The activation and fusion of macrophages and of osteoclasts require the adaptor molecule DNAXactivating protein of 12 kD (DAP12), which contains immunoreceptor tyrosine-based activation motifs (ITAMs). TREM2 (triggering receptor expressed on myeloid cells-2) is the main DAP12-associated receptor in osteoclasts and, similar to DAP12 deficiency, loss of TREM2 in humans leads to Nasu-Hakola disease, which is characterized by bone cysts and dementia. Furthermore, in vitro experiments have shown that deficiency in DAP12 or TREM2 leads to impaired osteoclast development and the formation of mononuclear osteoclasts. Here, we demonstrate that the ligation of TREM2 activated phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase 1 (ERK1) and ERK2, and the guanine nucleotide exchange factor Vav3; induced the mobilization of intracellular calcium (Ca 2+ ) and the reorganization of actin; and prevented apoptosis. The signaling adaptor molecule DAP10 played a key role in the TREM2-and DAP12-dependent recruitment of PI3K to the signaling complex. Src homology 2 (SH2) domain-containing inositol phosphatase-1 (SHIP1) inhibited TREM2-and DAP12-induced signaling by binding to DAP12 in an SH2 domain-dependent manner and preventing the recruitment of PI3K to DAP12. These results demonstrate a previously uncharacterized interaction of SHIP1 with DAP12 that functionally limits TREM2-and DAP12-dependent signaling and identify a mechanism through which SHIP1 regulates key ITAM-containing receptors by directly blocking the binding and activation of PI3K.

Nedd4 E3 ligases are members of the HECT E3 ubiquitin ligase family and regulate ubiquitination-mediated protein degradation. In this report, we demonstrate that calcium releases the C2 domain-mediated auto-inhibition in both Nedd4-1 and Nedd4-2. Calcium disrupts binding of the C2 domain to the HECT domain. Consistent with this, calcium activates the E3 ubiquitin ligase activity of Nedd4. Elevation of intracellular calcium by ionomycin treatment, or activation of acetylcholine receptor or epidermal growth factor receptor by carbachol or epidermal growth factor stimulation induced activation of endogenous Nedd4 in vivo evaluated by assays of either Nedd4 E3 ligase activity or ubiquitination of Nedd4 substrate ENaC-␤. The activation effect of calcium on Nedd4 E3 ligase activity was dramatically enhanced by a membrane-rich fraction, suggesting that calcium-mediated membrane translocation through the C2 domain might be an activation mechanism of Nedd4 in vivo. Our studies have revealed an activation mechanism of Nedd4 E3 ubiquitin ligases and established a connection of intracellular calcium signaling to regulation of protein ubiquitination.Protein ubiquitination is a major intracellular signaling event. E3 ubiquitin ligase (E3), 3 including the HECT (homologous to E6-AP carboxyl terminus) domain containing and the RING (the really interesting new gene) domain containing E3 ligases, is the key enzyme that catalyzes ubiquitination and confers specificity of ubiquitination substrates (1-3). Nedd4 E3 ubiquitin ligases are members of the WW domain-containing HECT E3 ubiquitin ligase subfamily (4). There are two Nedd4 E3 ligases, Nedd4-1 and Nedd4-2, in mammalian cells (5). Human Nedd4-1 gene (Nedd4) is localized on chromosome 15, and Nedd4-2 gene (Nedd4L) is on chromosome 18 (5). Both Nedd4-1 and Nedd4-2 have the same domain structure, with the C2 domain at the N terminus, followed by four WW domains, and the HECT domain at the C terminus. The primary peptide sequences of human Nedd4-1 and Nedd4-2 are ϳ65% identical. The most unconserved regions are located between the WW1 and the WW3 domains.

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Activation of NHE via calcium-dependent calpain contributes to hyperglycaemia-induced endothelial dysfunction through dissociation of hsp90 from eNOS.