Abstract
Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell adhesion protein involved in the regulation of cell adhesion and migration. Interestingly, several PECAM-1-deficient hematopoietic cells exhibit impaired chemotactic responses to stromal cell-derived factor 1 (SDF-1), a chemokine essential for B lymphopoiesis and bone marrow myelopoiesis. However, whether PECAM-1 is involved in SDF-1-regulated chemotaxis is unknown. We report here that SDF-1 induces tyrosine phosphorylation of PECAM-1 at its immunoreceptor tyrosine-based inhibition motifs in several hematopoietic cell lines via the Src family kinase Lyn, Bruton's tyrosine kinase, and JAK2 and that inhibition of these kinases reduced chemotaxis. Overexpression and knockdown of PECAM-1 enhanced and down-regulated, respectively, SDF-1-induced Gαi-dependent activation of the PI3K/Akt/mTORC1 pathway and small GTPase Rap1 in hematopoietic 32Dcl3 cells, and these changes in activation correlated with chemotaxis. Furthermore, pharmacological or genetic inhibition of the PI3K/Akt/mTORC1 pathway or Rap1, respectively, revealed that these pathways are independently activated and required for SDF-1-induced chemotaxis. When coexpressed in 293T cells, PECAM-1 physically associated with the SDF-1 receptor CXCR4. Moreover, PECAM-1 overexpression and knockdown reduced and enhanced SDF-1-induced endocytosis of CXCR4, respectively. Furthermore, when expressed in 32Dcl3 cells, an endocytosis-defective CXCR4 mutant, CXCR4-S324A/S325A, could activate the PI3K/Akt/mTORC1 pathway as well as Rap1 and induce chemotaxis in a manner similar to PECAM-1 overexpression. These findings suggest that PECAM-1 enhances SDF-1-induced chemotaxis by augmenting and prolonging activation of the PI3K/Akt/mTORC1 pathway and Rap1 and that PECAM-1, at least partly, exerts its activity by inhibiting SDF-1-induced internalization of CXCR4.
Highlights
Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell adhesion protein involved in the regulation of cell adhesion and migration
PECAM-1 was constitutively tyrosine-phosphorylated along with a well established substrate of JAK2, STAT5, in 32D/EpoR cells expressing JAK2-V617F or in human leukemic PVTL-2 cells expressing this mutant, and this was inhibited by ruxolitinib (Fig. 2, D and E). These results suggest that JAK2 as well as Lyn and BTK may be involved in tyrosine phosphorylation of PECAM-1 in the inhibition motifs (ITIMs) in hematopoietic cells stimulated with stromal cell-derived factor 1 (SDF-1)
We have reported for the first time that SDF-1 induced tyrosine phosphorylation of PECAM-1 in the murine model hematopoietic cell lines 32Dcl3 and BaF3 as well as in other hematopoietic cell lines, including leukemic cells, such as UT7 and U937 (Fig. 1)
Summary
Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell adhesion protein involved in the regulation of cell adhesion and migration. The cytoplasmic domain of PECAM-1 contains two immunoreceptor tyrosine-based inhibition motifs (ITIMs) that encompass Tyr663 and Tyr686, which become tyrosine-phosphorylated mainly by the Src family tyrosine kinases in response to various stimuli to recruit several SH2domain containing signaling molecules, such as the proteintyrosine phosphatase SHP2 and the Src family tyrosine kinases Through binding with these and various other signaling molecules, PECAM-1 plays important roles in modulation of intracellular signaling mechanisms, including activation of the PI3K/Akt pathway and Rap1 [17,18,19], to regulate a variety of cellular events, such as cell adhesion, through integrin activation, chemotaxis, and apoptosis [15, 16]. Overexpression or knockdown of PECAM-1 in hematopoietic 32Dcl cells enhanced or down-regulated, respectively, SDF-1– induced activation of the PI3K/Akt/mTORC1 pathway and Rap, which was required for and correlated with chemotaxis. These data suggest that PECAM-1 may enhance CXCR4-mediated chemotactic signaling pathways involving PI3K/Akt/mTORC1 and Rap, at least partly, by inhibiting internalization of CXCR4
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