CIN85 Modulates the Down-regulation of FcγRIIa Expression and Function by c-Cbl in a PKC-dependent Manner in Human Neutrophils

Abstract

We previously described a non-classical mechanism that arrests Fc␥RIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of Fc␥RIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated Fc␥RIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the Fc␥RIIa degradation process because the cholesterol-depleting agent methyl-␤-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented Fc␥RIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of Fc␥RIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate Fc␥RIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated Fc␥RIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of Fc␥RIIa signaling. Fc␥ receptors (Fc␥Rs)3 represent a family of membrane proteins involved in the recognition of the Fc portion of immunoglobulin G. They act as sensors for opsonized pathogens or immune complexes, and their engagement initiates intracellular signals that lead to multiple cell functions, such as degranulation, activation of the respiratory burst, and phagocytosis (1-3). Fc␥R isoforms either express an immunoreceptor tyrosine-based activating motif (ITAM) in their intracellular portion or associate with ITAM-containing accessory proteins with the exception of Fc␥RIIb, the unique inhibitory member of the Fc␥R family that transmits inhibitory signals through an immunoreceptor tyrosine-based inhibition motif (ITIM) (4, 5). In the case of antibody-mediated responses, such as IgG-dependent phagocytosis, neutrophils and other immune effectors, such as macrophages, have the capacity to trigger strong proinflammatory responses, which must be tightly controlled. On human neutrophils, only two Fc␥Rs, namely Fc␥RIIa (CD32a) and Fc␥RIIIb (CD16b), are constitutively expressed, neither of which possess an ITIM. The available data indicate that in contrast to other human phagocytes, including macrophages, human neutrophils express very little (6) or no (7) Fc␥RIIb.Several lines of evidence indicate that Fc␥RIIa is directly involve...

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Influenza virus H1N1 activates platelets through FcγRIIA signaling and thrombin generation

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CIN85 is required for Cbl-mediated regulation of antigen receptor signaling in human B cells

“…21 A recent study showed that CIN85 modulates c-Cbl-mediated down-regulation of Fc␥RIIa in human neutrophils. 22 It is thus of interest to determine whether CIN85 regulates the signaling pathways of other multimeric immune receptors, such as the T-and B-cell receptors.…”

“…21 A recent study showed that CIN85 modulates c-Cbl-mediated down-regulation of Fc␥RIIa in human neutrophils. 22 It is thus of interest to determine whether CIN85 regulates the signaling pathways of other multimeric immune receptors, such as the T-and B-cell receptors.Here, we demonstrate that CIN85 is constitutively associated with c-Cbl, Cbl-b, and BLNK in human B cells. Gain-of-function and loss-of-function experiments revealed that CIN85 up-regulated c-Cbl phosphorylation and inhibited BCR-induced calcium flux and phosphorylation of Syk and PLC␥2, without affecting BCR internalization.…”