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

Abstract

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 downregulated 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. Molecular & Cellular

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

“…The procedure we have used in relation to primary patient material is well documented for the study of TKI effects, and as such applies to a wider audience given the likelihood of access to biobanked primary cell material. This builds on earlier published work using CML patient material 5,6 , and makes a case for focusing this …”

“…5,6 In one of these studies, Brown et al 6 focused on the defective function of BCR-ABL expressing mature dendritic cells derived from CML patient monocytes and found that dysregulation of ABL1 protein distribution in CML dendritic cells (CML-DCs) resulted in changes to CrkL (a substrate protein of ABL1), altering the formation of complexes involved in the control of F-actin responses. Specifically, CrkL displayed elevated levels of phosphorylation in CMLDCs when compared with normal DCs 6 .…”

“…Phosphorylation at this site was shown to be upregulated in the presence of BCR-ABL in CD34 + cells derived from patients with CML when compared with control samples (from patients without CML), using cIEF with both commercially available and in-house antibodies, respectively. 5 Using the fully optimised PTPRC/CD45 assay developed in our laboratory (for assay development data see Supplementary Fig.2), we validated the approach using clinical samples, specifically looking at novel signaling differences between more primitive CD34 + CD38 -CML cells that co-enrich with stem cells and more mature CML CD34 + CD38 + progenitor populations, both in TKItreated samples and in untreated controls. The quantity of clinical starting material needed for this approach is significantly smaller than those required in previous studies using a cIEF platform 1,2 , and this technique also enables a more detailed analysis of protein phosphorylation than was possible with earlier technologies.…”

“…Different iterations of the technology have subsequently been employed in a range of studies, starting with the Firefly instrument (Protein Simple, formerly Cell Biosciences) in which imatinib induced changes in ERK1 and ERK2 activation status were assessed in human CML cells isolated from total blood, whilst a range of phosphorylation changes in proteins including STAT3 and STAT5 were defined for the K562 CML cell line 2 . Next generation systems (NanoPro 1000, formally CB1000) have been used to study proteins including AKT, 4EBP1, MET, PTPRC/CD45 and CrkL in a number of leukemias, using different primary cell types including murine CD138 + isolated from bone marrow (BM), human CD34-selected acute myeloid leukemia (AML) cells, CML-derived CD34+ and dendric cells [3][4][5][6] .…”

Chemical Proteomic Approaches Targeting Cancer Stem Cells: A Review of Current Literature

“…Phosphorylated peptides are then analyzed by reverse phase (RP)-LC-MS/MS. From these analyses, a number of phosphoevents appear to be differentially regulated by treatment with CXCL12 and NSC23766 (79). Interpretation of proteomic datasets has led to the identification of phosphorylation event at S962 of PTPRC/CD45, a protein tyrosine phosphatase.…”