analysis of acute myelogenous leukemia: preparation of samples for genomic and proteomic analyses

Current Pharmaceutical Biotechnology, 2006, 7, 000-000 1

1389-2010/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

Proteomics Approaches to Elucidate Oncogenic Tyrosine Kinase Signalingin Myeloid Malignancies

Eystein Oveland1,2, Kari E. Fladmark1,3, Line Wergeland4, Bjørn Tore Gjertsen4,5 andRandi Hovland1,6,*

1Proteomic Unit in Bergen, University of Bergen, Bergen, Norway; 2Department of Biomedicine, University of Bergen,Bergen, Norway; 3Department of Molecular Biology, University of Bergen, Bergen, Norway; 4Institute of Medicine,Hematology Section, University of Bergen, Haukeland University Hospital, Bergen, Norway; 5Department of Medicine,Hematology section, Haukeland University Hospital, Bergen Norway and 6Center for Medical Genetics and MolecularMedicine, Haukeland University Hospital, Helse Bergen HF, Norway

Abstract: Myeloid malignancies frequently harbor specific mutations in protein tyrosine kinases leading to oncogenic cellsignaling. The most extensively investigated example is chronic myeloid leukemia, where the pathogenic tyrosine kinasefusion protein Bcr-Abl is a successful target for disease control by the specific inhibitor imatinib mesylate. In acute mye-loid leukemia the receptor tyrosine kinase Flt3 is frequently mutated and inhibitors to impair the oncogenic signaling arein development. In this review we exemplify oncogenic signaling and how signal pathways can be unraveled with helpfrom proteomics-based technologies. The distinction between cell extract and single cell approaches aiming at rigorousstandardization and reliable quantitative aspects for future proteomics-based diagnostics is discussed.

Key Words: Proteomics, biosignature, receptor tyrosine kinase (RTK), Flt3, Kit, PDGFR, Bcr-Abl, JAK2, cell signaling.

INTRODUCTION

Dysregulated tyrosine kinase activity has been implicatedas a central pathogenic event in a number of human malig-nancies [1]. In a subset of hematological malignancies,myeloproliferative diseases (MPD), chronic myeloid leuke-mia (CML) and acute myeloid leukemia (AML), mutatedand putatively constitutive active protein tyrosine kinases(PTK) are striking frequent features. These malignanices arecharacterized by dysplastic, pre-malignant and malignantfeatures in the myeloid cell linage of bone marrow progeni-tors [2-4]. These malignant progenitors have their normalcounterpart in cells that will develop into mature red bloodcells, thrombocytes, granulocytes and monocytes. The dis-ease characteristics are typically caused by excess, depletionor dysfunction of these specialized cells. In some diseases,like in CML, several myeloid cell subsets are affected. InAML the mutated PTK is frequently accompanied by a de-fect in transcriptional regulation, introducing a block in dif-ferentiation along with the increased proliferation [5, 6].Clinically, MPDs are smouldering diseases that in somecases may develop into an AML, within stable phase thesediseases have a median survival beyond five years. AML hasa median survival of approximately three months if left un-treated, and even in patients treated with high dose chemo-therapy the over-all five years survival hardly exceeds 50%.Inspired by the fact that many myeloid malignancies com-prise a mutated TK, and a recent proof-of- principle workthat indicates that AML may be classified according to its

*Address correspondence to this author at the Center for Medical Geneticsand Molecular Medicine, Haukeland University Hospial, Helse-Bergen HF,N-5021 Bergen, Norway; E-mail: [email protected]

signal pathway response [7], the concept that diagnosticscould be based on protein analysis of the signaling moleculeshas been spawned. The WHO revised classification of mye-loid malignancies [8] and the recent discovery of JAK2 mu-tations in polycytemia vera may initiate further developmentof a more therapy-relevant classification of these diseases[3]. A substantial number of new agents perturbing signalingpathways are under development for clinical use. All thesenew therapeutics are targeting proteins and perturbing pro-tein signaling networks, thus highlightening the relevance ofprotein-based studies.

TYROSINE PHOSPHORYLATION INCELL SIGNALING

The human kinome consists of more than 500 proteinkinases, which can be clustered into nine main groups, de-pending on the sequence similarities in their catalytic do-mains [9]. The most common human cancer genes encodeproteins with a kinase domain and the majority is associatedwith somatic mutations [1].

PTKs define one of these groups and they are character-ized by their ability to catalyze the transfer and covalent at-tachment of the terminal phosphate group of ATP to hy-droxyl groups of tyrosine residues on substrate proteins. ThePTKs are substrate specific, highly regulated, rapid, shorttermed and reversible [10]. This makes PTKs important inthe initializing phase of signaling events as a rapid andstrong response to extracellular stimuli e.g. by growth fac-tors. The PTK group can be divided into 10 non-receptortyrosine kinase families and 20 receptor tyrosine kinase(RTK) families [11]. Even if tyrosine phosphorylation com-prise only about 0.05% of the total protein phosphorylation

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in the cell, this signal event is considered to be essential, forexample with respect to cellular growth and differentiationthrough its regulation of cell cycle and transcription [12-14].PTKs are considered to be prototypical oncogenes in humanmalignancies as c-Src was identified in the early 1970ies asthe first human proto-oncogene [15].

There are about 80 specific protein tyrosine phosphatases(PTPs) of which many are able to act as PTK counterparts[16] by competitively removal/hydrolysis of phosphorylgroups of the tyrosine residues. Even if PTPs in general arepotential tumor suppressors, some PTPs also have oncogenicpotential (reviewed by Östman et al. 2006) [17].

ACTIVATION OF TYROSINE KINASES

Oncogenic signaling by the Type III RTK family, such asPDGFRα [18], PDGFRβ [19], Kit [20], Flt3 [21] and CSF1R[22] is often associated with myeloid hematological malig-nancies. Type III RTKs are transmembrane PTKs that trans-duce signals from the cell exterior to the interior [23, 24](Fig. 1). The Type III RTK receptors are activated upon di-merization which is stabilized by binding of their respectiveligands, the ligand: receptor stochiometry to a ratio of 1:2[25, 26]. For example the Flt3 ligand (FL) and Kit ligand(KL) are structurally similar to type I transmembrane pro-teins but can also occur in a less active soluble form [27-29]and function both in a paracrine and autocrine manner [30,31]. Further, FL and KL are active as homodimers linkedtogether by noncovalent interactions [28]. The five ligandsfor PDGFRα and β form homo-and heterodimers [32, 33]and have been shown to have different signaling properties[34].

