GleevecSTI571 K562 Example

Human leukemia cells (K562) derived from a pleural effusion associated with chronic myelogenous leukemia, and known to express the activated protein tyrosine kinase BCR-ABL, were studied by the methods described above for the effects of BCR-ABL inhibition by the drug STI571/Gleevec (17,18). Cultured cells (108 cells) were treated for 24 h with or without 1 ^M STI571 and processed as depicted in Fig. 3. Table 1 presents a partial listing of phosphopeptides found in lysates from these cultures, and their ratios measured in two such experiments. From analysis of total cell lysates from K562 cells in these experiments, some 274 phosphorylation sites were mapped from 137 different peptides derived from 93 distinct proteins. The majority of sites are phosphoserine, followed by phos-phothreonine, and then phosphotyrosine, each less abundant by roughly an order of magnitude. We presume this to reflect the true in vivo abundances of these three phosphoamino acid species. The average of two phosphorylated hydrox-yamino acids per peptide may reflect a bias for doubly phosphorylated peptides in the IMAC procedure, but this remains to be determined.

Immunoprecipitation of BCR-ABL protein from K562 cells afforded a direct measure of its phosphorylation and protein-protein interactions. As summarized in Table 2 (A and B), treatment of K562 cells for 24 h with STI571 caused changes in the phosphorylation and associated proteins of BCR-ABL. Most notable is the STI571-induced loss of tyrosine phosphorylation at BCR residue 177. Phosphotyrosine 177 is a known binding site for the Ras-linked adaptor protein Grb2, and this association is known to be required for BCR-ABL to transform various cells in culture and to cause myeloproliferative disease in mouse models (19,20). Curiously we did not observe tyrosine 393-containing peptides in our analysis. This residue resides in the activation loop of the Abl kinase domain. In addition to BCR residue Y177 as noted above, STI571 treatment effected the loss of phosphorylation at Abl residues Y185, Y232, and S569. Curiously, S459 was found to become phosphorylated in response to STI571 treatment. We did not observe phosphorylation at Abl Y257, which was reported to be phosphorylated in K562 cells in response to STI571 (21). The Grb2-related adaptor protein GRAP-2, the phosphoprotein p62 DOK, and the lipid phosphatase SHIP2 were found to co-immunoprecipitate with BCR-ABL recovered from K562 cells. As

K562 cells ljxM STI571 24 hrs

Control 24 hrs

Enzymatic digest

Extract proteins

Extract proteins Enzymatic digest

Heavy-Isotope label

Isolate phosphopeptides

LC/MS analysis on FTMS for quantitation

Fig. 3. Global protein phosphorylation analysis. Schematic outline of the experimental approach to global phosphoprofiling. Total cell/tissue extracts are denatured and digested, typically by using trypsin. Carboxylate groups are converted to methyl esters, and during this derivatization process a 3-Da mass tag may be added such that comparative analysis can be conducted as described in the text. Phosphate-containing peptides are enriched by using immobilized metal affinity chromatography (IMAC). Phosphopeptide mixtures are subjected to LC-MS/MS analysis for peptide identification and phosphorylation mapping. Differential analysis by Fourier transform mass spectrometer is used to quantify relative changes in the abundance of phosphopeptides. A variety of software tools are used to align the protein differential and phosphorylation/identification data sets to enable the identification of phosphopeptides modulated by the drug treatment.

expected from the loss of phosphorylation at BCR Y177, the GRAP-2 interaction, and that of DOK were abolished by STI571 treatment (Table 2). The phosphopro-tein p62 (Dok) is a strong marker of Ph1-positive BCR-ABL-expressing blast cells in crisis phase (22), and is in fact a most prominent phosphotyrosine-containing protein substrate of a variety of activated and oncogenic tyrosine kinases (6). These findings illustrate the potential of a systematic phosphopro-teomics approach to discover pharmacodynamic biomarkers to assist in the discovery and clinical development of protein kinase inhibitor drugs.

Fig. 2. Protein interactions in Saccharomyces cerevisiae. Protein interactions of protein kinases (red dots), and protein phosphatases (green dots) are represented. Protein interactions were measured by co-immunoprecipitation from yeast lysates epitope-tagged kinases and phosphatases, followed by LC-MS/MS analysis of recovered proteins. Yellow dots represent interacting proteins. Arrows point from bait proteins to the prey proteins they were found to interact with (14).

