Syk-mediated Tyrosine Phosphorylation Is Required for the Association of Hematopoietic Lineage Cell-specific Protein 1 with Lipid Rafts and B Cell Antigen Receptor Signalosome Complex

Abstract

Reintroducing a Syk construct into the Syk knock-out cells recovered effectively both tyrosine phosphorylation and translocation of HS1 into lipid rafts. In contrast, translocation of HS1 into rafts was normal in a Lyn knock-out B cell line, and an HS1 mutant at the tyrosine residue Tyr 222 targeted by Lyn maintained the ability to partition into rafts upon BCR cross-linking. These data indicate that Syk plays an important role in the translocation of HS1 into lipid rafts and may be responsible for actin assembly recruitment to rafts and subsequent antigen presentations.

Syk Is Required for Monocyte/Macrophage Chemotaxis to CX3CL1 (Fractalkine)

“…Syk has been reported to directly phosphorylate at least two actin-binding proteins: the cortactin-related hemopoietic lineage cell-specific protein 1, which binds to the Arp2/3 complex and promotes actin assembly and branching (51,52), and SH3P7, a protein of unknown function, isolated from the phosphotyrosine immunoreactive fraction induced after lymphocyte activation (53). Notably, Syk-mediated phosphorylation of hemopoietic lineage cell-specific protein 1 was required for its translocation to lipid raft microdomain together with Arp2/3 and the actin regulator Wiskott-Aldrich syndrome protein (WASp) after BCR cross-linking (54). In addition, Syk has been shown to bind WASp through the adapter CrkL in platelets (55), and an interaction between Syk and the WASp-interacting protein has recently been reported upon Fc⑀RI activation in mast cells (56).…”

T-cell-receptor-dependent actin regulatory mechanisms

“…In B cells, tyrosine phosphorylation drives HS1 into lipid rafts, where it colocalizes with B-cell receptor, WASp and Arp2/3 complex (Hao et al, 2004;Yamanashi et al, 1997). In T cells, HS1 is rapidly phosphorylated at tyrosine 378 and 397 after TCR engagement.…”

The Coiled-coil Domain Is Required for HS1 to Bind to F-actin and Activate Arp2/3 Complex

“…DNA Constructs-HS1 constructs based on MGIN viral vector were prepared as previously described (12). To prepare glutathione S-transferase (GST)-tagged HS1 constructs, including GST-HS1, GST-HS1-* This work was supported by Grant HL52753 from the National Institutes of Health and Grant CA91984 from the NCI, National Institutes of Health (to X.…”

“…All mutations were verified by DNA sequencing. The MGIN-based retroviruses carrying HS1-GFP or HS1 mutants were prepared and infected to cells as described previously (12). Protein Preparation and Purification-Plasmids encoding GST-HS1 constructs were transformed into Escherichia coli strain BL21(DE3) pLysS.…”

“…Because endothelial cells express few endogenous HS1 proteins (data not shown), we also evaluated the interaction between HS1 mutants and F-actin in B cells where HS1 is abundant (17). The murine WEHI-231 B cells expressing GFP-tagged HS1 mutants were established similarly by retroviral infection (12), and the cell lysates were immunoprecipitated by GFP antibody. The pellets were blotted with antibody either against ␤-actin or HS1.…”