Importantly, the Type III RTKs are normally held in amonomeric inactive state due to an autoinhibitory mecha-nism. With respect to Flt3 the crystal structure revealed thatthe juxta membrane (JM) domain buries the catalytic centreof the kinase and stabilize the inactive confirmation. In gen-eral, ligand stimulation is thought to induce conformationalchanges, receptor dimerization and autophosphorylation ofkey tyrosine residues in the JM domain which is needed tostabilize the open confirmation [35]. Shortly after ligandactivation the RTKs are ubiquinated by Cbl and internalized[36]. Finally, specific receptor and non-receptor PTPs con-tribute to keep the RTKs and other PTKs in an inactive stateas discussed above. Exposure of tyrosine residues in the JM,kinase insert (KI) and C-terminal tail results in autophos-phorylation. These phosphotyrosine residues attract adaptorproteins with tyrosine binding domains (PTBs) and proteinswith src homology 2 (SH2) domains [35].

The mechanism of activation for non-receptor PTKs isquite similar to that of Type III RTKs. Src family membersfor example are kept inactive by masking the SH2 and SH3domains through cis-interaction with the C-terminal phos-photyrosine (Y527). Activation of Src can occur 1) uponbinding of a protein to the SH2 or SH3 domains, 2) bydephosphorylation of the C-terminal Y527 and 3) throughphosphorylation of a tyrosine residue (Y416) in the tyrosinekinase (TK) domain [37]. The PTK Abl, on the other hand,can be kept inactive by trans-interaction with the retino-blastoma protein (Rb) inhibiting the Abl TK domain, thusprohibiting the nuclear survival signaling by Abl [38].

ONCOGENIC TYROSINE KINASES IN MYELOIDMALIGNANCIES

Genetic Aberrations

The most common mechanism for generation of consti-tutive active PTKs in hematological malignancies is genefusions as a consequence of specific acquired chromosomaltranslocations [39]. The resultant chimeric PTK proteinscontain a partner protein at their N-terminal and a PTK do-main at the C-terminus. The partner proteins often harbor adimerization/oligomerization domain responsible for consti-tutive activation of the chimeric PTKs. It is also the controlelements of the translocation partner that regulate the expres-sion of the fusion PTK.

The best known example of gene fusions is the translo-cation of the bcr gene on 22q11 to the 5’ of the abl gene on9q34 [40]. The Bcr part of the fusion kinase contains acoiled-coil oligomerization domain which induce tetrameri-zation of the Bcr-Abl, resulting in constitutive activation[41]. Bcr is also found to be the partner protein of other on-cogenic kinases (Table 1). Another protein often implicatedas a fusion partner in creation of oncogenic PTKs is the geneencoding ETV6/TEL. This gene has been reported to fuse to22 different genes, among them the 6 PTKs in myeloid ma-lignancies (Table 1). Again constitutive activity is mostlikely created due to the HLH oligomerization domain inETV6 [42].

The majority of the RTK fusions lack the transmembraneregion of the RTK (except PDGFRβ fusions) which mightinfluence signaling properties. Centrosomal localizationwhen the partner proteins is a centrosomal protein is for in-stance seen for the oncogenic FGFR1 fusions found in theaggressive 8p11 myeloproliferative syndrome/stem cell leu-kemia-lymphoma syndrome [43]

A second important aberrancy responsible for oncogenicPTKs is submicroscopic mutations that may disrupt theautoinhibition of kinase activity. The most common kinasebecoming oncogenic by this mechanism in myeloid leukemiais Flt3. Two different classes of Flt3 mutations have beendescribed: 1) mutations in the JM domain (internal tandemduplications (ITD) and/or missense mutation) and 2) muta-tions in the activation loop in the TK2 domain (missense/ in-frame deletions/ insertions) [44-46]. The ITD is the mostfrequent Flt3 mutation and is associated with shorter event-free and overall survival, whereas the prognostic significanceof activation loop mutations is so far unclear [47, 48]. Dif-ferences in signal transduction are also found between thesetwo types of mutations [49, 50]. Submicroscopic mutationsare also found in other members of the type III RTK in AML(Table 1). Kit mutations commonly found in core-bindingleukemias harboring t(8;21)(q22;q22) or inv(16) [51-53],appear to adversely affect this otherwise good prognosticsubgroup [53-56]. In the non-receptor PTK family, activat-ing mutations situated in the pseudokinase domain of JAK2has recently been described. The V617F mutation is a fre-quently found in polycythaemia vera (PV), essensial trombo-cytosis (ES) and myelofibrosis (MF) [57, 58], and has alsobeen found in chronic myelomonocyte leukemia, myelodys-platic syndrome (MDS) and AML [59]. The patients withthis mutations have a significant higher rate of complications[58, 60].

Proteomics to Elucidate Oncogenic Tyrosine Kinase Current Pharmaceutical Biotechnology, 2006, Vol. 7, No. 3 3

A third oncogenic event resulting in constitutively activePTKs is increased or aberrant expression of the RTK itself,its ligand or both. FL stimulation of AML blasts results inincreased proliferation and reduced apoptosis in the majorityof patient samples. FL has a growth enhancing effect evenfor AML blasts with Flt3-ITDs [61]. Phosphorylation of Flt3as a result of autocrine stimulation is a common event inAML [30]. Co-expression of Kit and CSF1R with their re-spective ligands has also been found in this disease [62]. Inaddition, over expression of RTKs can result in constitutive

activation. Elevated levels of the Flt3 transcript has beenidentified and associated with constitutive activation of Flt3in AMLs without Flt3 mutations or the presence of FL [63].This is thought to be an unfavorable prognostic factor foroverall survival. However, cells with over-expression of Flt3have the same sensitivity towards Flt3 inhibitors as withcells harboring Flt3 mutations. High Kit expression is foundin core-binding AMLs [64] and a microarray study showover expression of FGFR1 and CSF1R in AML subgroups[65].

