A Specific PTPRC/CD45 Phosphorylation Event Governed by Stem Cell Chemokine CXCL12 Regulates Primitive Hematopoietic Cell Motility

CXCL12 governs cellular motility, a process deregulated by hematopoietic stem cell oncogenes such as p210-BCR-ABL. A phosphoproteomics approach to the analysis of a hematopoietic progenitor cell line treated with CXCL12 and the Rac 1 and 2 inhibitor NSC23766 has been employed to objectively discover novel mechanisms for regulation of stem cells in normal and malignant hematopoiesis. The proteomic data sets identified new aspects of CXCL12-mediated signaling and novel features of stem cell regulation. We also identified a novel phosphorylation event in hematopoietic progenitor cells that correlated with motile response and governed by the chemotactic factor CXCL12. The novel phosphorylation site on PTPRC/CD45; a protein tyrosine phosphatase, was validated by raising an antibody to the site and also using a mass spectrometry absolute quantification strategy. Site directed mutagenesis and inhibitor studies demonstrated that this single phosphorylation site governs hematopoietic progenitor cell and lymphoid cell motility, lies downstream from Rac proteins and potentiates Src signaling. We have also demonstrated that PTPRC/CD45 is down-regulated in leukemogenic tyrosine kinase expressing cells. The use of discovery proteomics has enabled further understanding of the regulation of PTPRC/CD45 and its important role in cellular motility in progenitor cells. CXCL12 governs cellular motility, a process deregulated by hematopoietic stem cell oncogenes such as p210-BCR-ABL. A phosphoproteomics approach to the analysis of a hematopoietic progenitor cell line treated with CXCL12 and the Rac 1 and 2 inhibitor NSC23766 has been employed to objectively discover novel mechanisms for regulation of stem cells in normal and malignant hematopoiesis. The proteomic data sets identified new aspects of CXCL12-mediated signaling and novel features of stem cell regulation. We also identified a novel phosphorylation event in hematopoietic progenitor cells that correlated with motile response and governed by the chemotactic factor CXCL12. The novel phosphorylation site on PTPRC/CD45; a protein tyrosine phosphatase, was validated by raising an antibody to the site and also using a mass spectrometry absolute quantification strategy. Site directed mutagenesis and inhibitor studies demonstrated that this single phosphorylation site governs hematopoietic progenitor cell and lymphoid cell motility, lies downstream from Rac proteins and potentiates Src signaling. We have also demonstrated that PTPRC/CD45 is down-regulated in leukemogenic tyrosine kinase expressing cells. The use of discovery proteomics has enabled further understanding of the regulation of PTPRC/CD45 and its important role in cellular motility in progenitor cells. Localization of the hematopoietic stem cell (HSC) 1The abbreviations used are: HSC/Phematopoietic stem and progenitor cellsLCliquid chromatographyLSKLin− Sca+ c-kit+ cells[pS]phosphorylated serine. 1The abbreviations used are: HSC/Phematopoietic stem and progenitor cellsLCliquid chromatographyLSKLin− Sca+ c-kit+ cells[pS]phosphorylated serine. in its niche microenvironment is vital for stem cell maintenance (1Cancelas J.A. Lee A.W. Prabhakar R. Stringer K.F. Zheng Y. Williams D.A. Rac GTPases differentially integrate signals regulating hematopoietic stem cell localization.Nat. Med. 2005; 11: 886-891Crossref PubMed Scopus (232) Google Scholar, 2Whetton A.D. Lu Y. Pierce A. Carney L. Spooncer E. Lysophospholipids synergistically promote primitive hematopoietic cell chemotaxis via a mechanism involving Vav 1.Blood. 2003; 102: 2798-2802Crossref PubMed Scopus (61) Google Scholar, 3Williams D.A. Zheng Y. Cancelas J.A. 