Table 1

Phosphoprofile of STI571/Gleevec™ in Human K562 Cells

Table 1

Phosphoprofile of STI571/Gleevec™ in Human K562 Cells

Peptide sequence

Protein name

Acc. #

Ratio ± Gleevec

SSpTPLHpSPSPIR

BAG-FAMILY MOLECULAR CHAPERONE REGULATOR-3

095817

2.23:1

2.5:1

EAETKpSPLVpSPSK

BETA ADDUCENT

P35612

0:7.4e6

0:4.Ie6

SEDARpSpSPSQR

DNA REPLICATION LICENSING FACTOR MCM4

P33991

1.7:1

1.5:1

TGpSESpSQTGTSTTSSR

EUKARYOTIC TRANSLATION INITIATION

FACTOR 4B

P23588

4.7:1

4.5:1

pSRpSRpTPPAIR

hypothetical protein KIAA0324 - human

T02345

2.3:1

2.2:1

pSRpTPPVTR

hypothetical protein KIAA0324 - human

T02345

1.3:1

1.6:1

HGpTPDPpSPR

hypothetical protein MGC13125

NP_1 16114.1

4.8:1

5.4:1

YpSPSQNpSPIHHIPSR

KIAA0164 gene product

NP_055554.1

1.6:1

1.5:1

LRLpSPpSPTSQR

LAMENT A/C

P02545

1.2:1

1.4:1

LKLpSPpSPSSR

LAMENT B1

P20700

12:1

1.2:1

YRpTPpSRpSR

peptidyl-propyl isomerase G (cyclophilin G)

NP_004783.1

8.5:1

1.4e 7:0

GPPpSPPAPVMHpSPSR

NUCLEAR PROTEIN SKIP (SKI-INTERACTING

PROTEIN)

Q13573

1.7:1

1.9:1

YHGHpSMS DPG VpS YR

PYRUVATE DEHYDROGENASE El

COMPONENT ALPHA SUB UNIT

P08559

1.46:1

1.35:1

SRSpSSpSPPPK

RNA binding protein

BAA83718

2.2:1

1.95:1

pSPpSPKPTK

RNA-binding protein SI, serine-rich domain

NP_006702.1

2.9:1

1.7:1

RQpSPSPSpTRPIR

SER/ARG -RELATED NUCLEAR MATRIX

PROTEIN

NP_005830

1.75:1

1.5:1

TRHpS PpTPQQSNR

SER/ARG-RELATED NUCLEAR MATRIX

PROTEIN

NP_005830

2.2:1

{Continued)

Table 1 (Continued)

Peptide sequence

Protein name

Acc. #

Ratio ± Gleevec

RRpSFpSIpSPVR

SON_HUMAN SON PROTEIN (SON3)

P18583

2.4:1

3.3:1

pSRpSGpSlKGSR

SPLICING FACTOR, ARG 1NINE/SERENE-RICH 7

Q16629

1.4:1

1.4:1

KHpS PpS PPPPTPTES R

SWI/SNF RELATED ACTIN DEPENDENT

REGULATOR OF CHROMATIN

XP_006719

1.35:1

1.3:1

KRpSPSPpSPTPEAK

SWI/SNF RELATED ACTIN DEPENDENT

REGULATOR OF CHROMATIN

XP_006719

1.4:1

1.4:1

pSVpSRpSPVPEK

SPLICING FACTOR, ARGININE/SERINE-RICH 5

Q13243

0:1.4e7

0:8e6

pSHpSPMSNR

TR2H_HUMAN TRANSFORMER-2 PROTEIN

HOMOLOG

Q13595

1.1:1

1.8:1

pSRpSYpTPEYR

TR2H_HUMAN TRANSFORMER-2 PROTEIN

HOMOLOG

Q13595

2.2:1

2.6:1

pSGpSNPNR

XRC1 _HUMAN DNA-REPAIR PROTEIN XRCC1

P18887

2.0:1

1.9:1

Phosphoproteins were characterized in K562 cells treated with or without STI571/Gleevec as indicated. One hundred thirty-seven phospho-peptides were characterized from 93 different proteins. A total of 274 sites of phosphorylation were mapped.

Phosphoproteins were characterized in K562 cells treated with or without STI571/Gleevec as indicated. One hundred thirty-seven phospho-peptides were characterized from 93 different proteins. A total of 274 sites of phosphorylation were mapped.

Table 2

Phosphoprofile of BCR-ABL and BCR-ABL-Interacting Proteins as a Function of STI571/Gleevec™ in Human K562 Cells

Table 2

Phosphoprofile of BCR-ABL and BCR-ABL-Interacting Proteins as a Function of STI571/Gleevec™ in Human K562 Cells

Panel A

Site

-ST1571

+ST1571

BCRpY177

+

-

BCRpS459

-

+

BCRpY644

+

+

AblpY185

+

-

AblpY226

+

+

AblpY232

+

-

AblpS569

+

-

Panel B

Protein

-ST1571

+ST1571

GRAP2

+

-

p62 Dokl

+

-

SHIP2

+

+

BCR-ABL was immunoprecipitated from K562 cells treated with or without STI571/Gleevec as indicated. Immuniprecipitates were analyzed by mass spectrometry to identify BCR-ABL phosphorylation (panel A), and to identify associated, co-immunoprecipitated proteins associated with BCR-ABL (panel b).

BCR-ABL was immunoprecipitated from K562 cells treated with or without STI571/Gleevec as indicated. Immuniprecipitates were analyzed by mass spectrometry to identify BCR-ABL phosphorylation (panel A), and to identify associated, co-immunoprecipitated proteins associated with BCR-ABL (panel b).

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

Get My Free Ebook


Post a comment