Table 1. Overview of the Different Genetic Aberrations Involving Tyrosine Kinases

Oncogenic kinase Rearranged with DiseaseSub-microscopic

mutationDisease

Over-expressionfound in

ABL 9q34 BCRETV6

22q1112p13

CML, AML [40]AML, aCML [42],

MDS [60]

ABL2 1q25 ETV6 12p13 AML [161]

JAK2 9p24ETV6BCR

PCM1

12p1322q118p23

aCML [162]aCML [163]

aCML, AML, MDS [164]

V617F PV, ET, MF, CMML,AML, JMML [57, 58]

FGFR1 8p11 FGFR1OPTRIM24CEP110

FGFROP2ZNF198

MYO18ALOC113386

BCR

6q277q329q3312p1113q1217q1119q1322q11

MPD [166]MPD [167]MPD [168]MPD [169]MPD [170]MPD [171]MPD [172]MPD [173]

AML [165]

PDGFRβ 5q33BCRETV6HIP1

CCDC6KIAA1509

TRIP11NIN

TP53BP1SPECC1RABEP1PDE4DIP

22q1112p137q1110q2214q3214q3214q2415q2217p1117p131q23

aCML [174]CMML /CEL [175]

MPD [176]aCML [177]MPD [178]AML [179]MPD [180]MPD [181]

JMML [182]CMML [183]MPD [184]

PDGFRα 4q12FIP1L1 4q12 HES/CEL [186]

exon 17, 19 AML [185]

NTRK3 15q25 ETV6 12p13 AML [187]

SYK 9q22 ETV6 12p13 MDS [188]

FLT3 13q12 exon 14, 20 AML [44-46] AML [63]

KIT 4q12 exon 8,11,17D816V

AML [51-53]SM [189-191]

AML [64]

CSF1R 5q33 exon 7, 22 MDS, AML [192] AML [65]

The frequency of the different aberrations is highly variable. Acute myeloid leukemia (AML), Myelodysplastic syndrome (MDS), Chronic myeloid leukemia (CML), atypicalchronic myeloid leukemia (aCML), Chronic myelomonocytic leukemia (CMML), Chronic eosionophilic leukemia (CEL), Myeloproliferative disease (MPD), Polycytemia vera (PV),Essensial trombocytosis (ET), Myelofibrosis (MF), Systemic mastocytosis (SM), Hypereosinophilic syndrome (HES), Juvenile monomyelocyte leukemia (JMML).


The fourth oncogenic event which induces constitutivePTK signaling is by indirectly deregulation due to reducedPTP activity or decreased expression of natural inhibitors.Epigenetic silencing of gene expression by hypermethylationof the promoter sequence is a general mechanism of inacti-vating tumor supressors. Hypermethylation of the Suppres-sor of Cytokine Signalling 1 (SOCS-1) has been found inMDS and AML [66]. The PTP, Shp1, is associated with re-duced expression in leukemias, lymphomas and multiplemyelomas due to hypermethylation of its promoter [67].Shp1 belongs to the SH2 domain-containing PTPs (Shps)which is a subfamily of non-transmembrane PTPs (Fig. 1)[68]. Shp2, another member of the family, is however sug-gested to have dual functions in myeloid malignancies. It isinvolved in promoting RTK signaling by removing phos-photyrosine residues that inhibits activation of the Ras-Erkpathway. Constitutively active Shp2 due to mutations is fre-quently observed in AML-M5 and, in this case, Shp2 ischaracterized as an oncogene [66].

Oncogenic Tyrosine Kinase Signaling

Signaling events induced by mutated PTKs and their wildtype counterparts often affect common signal pathways [71,72] (Fig. 1).

Fambrough et al. gave evidence for the hypothesis thatdifferent RTKs (e.g. both PDGFRβ and FGFR-1) essentiallyinduce identical immediate early genes [69]. However, it hasto be taken into account that this reflects studies on cell lines.The physiological response of a RTK in an intact organismcan only be understood by the knowledge of cell type, cellcompartmentalization and localization of signal molecules(“membrane rafts” enriched with signaling molecules), crosstalk between pathways, combinatorial control of specifictyrosine phosphorylated sites and their particular adaptors[25, 70]. Finally, signal duration and strength due to the in-hibitory mechanisms and redundant signaling molecules (su-perfluous molecules with similar functions) are believed tobe of importance.

Mutated PTKs frequently acquire constitutive activityand therefore confer factor independent growth, e.g. to Ba/F3and 32D cells and is an experimentally striking characteris-tics with oncogenic PTK signaling [6, 42]. In addition, theoncogenic fusion PTKs are subjected to regulations that re-side in protein domains and gene elements of the respectivefusion protein [73]. Quantitative differences are identifiedespecially with respect to elevated phosphorylation level andhigher transcriptional activation extent of target genes. Thus,oncogenic PTK signaling cannot be constrained by normalPTP activity and apoptotic mechanisms seems to be ignored.Some identified differences between mutated and wild typePTKs are discussed briefly.

Flt3-ITD cause transformation of Ba/F3 cells but mightin addition block myelopoiesis [74]. Flt3-ITD has been sug-gested to associate with the chaperone heat-shock protein 90and retain in the ER indicating aberrant signaling propertiesdue to dyslocalization [75, 76]. The MAPK/RSK1 and PKApathways are suggested as the principal pathways used byFlt3-ITD to trigger phosphorylation of the pro-apoptotic Badthereby inhibiting apoptosis [77]. It has also been indicatedthat Flt3-ITD activates transcription of Bcl-XL through sig-

nal transducer and activator of transcription (STAT)STAT5a [78] even though not proven for Flt3, STAT5 isdirectly phosphorylated by PDGFRβ [79]. Flt3-ITD wasshown to negatively regulate Foxo transcription factors andrepress the function of transcription factor CCAAT/enhancerbinding protein α (CEBPα), the latter through ERK1/2-mediated phosphorylation [80, 81]. Foxo and CEBPα arecrucial for cell survival and myeloid differentiation respec-tively [80, 81]. Over-expression of Flt3 as discussed aboveinduces the nuclear factor-kappa B (NF-κB) pathway andthus expression of survival genes [82].