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Rac guanosine triphosphatases represent integrating molecular therapeutic targets for BCR-ABL-induced myeloproliferative disease.Cancer Cell. 2007; 12: 467-478Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The increased survival was because of lower levels of tumor initiating Lin− Sca+ c-Kit+ (LSK) cells in vivo (24Sengupta A. Arnett J. Dunn S. Williams D.A. Cancelas J.A. Rac2 GTPase deficiency depletes BCR-ABL+ leukemic stem cells and progenitors in vivo.Blood. 2010; 116: 81-84Crossref PubMed Scopus (52) Google Scholar). Here we have constructed a series of phoshoproteomic experiments to investigate and discover novel regulators of motility in hematopoietic progenitor cells and how this is affected by the presence of the protein tyrosine kinase p210-BCR-ABL. The interpretation of the data sets led to the identification of a phosphorylation event at S962 of PTPRC/CD45. It was subsequently demonstrated that this phosphorylation event was involved in stem cell motility. This pathway is also impacted on and inhibited by the p210-BCR-ABL oncogenic tyrosine kinase. Ba/F3 cells expressing the leukemic oncogenes p210-BCR-ABL, NPM/ALK, TEL/PDGFRb, PIP1/PDGFRa, Flt3/ITD, and c-Kit D816V were cultured as described previously (26Pierce A. Williamson A. Jaworska E. Griffiths J.R. Taylor S. Walker M. O'Dea M.A. Spooncer E. Unwin R.D. Poolman T. Ray D. Whetton A.D. Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.PLoS One. 2012; 7: e38928Crossref PubMed Scopus (12) Google Scholar). The FDCP-Mix (Clone A4) cell line was cultured as described (27Spooncer E. Heyworth C.M. Dunn A. Dexter T.M. 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Murine Lin- cells were cultured under the same conditions as the FDCP-Mix cells however with the addition 10 ng/ml of SCF and Flt3 ligand. The Jurkat cell line was maintained in RPMI and 10% (v/v) fetal calf serum. Human CD34+ cells were derived from peripheral blood. Fresh leukapheresis samples obtained from patients with newly diagnosed CP CML with written informed consent. Non CML CD34+ cells were from autologous donors with non-stem cell disorders. After thawing, cells were cultured overnight in growth factors (SCF 0.2 ng/ml, G-CSF 1 ng/ml, GM-CSF 0.2 ng/ml, IL-6 1 ng/ml, LIF 0.05 ng/ml, MIP-α 0.2 ng/ml, Stem Cell Technologies, Vancouver, Canada). Cells were treated with a range of concentrations of small molecular weight inhibitors, cells were exposed to NSC23766 for 16 h before further experimentation, whereas they were exposed to PTP and SU6656 for 1 h. Nuclear and cytoplasmic fractions were prepared using the Nuclear Extract kit (Active Motif, Brussels, Belgium) as described previously (30Williamson A.J. Smith D.L. Blinco D. Unwin R.D. Pearson S. Wilson C. Miller C. Lancashire L. Lacaud G. Kouskoff V. Whetton A.D. Quantitative proteomics analysis demonstrates post-transcriptional regulation of embryonic stem cell differentiation to hematopoiesis.Mol. Cell.. Proteomics. 2008; 7: 459-472Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Membrane proteins were enriched for using sucrose density centrifugation as described previously (31Holland M. Castro F.V. Alexander S. Smith D. Liu J. Walker M. Bitton D. Mulryan K. Ashton G. Blaylock M. Bagley S. Connolly Y. Bridgeman J. Miller C. Krishnan S. Dempsey C. Masurekar A. Stern P. Whetton A. Saha V. RAC2, AEP, and ICAM1 expression are associated with CNS disease in a mouse model of pre-B childhood acute lymphoblastic leukemia.Blood. 2011; 118: 638-649Crossref PubMed Scopus (47) Google Scholar). Peptides were identified by RP-LC-MS/MS on a QStar® XL mass spectrometer (Applied Biosystems) as described previously (32Griaud F. Williamson A.J. Taylor S. Potier D.N. Spooncer E. Pierce A. Whetton A.D. BCR/ABL modulates protein phosphorylation associated with the etoposide-induced DNA damage response.J. Proteomics. 