The adaptor protein CIN85 assembles intracellular signaling clusters for B cell activation

Abstract

The adaptor molecule Cbl-interacting protein of 85 kD (CIN85) regulates signaling from a number of cell surface receptors, such as growth factor receptors and antigen receptors on lymphocytes. Because of its multidomain structure, CIN85 is thought to act as a classical adaptor protein that connects functionally distinct components of a given signaling pathway through diverse protein domains. However, we found that in B lymphocytes, CIN85 functions to oligomerize SLP-65, which is the central effector protein of the B cell receptor (BCR). Therefore, CIN85 trimerizes through a carboxyl-terminal, coiled-coil domain. The multiple Src homology 3 (SH3) domains of trimeric CIN85 molecules associated with multiple SLP-65 molecules, which recruited further CIN85 trimers, thereby perpetuating the oligomerization process. Formation of this oligomeric signaling complex in resting B cells rendered the cells poised for the efficient initiation of intracellular signaling upon BCR stimulation. Our data suggest that the functionality of signaling cascades does not rely solely on the qualitative linkage of their various components but requires a critical number of effectors to become concentrated in signaling complexes.

Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness

“…Tripartite phase separation at physiological concentrations. In accordance with previously published data 13,15 , citrine-tagged wild-type SLP65 but not mutant versions of SLP65 lacking either the N-terminal lipid binding domain or the CIN85-interacting PRMs formed submicrometer granules in resting B cells (Fig. 1a).…”

“…1). Hence, the formation of granules through coordinated interactions of SLP65 with both vesicles and CIN85 appeared requisite for proper SLP65 signaling as suggested previously 13,15 . Next, we investigated by confocal fluorescence microscopy, whether granule formation can be recapitulated in vitro using recombinantly expressed SLP65 and CIN85 and synthetic lipid vesicles in different combinations.…”

“…However, the vesicular compartmentalization of SLP65 appeared to be critical for the stimulation-independent (i.e. constitutive) association of SLP65 with CIN85 13,15 . The association of SLP65 and CIN85 is caused by atypical SH3 domain docking motifs in SLP65 that associate in a promiscuous manner with the three N-terminal SH3 domains of CIN85 15 .…”

“…constitutive) association of SLP65 with CIN85 13,15 . The association of SLP65 and CIN85 is caused by atypical SH3 domain docking motifs in SLP65 that associate in a promiscuous manner with the three N-terminal SH3 domains of CIN85 15 . A Cterminal coiled coil domain of CIN85 forms stable CIN85 trimers that drive multimerization of SLP65.…”

Human Cytomegalovirus UL135 Interacts with Host Adaptor Proteins To Regulate Epidermal Growth Factor Receptor and Reactivation from Latency

“…Like Abi-1, CIN85 and CD2AP regulate trafficking and signaling of receptor tyrosine kinases (RTKs) (34-39) by recruiting endocytic components (e.g., Alix [40], Dab2 [41], clathrin adaptor AP-2 [42], endophilins [43,44], and Cbl ubiquitin ligase [37,[44][45][46][47][48][49]). CD2AP is highly homologous to CIN85 in regard to structure and function (39,(45)(46)(47)(50)(51)(52). Because CD2AP and CIN85 bind to the same polyproline consensus sites and have similar interactomes and similar functions, we cannot genetically distinguish the contribution of CIN85 and CD2AP interactions solely by mutating their common SH3 ligands in pUL135.…”