STATs are activated by oncogenic PTKs (Flt3, Bcr-Abl,TEL-PDGFRβ, TEL-JAK2) in leukemias and differentSTATs seem to be associated with different oncogenic PTKs[72, 83]. STATs dimerize upon tyrosine-phosphorylation andenter the nucleus where they activate transcription of sur-vival genes [84]. Src PTK family members mediate the sig-naling from oncogenic Type III RTKs and Bcr-Abl toSTATs, however, direct interactions between Type III RTKs,e.g. PDGFRβ, and STATs has also been shown [73]. JMtyrosine-phosphorylated residues are necessary for TEL-PDGFRβ full-activation of STAT5. Activation of phosphati-dylinositol 3-kinase (PI3K) and phospholipase Cγ (PLCγ), inaddition, is needed for transformation [85]. The K641E andthe del559 mutants of Kit differ in their constitutive activa-tion of Akt, MAPK and STATs pathways [86]. Phosphory-lation of STAT3 has been associated with mutations of theD816 in Kit and activation of this pathway has been postu-lated to be required for its transforming ability [87].

The Abl kinase is related to the Src PTK family but con-tains additional regulatory domains (Fig. 1). Abl is importantin cell cycle regulation and is normally inhibited by Rb inthe G1 state. In contrast, the oncogenic Bcr-Abl chimericproteins resulting from chromosomal translocations induceboth constitutive and elevated tyrosine kinase activity [88].The Bcr part of the CML-associated p210 Bcr-Abl protein isessential for full oncogenic activation, and p210 Bcr-Abl isinvolved in induction of Ras and PI3K signaling [88, 89].STAT5 and NF-κB are constitutively activated by Bcr-Abl[88-91]. Moreover, Bcr-Abl inhibits apoptosis by blockingrelease of cytochrome C due to direct phosphorylation ofBad but can also mediate survival signaling independent ofBad, through phosphorylation of MAPK and PI3K pathways[92]. Furthermore, Bcr-Abl is postulated to modulate tran-scription possibly due to its impact on chromatin remodelingat promoters of survival genes [93].

PROTEOMICS IN STUDYING ONCOGENICTYROSINE KINASE SIGNALING

A wide variety of genetic and epigenetic events contrib-ute to dysregulation of PTK in myeloid hematological ma-lignancies. Until now the diagnostic techniques are DNAbased and numerous gene specific tests are needed to detectall the mutations. As a number of new clinical drugs are de-signed to inhibit signaling pathways it would be more effi-cient to determine the activity of PTKs rather than searchingfor all the possible mutations in the different kinase genes.As reviewed in the previous section different mutations in-duce different signaling. By using proteomic techniques,dysregulation of specific PTKs can be detected as specific


biosignatures, as recently shown by Irish et al. [7]. However,increased understanding of how the oncogenic PTKs affectsignaling pathways is needed in order to define these specificbiosignatures. In the next sections we will describe differentmethods that can be used for such proteomic studies (over-viewed in Fig. 2).

Cellular Model System

Most signaling pathways are mapped and characterizedin cell lines, and only to a limited extent examined in pri-mary cells or intact organisms. Studies of primary AML cellsindicate substantial heterogeneity in signal responses be-

Fig. (1). Signaling by oncogenic protein tyrosine kinases.

The signaling events presented in the figure are highly simplified and all intermediates are not shown. Type III RTKs, activated by mem-brane bound or soluble ligand-dimer(s) are characterized by an extracellular domain (EC) consisting of five immunoglobuline folds withligand binding properties, a transmembrane region (TM), an intracellular juxtamembrane region (JM) and two intracellular kinase domains(TK1 and TK2) separated by a kinase insert domain (KI). Bcr harbors N-C terminally a CC domain (oligomerization, critical for transforma-tion) a Ser/Thr kinase domain (S/T, with a SH2 binding domain), a Dbl domain and a pleckstrin homology (PH) domain. Abl consists of N-to C-terminally a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain, a PTK domain (TKD), a proline rich nuclear localizationsignal (PPP), a DNA binding motif (DNA) and an actin interacting site (Actin). Src family PTKs and Shp family PTPs both are attracted tothe membrane upon activation.

General signaling components involved are: 1) Adaptor proteins (Grb2, Dok1, Cbl, Crk1) [193], PTKs and PTPs [194] (Src PTK familymembers, the SH2 domain of SHP2 PTP and RasGAP a GTPase-activating protein); 2) phospholipase Cγ (PLCγ) involved in phosphoinosi-tol metabolism [126]; 3) phosphatidylinositol 3-kinase (PI3K) pathway by binding of its p85 regulatory subunit to two phosphotyrosine resi-dues through its SH2 domain [195]; 4) NF-κB transcription factor (Inhibitor of NF-κB, IκB is phosphorylated and degraded by the protea-some) [196]; 5) MAPK pathway (through the adaptor molecule Grb2 which forms a complex with the nucleotide exchange factor acting onRas) [197]; 6) Signal transducer and activator of transcription (STAT), promotes transcription of survival genes [71, 79, 198].

The lines show signaling events and double arrow heads at the ends indicate interactions, single arrow head indicates activation and dot indi-cates inhibition. Yellow circle indicates tyrosine phosphorylation stretches/sites of regulatory importance. Red star indicates oncogenic mu-tations.


tween different patients [7, 61]. This heterogeneity is re-flected in clinical presentation by pronounced variations inboth karyotypes and mutations of signaling molecules in thecancer cells [94-96]. Cryopreserved AML cells in vitro mayrepresent a powerful disease model. As in AML, the malig-nant primary cells are easy to harvest from the patient by asimple blood sample and the cells can be stored in liquidnitrogen after a density separation [97-99]. However, limitedsample material and difficulty with in vitro culturing [100]complicates the use of patient cells for mass spectrometry(MS)-based proteomic approaches. Flow cytometry (de-scribed in STUDIES ON SINGLE CELLS) on the otherhand, is well suited for identification of patient specificbiosignatures [7]. In MS-based proteomics the amount ofprotein needed for analysis may be substantial, exemplifiedby the phosphopeptide studies of growth factor signaling

where 10 to 1000 million cells have been used [101, 102].These studies have been performed on cell lines, which incontrast to primary cells, contain a far higher amount ofprotein per cell due to perturbed size regulation in vitro[102]. A further problem with cell lines is the possibility forcell contamination and selection for in vitro culturing de-pendent features that are not observed in vivo, e.g. mutationsin the antioncogene TP53 [103-105].

