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

Andrew J.K. Williamson,Anthony D. Whetton,Cong Zhou,Ewa Jaworska,Mark Aspinall-O'Dea,Lee Lancashire,Andrew Pierce,Sara Cadecco,Richard D. Unwin,Michael J. Walker,Tessa L. Holyoake,Elaine Spooncer,Sheela A. Abraham

doi:10.1074/mcp.m112.024604

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 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.

Highlights

From the ‡Stem Cell and Leukaemia Proteomics Laboratory, School of Cancer and Enabling Sciences, Manchester Academic Health Science Centre, University of Manchester, 27 Palatine Rd, Manchester, M20 4QL; §Clinical Experimental Pharmacology Laboratory, Paterson Institute for Cancer Research, Manchester Academic Health Science Centre, University of Manchester, M20 4BX; ¶Paul O’Gorman Leukaemia Research Centre, Institute of Cancer Studies, University of Glasgow, Gartnavel General Hospital, Glasgow, G12 0XB
Using the NanoPro 1000 instrument we have demonstrated a decrease in the level of pS962 PTPRC/CD45 in CD34ϩ cells expressing BCR-ABL from 3 patients with chronic myelogenous leukemia (CML) compared with a nonCML control (n ϭ 3) (Fig. 6D)
Using the hematopoietic progenitor cell line FDCP-Mix; we sought to uncover a mechanistic event that is relevant in primitive hematopoietic cell motile responses

Summary

Introduction

Phosphoproteomic Analysis of CXCL12 Action on Multipotent Cells—The phosphoproteomic experiment employed the multipotent FDCP-Mix hematopoietic cell line [27] pretreated with or without the Rac1/2 inhibitor NSC23766 and CXCL12 (Fig. 1A). 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 Table S1B).

Results

Conclusion