2012; 77: 14-26Crossref PubMed Scopus (7) Google Scholar). One hundred micrograms of CXCL12 treated FDCP-Mix cells was spiked with 250 fmol of heavy labeled peptide N[pS]NVVPYDFNR before being fractionated using high pH reverse phase liquid chromatography. Fractions were loaded onto a 15cmX75 μm PepMap C18 3 μm column (LC Packings) using a standard LC Packings UltiMate pump and FAMOS autosampler. Samples were desalted on line before separation using a micro-precolumn (5mmX300 μm) cartridge. The washing solvent was 0.1% formic acid delivered at a flow rate of 30 μl/mins for 3 mins. Peptides were separated over 60mins solvent gradient at 300 nl/mins, over 25 mins buffer B (Buffer A: 2% Acetonitrile, 0.1% Formic acid; Buffer B: 80% Acetonitrile, 0.1% Formic acid) was increased from an initial 8% to 40%. The content of buffer B was increased to 60% over the next 10mins and finally to 80% over the subsequent 2 mins. Chromatography was performed on line to a 4000 Q-TRAP mass spectrometer (AB Sciex). All voltages and gas settings used are described previously (33Unwin R.D. Griffiths J.R. Leverentz M.K. Grallert A. Hagan I.M. Whetton A.D. Multiple reaction monitoring to identify sites of protein phosphorylation with high sensitivity.Mol. Cell. Proteomics. 2005; 4: 1134-1144Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The transitions for MRM analysis are presented in Table I.Table IParameters required for MRM analysis of the heavy labelled pS962 PTPRC/CD45 peptide and the endogenous pS962 PTPRC/CD45 peptideQ1 transition (m/z)Q3 transition (m/z)Dwell time (mSec)Collision energy (mV)707.8496.230035707.8821.430035707.8840.430035707.8920.430035702.8496.230035702.8811.430035702.8910.430035717.8840.430035717.8811.430035 Open table in a new tab Data was processed by a “Thorough” search against a mouse CDS database (mouse_KBMS5_0_20050302) containing 115,658 protein entries using ProteinPilot v2.0 software with default settings; for which the specificity of trypsin, the number of allowed missed cleavages and mass tolerances are preset. The software also does probability based analysis of the majority of the known modifications, however 8plex iTRAQ and MMTS modifications were specified (Applied Biosystems, Warrington, UK). Within Microsoft Excel, data was filtered on Modification and Theoretical MW then sorted on modification to separate phosphorylated and non-phosphorylated peptides. This process gave the number of unique phosphoentities (different mass spectra, they can be the same peptide “backbone” with the same site but other modifications) and phosphopeptides (unique sequences, does not account for different sites or missed cleavages). Subsequent manual analysis of spectral data was undertaken to confirm phosphorylation sites of interest. False discovery rate (FDR) was calculated using a reverse database and the ProteinPilot v.2.0 software. Ratio values for each phosphoentity were obtained from weighted averages of multiple spectra when appropriate, similarly to the way ProteinPilot™ calculate ratios for proteins. The minimum iTRAQ™ reporter ion area used for quantification was set at 20 (arbitrary units). ProteinPilot™ biases were used to correct for any sampling error so that the median value of log2 (ratios) of the phosphoentity distribution is equal to 0. Data is available to search and visualize at www.scalpl.org/hank/PhosphoHexPage?me=HSC%20Chemotaxis, it has also been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD000432. Rabbit anti-mouse pS962 PTPRC/CD45 specific antibody was generated by Eurogentec and used at a dilution of 1:100 for Western blotting, total PTPRC/CD45 antibody (Becton Dickinson UK Ltd) used at 1:1000, Histone H3, Na+K+ ATPase pump, Src and pY416 Src (Cell Signaling Technologies, Danvers, MA) were used at 1:1000, α-Tubulin and actin (Santa Cruz Biotechnology, Santa Cruz, CA) 1:1000. Anti CD45 PE-Cy7 antibody for flow cytometric analysis (ebiosciences)was