STUDIES ON CELL EXTRACTS

A biosignature specific for an oncogenic PTK can consistof a phosphorylation site on the kinase together with activa-tion of a adaptor protein and other downstream target. Inorder to decide the best signature thorough mapping of theoncogenic PTK signaling pathway is needed. 30% of all

Fig. (2). Overview of different techniques used for analysis of phosphorylated proteins.

Phosphorylation events can be studied on single cells using flow cytometry. Protein extracts are used for mass spectrometry (MS)-basedproteomic studies. The general strategy is enrichment and reduction of complexity. The reduction of complexity is either though gel separa-tion or through HPLC fractionation. Western blot can be used for verification of MS results and to check whether the environment procedurehas worked. Here we show Flt3, Src and Grb2 in total lysate and after immunoprecipitation of Flt3. None of these proteins bind unspecifi-cally to the beads (preclear). The majority of Flt3 is immunoprecipitated (unbound vs. eluate). Both Scr and Grb2 coimmunoprecipitate withFlt3.


proteins in a cell are estimated to be phosphorylated at anygiven time [24]. Most proteins have several phosphorylationsites and which site that is phosphorylated depends on thesignal [106]. Furthermore, only 0.05% of all phosphorylationevents involve tyrosine. Thus, each signal specific modifica-tion is present at a very small amount. Therefore, a centralproblem when identifying phosphoproteins involved in asignal transduction pathway, is to obtain sufficient amount ofphosphorylated material for analysis. In the following, wewill present different considerations and methods for en-richment, and discuss how these can be coupled to variousmethods for detection/identification.

Protein Extraction and Enrichment of PhosphorylatedProteins and Peptides

Three major obstacles in phospho-proteomics in myeloidcells are 1) dephosphorylation by released phosphatases, 2)requirement of protein/peptide enrichment due to low abun-dance of phosphoproteins and 3) that myeloid/leukemic cellsare complex and fragile.

In order to preserve all phosphorylations during the proc-essing of the sample it is recommended to carefully plan thesuitable conditions for cell lysis and buffer composition. Cellmembranes can be disrupted chemically using detergents ormechanically like homogenization, and the method of choicedepends on the downstream analysis. When using detergentstitration of the detergents are often necessary since the nu-clear membranes of myeloid cells are easily disrupted uponlysis. In order to reduce sample complexity and enrich low-abundance proteins general subcellular fractionation tech-niques can be used, reviewed by Huber et al. [107]. This canbe followed by conventional chromatography methods suchas gel filtration, ion exchange, immunoaffinity and metal-chelate affinity chromatography. The immunoaffinity me-thod, immunoprecipitation, is widely used for proteomicstudies. Proteins are isolated by binding to a specific anti-body attached to a sedimentable solid matrix. The antibodyused can be protein specific or modification specific. Thepool of tyrosine-phosphorylated proteins is efficiently en-riched using immunoprecipitation with phospho-tyrosineantibodies. A broader spectrum of tyrosine-phosphorylatedproteins can be achieved by combining different phospho-tyrosine antibodies since their binding specificities are notcompletely overlapping [108]. Currently, no antibody is suit-able for enrichment of serine/threonine phosphorylated pro-tein. For enrichment of these proteins immobilized metalaffinity chromatography (IMAC) or commercial columnscan be used [109].

Affinity purification is also a useful method to studyprotein-protein interaction. Co-immunoprecipitation relieson the ability of an antibody to specifically bind complexescontaining the epitope. However, it is difficult to separatebackground binding and still preserve functional importantinteractions. Because of the biochemical diversity of protein-protein interactions, the conditions for co-immunoprecipi-tation will differ depending on the protein-complex of inter-est.

Protein-protein interactions can also be studied usingpeptides constructed based on the binding site of a specificprotein. This is nicely shown by Blagoev et al. 2003 who

used Grb2s SH2 domain coupled to GST to identify 28 pro-teins with increased peptide interaction after EGF stimula-tion [110]. Phosphorylated peptides can also be used in thisapproach [111]. Other strategies for identification of peptide-binding partners are phage display [112], array technologies[113] and peptide library [114].

To facilitate purification of a specific protein this mole-cule can be engineered to include a short stretch of residuescorresponding to an epitope. This is achieved by appendingthe sequence of the epitope tag (FLAG, HA, c-myc, His,TAP, etc) to the protein coding sequence in an appropriatevector. The epitope-tagged proteins are then purified by im-munoprecipitation or other tag-specific affinity methods.Adaptor proteins and associated binding partners will bepulled down simultaneously with the tagged proteins. Thetandem affinity purification (TAP) tag system is a methoddeveloped to identify protein complexes in cells in a nativestate [115] and results in a pure sample of the protein com-plex for MS analysis.

Enrichment of proteins and removal of components in-fluencing the downstream application can be done by variousprecipitations. Solvents used are e.g. acetone, methanol,chloroform, thrichloracetic acid, doxocholate and mixturesof these. With respect to PTKs, acetone precipitation mightbe the method to start with (For Flt3, see Fig. 3), but optimi-zation is probably required. The protein precipitates are re-solubilized in an appropriate buffer compatible with the nextstep of analysis.

Fig. (3). Immunoprecipitation to enhance Flt3 protein concen-tration.

Immunoprecipitated Flt3 upconcentrated with (A) spin column (10kDa cut-off), (B) acetone precipitation, (C) TCA precipitation. Theprotein samples were separated on SDS-PAGE and blotted againstFlt3 (Unpublished results). The two typical bands are indicated byarrows.

For identification of the phosphorylation sites in a spe-cific protein by MS it is recommended to have a Coomassie-

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stainable amount. Still, enrichment of the phosphopeptidesmight be needed. Many methods have been suggested forenrichment of phosphopeptides prior to MS and to date ageneral approach satisfying yield and purity has not beenestablished. However, several dimensions of enrichment canbe applied in order to have the sample ready for MS analysisand these will be introduced briefly.

