SLP-76 couples integrin receptor ligation with ERK1/2 activation and podosome distribution in dendritic cells
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The SH2 domain-containing leukocyte protein of 76kDa (SLP-76) adaptor is an important molecular intermediate in multiple signaling pathways governing immune cell function. Here we report that SLP-76 is expressed in CD11c + B220 - dendritic cells (DCs) isolated from murine thymus or spleen and in CD11c + bone marrow-derived DCs (BMDCs). SLP-76 is rapidly phosphorylated on tyrosine residues and associates with the guanine nucleotide exchange factor (GEF) Vav in a Syk-dependent manner upon plating of BMDCs on integrin agonists. Furthermore, SLP-76 and Vav are recruited to lipid rafts in an apparently Syk-dependent, integrin-specific manner. SLP-76 is not strictly required for the in vitro or in vivo generation of DCs, but SLP-76 deficient BMDCs adhere poorly to fibronectin (FN) suggesting impaired integrin function. In the absence of SLP-76, BMDCs also perform poorly in a phagokinetic assay indicating impaired random motility on an integrin substrate. In addition, the pattern and distribution of actin-based podosome formation is markedly altered in BMDCs lacking SLP-76 following integrin engagement. Consistent with impaired adhesion, cutaneous SLP-76 deficient DCs exit ear tissue at an elevated frequency compared to wild type DCs. SLP-76 deficient BMDCs manifest multiple signaling defects following integrin ligation; including reduced global tyrosine phosphorylation and markedly impaired phosphorylation of p44/42 MAP kinase (ERK1/2). These data implicate SLP-76 as an important molecular intermediate in the signaling pathways regulating multiple integrin-dependent DC functions, and add to the growing body of evidence that hematopoietic cells employ unique molecular intermediates and mechanisms for regulating integrin signaling.