CD2AP/SHIP1 complex positively regulates plasmacytoid dendritic cell receptor signaling by inhibiting the E3 ubiquitin ligase Cbl

Abstract

The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA-induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.

FIGURE 1

CD2AP is highly expressed in pDCs and is indispensable for BDCA2 signaling. (A) The relative expression level of CD2AP from the human expression microarray database. The relative expression of each gene was compared by plotting the values extracted from the gene expression database. A value < 1 indicated the absence of gene expression. (B) Human pDCs, myeloid dendritic cells (mDCs), monocytes, NK cells, T cells, and B cells were isolated from PBMCs, and total RNA was purified and reverse transcribed. The cDNAs were subjected to real-time PCR analysis, and the expression of CD2AP was normalized to the level of β-actin, whose value was multiplied by 1000. (C) Human pDCs, as well as other major immune cell types in human PBMCs, were isolated, and protein extracts of 1 × 10⁵ cells/lane were separated by SDS-PAGE and subjected to immunoblot analysis using anti-CD2AP Ab. β-actin was used as a loading control. (D) Using siRNA, CD2AP was knocked down in Gen2.2 cells, and the cells were stimulated with 1 µM CpG-A in the presence of plate-bound anti-BDCA2 Ab for 24 h. IFN-a in the supernatants was determined by ELISA. Each circle indicates an independent experiment.

FIGURE 2

CD2AP binds to SHIP1 and the CD2AP/SHIP1 complex is needed for BDCA2 signaling. (A) Coomassie blue staining of CD2AP-binding proteins purified by immunoprecipitation of CD2AP from Gen2.2 cell lysate. Proteins identified by mass spectrometry are as indicated. (B) CD2AP was immunoprecipitated from human primary pDC lysate, and CD2AP-binding proteins were subjected to SDS-PAGE and subsequent immunoblot analysis by anti-SHIP1 Ab. (C) CD2AP, SHIP1, FcεR1γ, and Syk were knocked down using siRNA in Gen2.2 cells. The nontargeting siRNA (siNS) was used as a control. The siRNA knockdown Gen2.2 cells were stimulated with 1 µM CpG-A in the presence of plate-bound BDCA2. The IFN-a level in the supernatant was measured by ELISA. (D) The siRNA knockdown Gen2.2 cells were cross-linked using anti-BDCA2 Ab for 2 or 10 min. The total protein phosphorylation and phosphorylation of Syk, as well as total protein levels of CD2AP, SHIP1, FcεR1γ, and Syk, were analyzed by immunoblotting. β-actin was used as a loading control. Data are representative of three independent experiments.

FIGURE 3

The first SH3 domain of CD2AP binds to the P-rich domain of SHIP1. (A) Lysates from 293T cells transfected with full-length GFP-SHIP1 and HA-tagged full-length CD2AP or deletion mutants of CD2AP were immunoprecipitated with HA-agarose beads, followed by immuno-blotting with anti-SHIP1 Ab or with anti-HA Ab. (B) Lysates from 293T cells transfected with HA-tagged full-length CD2AP and full-length or deletion mutants of GFP-SHIP1 were immunoprecipitated with anti-GFP Ab, followed by immunoblotting with anti-HA tag Ab or anti-GFP Ab.

FIGURE 4

Knockdown of CD2AP or SHIP1 using shRNA reduces ITAM signaling and enhances the degradation and ubiquitination of Syk. (A) CD2AP knockdown Gen2.2 cells (shCD2AP), SHIP1 knockdown Gen2.2 cells (shSHIP1), or scramble shRNA knockdown Gen2.2 cells (sc) were cross-linked using anti-BDCA2 Ab for 5 or 20 min. The total protein phosphorylation and phosphorylation of Syk, Vav1, and SHIP1, as well as total protein levels of CD2AP and SHIP1, were analyzed by immunoblotting. β-actin was used as a loading control. (B) shRNA knockdown Gen2.2 cells were cross-linked by BDCA2, and the calcium influx was analyzed by FACS. (C) shRNA knockdown Gen2.2 cells were stimulated with 1 mM CpG-A in the presence of plate-bound anti-BDCA2 Ab. The IFN-α level in the supernatant was measured by ELISA. Data are representative of six independent experiments. (D) shRNA knockdown Gen2.2 cells were cross-linked by BDCA2 for 5 or 20 min. Protein levels of FcεR1γ, Syk, CD2AP, and SHIP1 were analyzed by immunoblotting. β-actin was used as a loading control. (E) shRNA knockdown Gen2.2 cells were cross-linked using anti-BDCA2 Ab for 5 or 20 min. Syk was immunoprecipitated, resolved by SDS-PAGE, and immunoblotted with anti-Ub Ab and anti-Ub (K48-specific) Ab.

FIGURE 5

CD2AP mediates the association of SHIP1 and Cbl, and the CD2AP/SHIP1 complex inhibits the E3 ubiquitin ligase activity of Cbl. (A) 293T cells were transfected with GFP-Cbl together with HA-CD2AP, 36 h after transfection, anti–HA-CD2AP immunoprecipitates from the lysate protein were immunoblotted with anti-Cbl. Aliquots of cell lysates were immunoblotted directly with an anti-Cbl Ab to visualize GFP-Cbl. (B) 293T cells were transfected with GFP-Cbl, together with HA-CD2AP and GFP-SHIP1; 36 h after transfection, SHIP1 was immunoprecipitated and immunoblotted with anti-Cbl and anti-HA Abs. Aliquots of cell lysates were immunoblotted directly with anti-Cbl and anti-HA Abs to visualize GFP-Cbl and HA-CD2AP. (C) 293T cells were transfected with the indicated expression plasmids together with HA-ubiquitin and Syk plasmids; 24 h after transfection, cells were incubated with 25 mM MG132 for 3 h. Syk was immunoprecipitated from the cell lysates and immunoblotted with anti-HA, anti-Syk, anti-SHIP1, and anti-Myc Abs. Aliquots of cell lysates were immunoblotted directly with indicated Abs. Data are representative of three independent experiments.

FIGURE 6

The CD2AP/SHIP1 complex and Cbl are recruited to BDCA2 and FcεR1γ complex after BDCA2 cross-linking in human primary pDCs. (A) pDCs were cross-linked using BDCA2 mAb for 0, 2, or 20 min. The pDCs were stained with anti-CD2AP and anti-SHIP1 Abs, as described in Materials and Methods. (B) pDCs were cross-linked using BDCA2 mAb for 0, 2, or 20 min. The pDCs were stained with anti-CD2AP, anti-FcεR1γ, and Alexa Fluor 647-con-jugated anti-Cbl Abs. Images were taken using a Leica confocal microscope. Original magnification ×1260.