used at 1:2000. Cells were lysed on ice with Bicine/CHAPS lysis buffer for 1 h with regular vortexing. Following this, lysates were centrifuged at 20,817 × g for 15mins at 4 °C. A NanoPro 1000 (Protein Simple, USA) instrument similar to that described (34O'Neill R.A. Bhamidipati A. Bi X. Deb-Basu D. Cahill L. Ferrante J. Gentalen E. Glazer M. Gossett J. Hacker K. Kirby C. Knittle J. Loder R. Mastroieni C. Maclaren M. Mills T. Nguyen U. Parker N. Rice A. Roach D. Suich D. Voehringer D. Voss K. Yang J. Yang T. Vander Horn P.B. Isoelectric focusing technology quantifies protein signaling in 25 cells.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16153-16158Crossref PubMed Scopus (168) Google Scholar) was used to analyze pS962 PTPRC/CD45 in clinical CD34+ samples. Each cell lysate was buffer exchanged with sample diluent plus DMSO Inhibitor mix using Amicon Ultra 3K centrifuge filters (Merck Millipore, Germany) and mixed in a 1:3 ratio with Premix containing pI standard. Each capillary loaded 400 nL sample mix. Primary anti-pS962 PTPRC/CD45 (Eurogentec) and used at 1:20 dilution, secondary (Goat-anti rabbit-biotin) and tertiary (Streptavadin-HRP) were used at a 1:100 dilution prepared with antibody diluent. XDR-peroxide/luminol was used for chemiluminescent detection. Instrument parameters were 25s sample load (400 nL load volume) with 40mins separation at 21000μW followed by optimized immobilization exposure of 90s. Primary, secondary and tertiary incubation times were 480, 60, and 10mins respectively. Detection profile included 11 exposures from 5–1200 s. Spectra analysis was undertaken using Compass software. The migration of FDCP-Mix cells and the primary hematopoietic cells in response to CXCL12 was performed essentially as previously described (35Pierce A. Lu Y. Hamzah H.G. Thompson S. Owen-Lynch P.J. Whetton A.D. Spooncer E. Differential effect of leukaemogenic tyrosine kinases on cell motility is governed by subcellular localisation.Br. J. Haematol. 2006; 133: 345-352Crossref PubMed Scopus (4) Google Scholar). Phospho mutants of S962 of PTPRC/CD45 were generated by site directed mutagenesis as per manufacturer's instructions in the Quikchange II kit (Stratagene, Cambridge, UK). Mutants were subcloned into MSCV-GFP retroviral vectors for transfection of Lin- cells. Transfection of Jurkat cells was undertaken by electroporation using program X-001 of the Amaxa II (Lonza) nucleofector reagent V. The phosphoproteomic experiment employed the multipotent FDCP-Mix hematopoietic cell line (27Spooncer E. Heyworth C.M. Dunn A. Dexter T.M. Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors.Differentiation. 1986; 31: 111-118Crossref PubMed Scopus (208) Google Scholar) pretreated with or without the Rac1/2 inhibitor NSC23766 and CXCL12 (Fig. 1A). Two experiments were performed to analyze the response of phosphopeptides to CXCL12. The first experiment was performed over a 30min time period in presence or absence of NSC23766 (Fig. 1A, Exp. 1) and the second experiment was a 6 h CXCL12 treatment (Figure 1A, Exp. 2). The concentration of NSC23766 used (100 μm) in this experiment was determined using the Boyden chamber chemotaxis assay and CXCL12, a known chemotactic agent (2Whetton A.D. Lu Y. Pierce A. Carney L. Spooncer E. Lysophospholipids synergistically promote primitive hematopoietic cell chemotaxis via a mechanism involving Vav 1.Blood. 2003; 102: 2798-2802Crossref PubMed Scopus (61) Google Scholar) (Fig. 1B). After treatment with CXCL12 and NSC23766, subcellular fractionation, iTRAQ labeling and phosphopeptide enrichment, samples were subjected to relative quantification LC-MSMS. Multipotent FDCP-Mix cells form cobblestones on irradiated long term bone marrow culture stromal cell layers (36Heyworth C.M. Alauldin M. Cross M.A. Fairbairn L.J. Dexter T.M. Whetton A.D. Erythroid development of the FDCP-Mix A4 multipotent cell line is governed by the relative concentrations of erythropoietin and interleukin 3.Br. J. Haematol. 