNA libraries to express large

amounts of candidate substrates, the major limitation ofthe present procedure is the limited abundance of can-

didate substrates. This problem can be circumvented

either by using selective chromatographic steps as pro-

tein enrichment steps [35,36] or by taking advantage of

more sensitive sequencing technologies (as discussed

above).

In conclusion, profiling substrate phosphorylation at

the phosphopeptide level together with sequence data-base searching may represent an interesting opportunity

in the forthcoming scenario of functional proteomics.

As a variety of protein array applications is progres-

sively becoming available, the adaptation of this exper-

imental approach to enable global analysis of substrate

phosphorylation is envisaged.

Acknowledgments

The experimental work for this project was carried

out in the laboratory of Dr. Jolinda A. Traugh. Many

thanks also to Barbara Walter for the purification of

baculovirus-expressed GST–c-PAK and to Kevin Orton

for the HPLC of phosphopeptide a.


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profiling substrate phosphorylation at the phosphopeptide level

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