IMAC columns complexed with Ga3+ or Fe3+ have beenwidely used with variable success [116, 117]. Unfortunately,beside phosphopeptides the material also binds acidic pep-tides, but this can be reduced by methyl esterification ofacidic residues [118]. IMAC material can be used to makenano-columns which followed by nano-reverse phase withsequentional hydrofobicity are useful for matrix-assistedlaser desorption ionization time of flight (MALDI-TOF)studies of phosphopeptides [106, 119]. The use of graphitecolumns to desalt and upconcentrate improves the detectionof hydrophilic phosphopeptides [120]. For liquid chromatog-raphy (LC)-MS studies, the IMAC columns can also be usedas part of two/multi-dimensional separation [121]. An inter-esting alternative to the IMAC procedure is the use of TiO2

for enrichment of phosphopeptides. This material shows lessbinding of the unphosphorylated acidic peptides than IMACand good compatibility with reverse-phase chromatography.An immunoprecipitation approach for enrichment of phos-photyrosine peptides are generally considered to be ineffi-cient [122], but a recent publication contradict the prevailingview [123].

High pressure liquid chromatography (HPLC) is a highthroughput separation technique and both strong cation ex-change (SCX) columns and strong anion exchange (SAX)columns have been used in order to enrich phosphopeptides.With SCX the first fractions contain most of the phos-phopeptides as they are negatively charged due to the phos-phoryl group [124]. The SAX column utilizes this chargeand captures the phosphopeptides till they are eluted in thelast fraction [125].

Hypothetically in silico predicted tyrosine phosphoryla-tion sites or sites identified by MS can be investigated invivo. By using site-directed mutagenesis amino acid substi-tutions e.g. tyrosine to phenylalanine (chemically and struc-turally similar to tyrosine but lacking the hydroxyl groupwhich can be phosphorylated) can be introduced and used intransfection studies [126].

Gel-Based Separation and Identification

Traditionally proteins involved in oncogenetic signalinghave been studied by separating cell extracts on sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [127] followed by Western blotting. Educatedguessing have been used to decide which proteins to lookfor. An increasing number of antibodies specifically raisedagainst activated PTKs, phosphorylated on specific tyrosineresidues, are available although the specificity and sensitivityof these proteins are very variable (www.antibodyresource.com). This approach is useful, however, for confirmation ofresults from MS-based methods.

Two-dimensional gel electrophoresis (2DE) [128] is apowerful tool to separate proteins by their isoelectric point(pI) (first dimension) and molecular weight (Mw) (second

dimension). Each spot on the gel normally corresponds to asingle protein with a certain pI/Mw. Depending on the sensi-tivity needed; the 2DE spots can be visualized by generalprotein stains (e.g. Sypro Ruby (1 ng/spot), silver nitrate (10ng/spot) or Coomassie (100 ng/spot) staining). By combiningmetabolic labeling using radio-labeled methionine (generalprotein labeling) or phosphate (phosphorylation specific)with 2DE gels the sensitivity can come down to 200 fg/spot.This method is even more sensitive than using phosphoty-rosine specific antibodies. There are commercially availablestains for detecting phosphoproteins in-gel, but these stainsare not very sensitive.

Modifications will alter the pI/Mw of the protein. Phos-phorylations decreases the pI of the precursor protein withone charge for each phosphate added, thus, multiphosphory-lated proteins may appear as “beads on a string” on the gel(See Ånensen et al. in this issue). However, it is important tobe aware of that such acidic shifts not always appear due toauthentic post translation modifications, but might be arti-facts due to sample preparation [129]. Alterations in the sig-nal pathway induced by oncogenic PTK are reflected in the2DE pattern when comparing gels from cells with activeversus inactive oncogenic PTKs. A combination of an effi-cient pre-fractionation of radioactively labeled proteins priorto 2DE separation seems, at present, to be the best choicewhen relying on a gel-based approach to identify phos-phorylated proteins involved in signal transduction path-ways. By incubation the gel with 1N KOH (55oC, 1-2 h) onecan remove Ser/Thr- phosphorylated proteins and focus onthe tyrosine phosphorylated [130]. Analysis of 2DE patternscan be performed using a number of different available soft-ware. Protein spots of interest are picked from the gel, cutinto peptides with a specific protease and identified usingMS.

A gel-based approach has some limitations. Not all pro-teins are separated well in the 2DE gels. This includes mem-brane proteins and proteins with a far basic or acid pI. Addi-tionally, the staining sensitivity of 2DE gels, are, at present,lower than what is possible to analyze by MS. On the con-trary, the large advantage of 2DE gels is that protein modifi-cations, such as phosphorylations, can be visualized.

Characterization of Phosphorylated Proteins by MassSpectrometry

Unknown proteins, separated by gel electrophoresis orLC can be identified by cleaving the proteins into peptidesby a protease such as trypsin. The absolute masses of theresulting peptides are measured by MS. These masses arethen compared in silico to theoretically cleaved genometranslates and a statistically best match is given. In the caseof phosphoproteins, trypsination will generate both phos-phorylated and unphosphorylated peptides which has to besorted by front-line chromatographic techniques or by themass spectrometer itself. The phosphopeptides to be ana-lysed are ionized in the mass spectrometer either by aMALDI or by electrospray ionization (ESI). The mass spec-trometer will determine the mass/charge (m/z) of the ionizedpeptides. Phosphopetides can be identified as phosphorylatedby analysing the sample before and after alkaline phospha-tase treatment, as it will result in a mass shift of -80 Da, cor-


responding to the loss off HPO3 [131]. Phosphatase treatmentcan also be used in combination with reverse-phase diagonalLC, as dephosphorylation renders the phosphopeptides morehydrophobic, so that they can be selected for [132]. In bothcases, the dephosphorylated peptides are submitted toMS/MS experiments to localize the phosphorylated residue.Both, MALDI- and ESI-based instruments can be used tolocalize the phosphorylation site(s) of the phosphopeptide,but ESI is preferred as it generates doubly and triply chargedions which will increase the possibility to identify the phos-phorylated residue.

Not all phosphopeptides are stable during MS. This in-cludes serine- and threonine-phosphorylated peptides. Thesephosphopetides will tend to loose their phospho moiety asmeta-phosphoric acid (-80 Da) and in a more intensive extentphosphoric acid (-98 Da) [133]. This is not the case for tyro-sine-phosphorylated peptides, which are stable during MS. Apossible way to identify and characterize tyrosine-phos-phorylated peptides in one run is to use a mass spectrometercapable of precursor ion scanning (PIS), e.g. a triple-quadrupole-instrument. In such, the first quadrupole is usedas a mass filter, the second as a collision cell to fragment thepeptides and the third to monitor certain fragment productsgenerated by the second quadrupole [134]. In the case oftyrosine-phosphorylated peptides the fragment product ofinterest is the immonium ion m/z = 216.043 Da. The instru-ment can then select the precursor ion (peptide) of this frag-ment product and obtain sequence information by MS/MS orMS3 to identify the phosphorylated tyrosine residue.