1995; 91: 15-22Crossref PubMed Scopus (42) Google Scholar, 37Choong M.L. Luo B. Lodish H.F. Microenvironment-driven changes in the expression profile of hematopoietic cobblestone area-forming cells.Ann. Hematol. 2004; 83: 160-169Crossref PubMed Scopus (5) Google Scholar) as seen with LSK cells and also respond to CXCL12 (2Whetton A.D. Lu Y. Pierce A. Carney L. Spooncer E. Lysophospholipids synergistically promote primitive hematopoietic cell chemotaxis via a mechanism involving Vav 1.Blood. 2003; 102: 2798-2802Crossref PubMed Scopus (61) Google Scholar). CXCL12 treatment with and without NSC23766 in the FDCP-Mix cells enabled the data to be considered in the context of Rac inhibitor sensitive proteins. This is especially pertinent as Rac proteins are key effectors in p210-BCR-ABL mediated transformation (24Sengupta A. Arnett J. Dunn S. Williams D.A. Cancelas J.A. Rac2 GTPase deficiency depletes BCR-ABL+ leukemic stem cells and progenitors in vivo.Blood. 2010; 116: 81-84Crossref PubMed Scopus (52) Google Scholar) and stem cell homing/retention (1Cancelas J.A. Lee A.W. Prabhakar R. Stringer K.F. Zheng Y. Williams D.A. Rac GTPases differentially integrate signals regulating hematopoietic stem cell localization.Nat. Med. 2005; 11: 886-891Crossref PubMed Scopus (232) Google Scholar, 3Williams D.A. Zheng Y. Cancelas J.A. Rho GTPases and regulation of hematopoietic stem cell localization.Methods Enzymol. 2008; 439: 365-393Crossref PubMed Scopus (55) Google Scholar, 24Sengupta A. Arnett J. Dunn S. Williams D.A. Cancelas J.A. Rac2 GTPase deficiency depletes BCR-ABL+ leukemic stem cells and progenitors in vivo.Blood. 2010; 116: 81-84Crossref PubMed Scopus (52) Google Scholar, 38Sanchez-Aguilera A. Lee Y.J. Lo Celso C. Ferraro F. Brumme K. Mondal S. Kim C. Dorrance A. Luo H.R. Scadden D.T. Williams D.A. Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 9607-9612Crossref PubMed Scopus (30) Google Scholar, 39Shang X. Cancelas J.A. Li L. Guo F. Liu W. Johnson J.F. Ficker A. Daria D. Geiger H. Ratner N. Zheng Y. R-Ras and Rac GTPase cross-talk regulates hematopoietic progenitor cell migration, homing, and mobilization.J. Biol. Chem. 2011; 286: 24068-24078Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The principal components of the experimental design are shown in Fig. 1A. Cells were fractionated into nuclear, cytosolic or membranous fractions (appropriate marker analyses confirmed this, Fig. 1C) and the enriched phosphopeptides analyzed on a tandem mass spectrometer. In the experiments to determine the role of CXCL12 in multipotent cell signaling mechanisms we identified 747 phosphopeptide observations in the cytosolic fraction, 92 in the membranous fraction and 557 in the nuclear fraction (supplemental Table S1A). In the 6 h CXCL12 analysis we identified 28 phosphopeptides in the membranous fraction and 250 in the nuclear fraction (supplemental Table S1B). There is no apparent and immediately obvious reason for the difference in the number of phosphopeptides identified between the 2 experiments; this could be because of sample preparation or the analysis. From the analyses that have been performed it can be seen that a number of phosphoevents appear to be differentially regulated by treatment with CXCL12 and NSC23766. From the data accumulated we were interested in selecting and taking forward phosphoevents and demonstrating their importance in motility. We next took observations and validated their role in primitive hematopoietic homing. A major aim of our study was to find CXCL12-stimulated phosphorylation events that are NSC23766 sensitive. pS962 of PTPRC/CD45 (NRN[pS]NVVPYDFNR) was seen to be increased in CXCL12-treated cells in an NSC23766 sensitive fashion over a 6 h period (Fig. 2A and supplemental