Reduced Expression of DOCK4 Leads to Increased Protein Phosphorylation and Migration of Hematopoietic Stem/Progenitor Cells

Dedicator of cytokinesis 4 (DOCK4) is a large, multi-domain signaling protein involved in modulating signaling networks in mammalian cells. Recent studies have highlighted its role across multiple tissue types in maintaining homeostasis. Deletion and/or mutations in the DOCK4 gene lead to malignant conditions and has been attributed to poor long-term prognosis. Within the hematopoietic compartment reduced expression of DOCK4 leads to dysplasia in the erythroid lineage (Sundaravel et al, PNAS, 2015). However, its functions during early stages of hematopoietic cell development have not been defined. Identification of signaling networks regulated by DOCK4 within the hematopoietic compartment will provide insights into developing strategies in overcoming functional deficits that arise due to reduced expression of DOCK4. In this study, using primary human hematopoietic stem/early progenitors (HSPCs), we identified the major downstream signaling networks regulated by DOCK4 and functional consequences of reduced expression of DOCK4.

Global phospho-proteomic analysis was performed on primary human CD34+/CD90+ hematopoietic cells expressing normal levels or reduced levels of DOCK4. A similar set of experiments were also performed on cells after they have committed to the erythroid lineage. These initial studies, using 1D and 2D phospho immunoblot analysis, we showed that cells expressing reduced levels of DOCK4 exhibit increased global tyrosine phosphorylation compared to cells expressing normal levels of DOCK4 regardless of whether cells exposed to hematopoietic cytokines. Further analysis of CD34+/CD90+ cells using liquid chromatography coupled mass spectrometry (LC/MS/MS) enabled us to precisely identify multiple proteins that were differentially phosphorylated. Among the proteins that were hyper-phosphorylated, phosphatases, PTPN6 (SHP1), INPP5D (SHIP1) and LYN kinase exhibited the greatest increase in their levels of tyrosine phosphorylation. These results were further validated by immunoblot analysis using site-specific phospho-antibodies against each of the three proteins. Co-immunoprecipitation experiments using HSPCs showed that DOCK4 interacts directly with INPP5D but not with PTPN6 or LYN suggesting that INPP5D is part of the DOCK4 protein complex but not PTPN6 or LYN. We then abrogated DOCK4-INPP5D interaction by knockdown of DOCK4, which led to increased localization of phospho-INPP5D (Y1021) from the cytoplasmic compartment to the cell membrane in CD34+/CD90+ HSPCs. To determine the functional pathways impacted by reduced expression of DOCK4, we performed in-silico DAVID/GO pathway analysis using our phospho proteomic data and found that cell migration function as one of the highly enriched pathways affected, when DOCK4 levels were reduced in HSPCs. We confirmed these findings by examining the migratory abilities of HSPCs expressing low levels of DOCK4 by an in vitro transwell assay, which showed approximately 2-fold increase in HSPC migration in the cell population where DOCK4 was expressed at fifty percent level. In addition, these cells also exhibited increased cell spreading and filopodia formation. Finally, pharmacologic inhibition of enzyme activities/phosphorylation of INPP5D, LYN or PTPN6 decreased the migration of HSPCs. Based on these results; we conclude that DOCK4 is a critical signaling intermediate, which acts within the hematopoietic stem/early progenitor compartment to regulate primary human HSPCs migration by modulating downstream kinases and phosphatases. Therefore using pharmacological inhibitors of INPP5D, LYN or PTPN6 may provide an avenue to restore proper migratory patterns of HSPCs within the hematopoietic niche and potentially reverse differentiation block as observed in dysplasia.