Electron capture dissociation (ECD) is an alternative ionactivation method. More recently, new ECD-based highresolution instrumentation dedicated for protein and peptidesequencing have been developed [135-137]. ECD methodsare very suitable for studying protein phosphorylation sincethe modification remains intact.

Quantitative Phosphoproteomics

The peptide mass signal generated by the most com-monly used mass spectrometers is not suitable for quantita-tive analysis of phosphorylation states. The reason being thationization efficiency of a phosphopeptide versus the respec-tive unphosphorylated peptide or between different peptidesin general is unpredictable [138]. At present the only possi-bility that combines peptide identification and quantificationof phosphorylation state is to compare to chemically similarpeptides. This can be done by incorporating stable-isotopesinto the peptides both in vitro and in vivo. In vitro labelingmethods includes the introduction of different tags contain-ing heavy atoms to the peptides [139, 140] or tryptic diges-tion in the presence of 18O-labelled water [141]. Alterna-tively, proteins may be labeled in vivo by culturing cells in amedium containing stable-isotope containing amino acids,i.e. the Stable Isotop Labeling with Amino acids in Cell cul-ture (SILAC) method [142]. Thus, in vivo labeling is re-stricted to cell culture-based model systems. A typical ex-periment using the SILAC method would then start by meta-bolic labeling of cells with either heavy or light isotope-containing amino acids prior to exposing the cell populationwith vehicle or the treatment factor of interest. Cell lysatesare then mixed 1:1 and separated by an SDS-gel. Appropri-

ate sized bands are excised from the gel, proteins are tryp-sinated and the tryptic digests are analyzed by LC-MS/MS.The intensity difference between the heavy and the lightisotope containing but otherwise identical peptides can thenbe determined. Enrichment or selection of phosphopro-teins/phosphopeptides may be performed after mixing thetwo lysates or after tryptic digestion. Such approach wasused to elucidate the signaling pathway of the epidermalgrowth factor receptor by using affinity purification over aSH2 domain selecting for the tyrosine-phosphorylated pro-teins [110]. It should be noted that not all amino acids aresuitable candidates for isotopic-labeling. The phosphoryla-tion targets serine, threonine, and tyrosine are examplethereof, as they all during catabolism can break down toother amino acids. Therefore, phosphopeptides to be ana-lyzed by SILAC method must contain amino acids fre-quently used for stable isotope labeling, as arginine or lysine.

There is, however, a method that is capable of accuratequantitation of phosphorylations without the use of stableisotopes. This element MS performed with inductively cou-pled plasma (ICP)-MS. This method enables simultaneousmeasurement of 31P and 32S to determine the phosphorylationdegree of intact proteins or peptides [143]. Unfortunately,after ICP-MS ionization no MS/MS-sequence informationcan be obtained. Therefore, the sample has to be split beforeICP-MS to obtain structural information. Interestingly, phos-phoproteins may also be screened “in gel” by laser ablationICP-MS [144].

STUDIES ON SINGLE CELLS

When cells are lysed, their proteins loose both cellularmilieu and subcellular locations. Utilizing the techniquesdescribed in the previous chapter important information ofthe different PTKs and their signaling partners is gained, butsome information is also lost. This can be information linkedto time spans in regulation or exact protein location in thecell at time of activation or inactivation. Techniques likephosphoprotein analyses by flow cytometry or fluorescenceresonance energy transfer (FRET) imaging in living cells canovercome these obstacles and elucidate signaling pathwaysboth in time and space. These methods will be briefly de-scribed in the following sections.

Using flow cytometry one can count, examine, and de-pending on the flow cytometer also sort living cells from astream of fluid. A laser light with specific frequency is di-rected onto a focused stream of fluid. In the flow cytometer anumber of detectors are aimed at the cells in the fluid; one inline with the laser beam (Forward Scatter or FSC) and sev-eral perpendicular to it (Side Scatter or SSC) and one ormore fluorescent detectors. As much as 19 parameters (17fluorescent colors and FSC/SSC) can be studied at the sametime [145]. Each cell passing through the beam scatters thelight in some way, and e. g. specific fluorescent antibodies(extracellular or intacellular labeling) attached to the cellmay be excited into emitting light. This information ispicked up by the detectors, and by analyzing fluctuations inbrightness at each detector it is possible to deduce variousfacts about size, granularity, cell surface markers and intra-cellular proteins of the cells. The use of phoshpospecificantibodies enables researchers to get in situ information


about the phosphoproteome state at a given time point in agiven population of cells. Single cell proteomics using flowcytometry show a different picture than conventional bio-chemical or proteomic methods and gives a more complexsignaling network more likely to be an authentic situation[7]. For a review on single cell proteomics in mapping ofsignaling pathways in cancer see Irish et al. [146]. Singlecell proteomics by the means of flow cytometry is utilized inprimary AML-cells in a recent paper [147].

FRET is a transfer of energy between two fluorescentmolecules. A donor molecule is excited at its specific excita-tion wavelength and the energy obtained is then transferrednon-radiatively to a second molecule, the acceptor, causingan emmition of fluorescence from the acceptor [148]. FRETcan be used to study signaling pathways by the use of in-tramolecular probes [149]. This is extensively reviewed inKiyokawa et al. [150]. A typical FRET probe for the studiesof PTK activity in a living cell consists of a fluorescent pro-tein (e.g. CFP), a phosphotyrosine binding domain, a con-sensus substrate for the relevant PTK and another fluorescentprotein (e. g. YFP) [151]. Activation of the PTK by additionof a cellular stimulus will lead to a decrease in yellow tocyan emission ration while inhibition of the kinase will leadto the inverse. The fluorescence is calculated using fluores-cence microscopy and imaging.

COMPUTATIONAL APPROACHES

Cell signaling induced by both native and oncogenicPTKs is a complex field of research and the overall eventsare not fully characterized. Web-servers and computer pro-grams specialized on signaling are tools available in order tosupport the unraveling of signaling events induced by proteinphosphorylation.

