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

Abstract

IntroductionB-cell receptor (BCR) signaling guides critical cell fate decisions in B cells during ontogeny. 1,2 BCRs can generate tolerogenic signals to purge or silence B cells that bind to self-antigens, and immunogenic signals to expand B cells that are specific for foreign antigens. Thus, BCR signaling must be properly regulated at the various stages of B-cell development, as aberrant regulation of BCR signaling potentially leads to autoimmunity and B-cell malignancies.On BCR ligation by antigens, the Src-family protein tyrosine kinase (PTK) Lyn and Syk are initially activated. Syk propagates the signal by phosphorylating downstream signaling molecules, causing the activation of critical signaling intermediates phosphoinositol 3-kinase (PI3K) and phospholipase C (PLC)␥2. PI3K activates Akt kinase, which is important for B-cell survival. 3 PLC␥2 activation induces the release of intracellular Ca 2ϩ and the activation of protein kinase C (PKC), which cause the activation of mitogen-activated protein kinases (MAPKs; ERK, JNK, and p38 MAPK) and of transcription factors, including NF-B and NF-AT. These molecules regulate further downstream molecules that are responsible for determining B-cell fates such as survival, growth, and differentiation. 1,2 Casitas B-lineage lymphoma (Cbl) proteins are E3 ubiquitin ligases that regulate signals of various receptors by promoting the ubiquitination of signaling components. 4,5 Tyrosine phosphorylation of Cbl proteins is critical for their function. 6 Mammalian Cbl proteins consist of 3 members, c-Cbl, Cbl-b, and Cbl-3, among which c-Cbl and Cbl-b are expressed in hematopoietic cells. 7 In B cells, Cbl proteins associate with Syk and B-cell linker (BLNK), and negatively regulate BCR signaling. 8,9 B cell-specific ablation of c-Cbl/Cbl-b proteins in mice causes aberrant BCR signaling as well as impaired B-cell anergy, culminating in the development of systemic lupus erythematosus (SLE)-like disease. 10 In addition, c-Cbl is hypophosphorylated on tyrosine in advanced stages of chronic lymphocytic leukemia (CLL). 11 These findings suggest that Cbl-mediated regulation of BCR signaling is critical for the fate decisions of self-reactive and malignant B cells.Adaptors are noncatalytic molecules that integrate the spatial and temporal assembly of multiprotein complexes involved in the survival, growth, and differentiation of B cells. We previously showed that the B lymphocyte adaptor molecule of 32 kDa (Bam32)/DAPP1 regulates BCR signaling/internalization and B-cell survival. 12,13 The SH3KBP1 (SH3-domain kinase-binding protein 1) gene, which is also known as CIN85 (c-Cbl interacting protein of 85 kDa), encodes an adaptor that is independently identified by several groups and contains 3 SH3 domains, a proline-rich region, and a coiled-coil domain. [14][15][16][17] Early studies showed that in nonimmune cells, CIN85 regulates the clathrindependent internalization of receptor tyrosine kinases (RTKs) such as epidermal growth factor receptors (EGFRs). 18,19 The formation of the ternary...

Cbl-family ubiquitin ligases and their recruitment of CIN85 are largely dispensable for epidermal growth factor receptor endocytosis

“…Recent studies using Hela, Hep2, B lymphoma cell line BJAB and porcine aortic endothelial cell lines have arrived at a similar conclusion using a CIN85 knockdown approach (Niiro et al 2012; Ronning et al 2011; Schroeder et al 2010; Schroeder et al 2012). …”

The autoinhibitory C-terminal SH2 domain of phospholipase C–γ2 stabilizes B cell receptor signalosome assembly

“…One could imagine that interactions between the cSH2 domain of PLC-γ2 and Syk and Btk act to stabilize the complex consisting of the BCR, pSyk, pBLNK, pPLC-γ2, and pBtk. We observed that Cbl, an inhibitor of PLC-γ2 (39), was aberrantly colocalized with the BCR in B cells expressing Δ20–22 PLC-γ2, but not in B cells expressing wild-type PLC-γ2. The altered association of Cbl with the BCR could affect PLC-γ2 activity by targeting Syk for ubiquitination and degradation, a function we previously described for Cbl (41).…”

“…Indeed, in human B cells, the Cbl-interacting protein of 85 kD (CIN85) constitutively associates with Cbl and BLNK, and functions to increase the phosphorylation of Cbl, which results in decreased phosphorylation of PLC-γ2 and inhibition of Ca 2+ flux (39). These observations raised the possibility that the failure of BLNK to form a stable complex with Syk, Btk, and PLC-γ2 directly or indirectly enables the increased association of Cbl with BLNK, further inhibiting PLC-γ2 activity.…”

Preserved in vivo reconstitution ability of PBSCs cryopreserved for a decade at −80 °C

“…mRNAs were then extracted from cells and reverse transcribed to cDNA as described previously. 12 The mRNA levels were quantified in triplicate using a real-time PCR (Applied Biosystems, Japan, Tokyo). β2-Microglobulin mRNA separately amplified in the same plate was used for internal control.…”