Mapping of Tyrosine Kinase Signaling Pathways

Interactive web-based databases are created in order tohelp to document the increasing amount of research dataemerging on signaling molecules. They often contain links toinformation about domains, interaction partners and otherdata in order to cross-link all the available information.Promising signal pathway documentation websites are theAlliance for Cellular Signaling (www.signaling-gateway.org) by the Nature Publishing Group and the Signal Trans-duction knowledge Environment (http://stke.sciencemag.org)by Science, Advancing Science Serving Society. Anothersite with a delicate user interface is the Biomolecular Inter-action Network Database (www.BIND.ca) from UnleashedInformatics, Canada [152]. The Protein Lounge (www.proteinlounge.com) among others offer, in addition to signalpathways, a tool to build pathways yourself (Pathwaybuilder). Cell Signaling Technology (www.cellsignal.com)has put a lot of effort into interactive signaling maps whichcan be very useful when looking for available products inyour favorite pathway.

The likelihood of protein interactions to occur might besimulated in silico and there are several prediction programs/databases available. Search Tool for the Retrieval of Inter-acting Genes/Proteins, String, (http://string.embl.de) is adatabase where probable interactions can be searched for

where the use is extended beyond the species in which origi-nally described [153]. (see Kleppe et al. this issue). How-ever, such databases should be frequently updated and in-clude oncogenic kinases.

Prediction of Protein Phosphorylation

Hypothetical tyrosine phosphorylation sites might bepredicted by NetPhos (www.cbs.dtu.dk/services/NetPhos) orPhospho.ELM (http://phospho.elm.eu.org) [154]. Targets forphosphorylations likely to occur can also be predicted byhomology searches or alignments with closely related pro-teins (e.g. PTKs of the same family). Predicted phosphoryla-tion sites can give valuable information about the likelihoodof docking sites for adaptor molecules and maybe indicateoncogenic potential .

CLINICAL IMPLICATIONS

As our insight on mutations involved in development ofmyeloid malignancies increases new diagnostic analysis aredeveloped to identify patients with these aberrations. Untilnow these diagnostic techniques aim at identifying the ge-netic aberration creating the oncogene and a number of dif-ferent analyses are needed. Gene expression studies usinggene arrays have shown to add new information to biologicaland clinical classification of these diseases, and may be usedfor diagnostic purposes in the near future [155].

Based on the knowledge gained by identifying oncogenicPTKs, the current trend in cancer drugs is to target the dys-regulated kinase by antagonists or antibody. Imatinib is oneof the first drugs in this class. Imatinib was initially designedto inhibit the tyrosine kinase activity of Bcr-Abl [156], but isalso identified to inhibit an increasing number of other tyro-sine kinases such as ETV6-PDGFRβ, Kit and FIP1L1-PDGFRα. Patients with hypereosinophilic syndrome andchronic eosinophilic leukemia with FIP1L1-PDGFRα fusionrespond well to imatinib treatment. However, FIP1L1-PDGFRα negative patients with the same disease also re-spond to imatinib treatment, indicating dysregulation ofother imatinib responsive oncogenic kinases in this disease[157-159].

From a therapeutic point of view when using these newdrugs, determination of the activity of PTKs is the most im-portant. By phosphoproteomics, specific biosignatures canbe detected and make the basis for kinase-directed treatmentof patients with myeloid malignancies. However, in order todesign these specific signatures more proteomic studies haveto be done. A quantitative MS based approach is useful whensearching for proteins that can be included in an oncogenespecific signature. The initial studies have to be preformedusing cell lines since this approach require quite large quan-tity of proteins due to low abundance of the phosphoproteins.Until know, the mass spectrometer has primarily been usedfor research purposes. To be able to use a mass spectrometerin a reliable clinical diagnostic platform, the methodologyfor isolation and enrichment of phosphoproteins has to bestandardized and the MS machines have to be more sensitiveand show even higher grade of reproducibility. Flow cy-tometry can be used to simultaneously measure multiple sin-gle cell phosphorylation events, and is a method that can


easily be performed in established routine diagnostics plat-forms. The method has the advantage that relative smallamount of cells are required for an assay. We have recentlyshown that by combining the basal phosphorylation level inpatients cells with the phosphorylation level after cytokinestimulation, we where able to create phosphoprotein profilesthat correlated with genetics and phenotypes of AML [7].However, standardization and reliable quantitative aspectshave to be solved before protein based diagnostics couldplay a major role in future diagnostics (see also Sjoholt et al.in this issue).

ACKNOWLEDGEMENTS

We thank Ann Krisitin Frøyset for providing pictures forFig. (2) and Fig. (3). The authors are financed by the HelseVest RHF, The Norwegian cancer society and The Norwe-gian Research Council Functional Genomics program fundedby Norwegian Research Council.

ABBREVIATIONS

2DE = Two dimensional gel electrophoresis

AML = Acute Myeloid Leukemia

CML = Chronic Myeloid Leukemia

ECD = Electron Capture Dissociation

ESI = Electrospray Ionization

FCS = Forward scatter

FL = Flt3-Ligand

FRET = Fluorescence Resonance Energy Transfer

HPLC = High Pressure Liquid Chromatography

ICP = Inductively Coupled Plasma

IMAC = Immobilized Metal AffinityChromatography

IP = Immunoprecipitation

ITD = Internal Tandem Duplication

JM = Juxta membrane

KI = Kinase Insert

KL = Kit ligand

LC = Liquid Chromatography

MALDI = Matrix-Assisted Laser Desorption Ionization

MDS = Myelodysplastic syndrome

MS = Mass Spectrometry

pI = Isoelectric point

PTB = Protein Tyrosin Binding

PTK = Protein Tyrosine Kinase

PTP = Protein Tyrosine Phosphatase

RTK = Receptor Tyrosine Kinase

SAX = Strong Anion exchange

SCX = Strong Cation exchange

SDS-PAGE = Sodium Dodecylsulphate polyacrylamidegel electrophoresis

SILAC = Stable Isotop Labeling with Amino acidsin Cell culture

SSC = Side scatter

TK = Tyrosine kinase

TOF = Time of flight

WHO = World Health Organization

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Biophys. Acta, 1378, F79-113.

analysis of acute myelogenous leukemia: preparation of samples for genomic and proteomic analyses

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