Tetraspanin microdomains control localized protein kinase C signaling in B cells

Abstract

Activation of B cells by the binding of antigens to the B cell receptor (BCR) requires the protein kinase C (PKC) family member PKCβ. Because PKCs must translocate to the plasma membrane to become activated, we investigated the mechanisms regulating their spatial distribution in mouse and human B cells. Through live-cell imaging, we showed that BCR-stimulated production of the second messenger diacylglycerol (DAG) resulted in the translocation of PKCβ from the cytosol to plasma membrane regions containing the tetraspanin protein CD53. CD53 was specifically enriched at sites of BCR signaling, suggesting that BCR-dependent PKC signaling was initiated at these tetraspanin microdomains. Fluorescence lifetime imaging microscopy studies confirmed the molecular recruitment of PKC to CD53-containing microdomains, which required the amino terminus of CD53. Furthermore, we showed that -deficient B cells were defective in the phosphorylation of PKC substrates. Consistent with this finding, PKC recruitment to the plasma membrane was impaired in both mouse and human-deficient B cells compared to that in their wild-type counterparts. These data suggest that CD53 promotes BCR-dependent PKC signaling by recruiting PKC to the plasma membrane so that it can phosphorylate its substrates and that tetraspanin-containing microdomains can act as signaling hotspots in the plasma membrane.

Dynamic regulation of CD45 by tetraspanin CD53

“…Since CD45 is known to function during early TCR signaling at the immunological synapse, we investigated whether CD53 was also recruited to sites of heavy TCR signaling. To achieve this, we examined the localization of CD53 during TCR signaling using the micro-contact printing technique 14 . This allows us to analyze the recruitment of membrane proteins to discrete areas of TCR signaling in live cells.…”

“…Quantification showed that the CD53 signal was significantly increased at the stamp contact site, in contrast to CD37 (Figure 5C). Based on our previous study that demonstrated a direct interaction between CD53 and PKCβ in B cells, we investigated whether such interaction was also present in T cells 14 . Using micro-contact printing we found that PKCθ was also recruited to the TCR signaling sites, which was contemporaneous with CD53 recruitment (Figure S3A-B).…”

“…This is in stark contrast to our findings, which showed a reduced proliferation of Cd53-/- T cells, suggesting that CD53 on T cells functions through a mechanism that is unique to this tetraspanin. Furthermore, we have previously reported that CD53 plays an important role in recruiting and stabilizing PKCβ in B cells 14 . In contrast, our observations show that the CD53 is not involved in the direct stabilization of PKCθ in T cells.…”

“…They interact with each other and with partner proteins, forming membrane structures known as tetraspanin enriched microdomains (TEMs). Tetraspanins have been shown to play important roles in numerous cell functions including, adhesion and migration, fusion and cell signaling 11, 12, 13, 14 . Though immune cells express many tetraspanin proteins, there are only two known immune-specific tetraspanins: CD37 and CD53.…”

“…Though immune cells express many tetraspanin proteins, there are only two known immune-specific tetraspanins: CD37 and CD53. We have previously shown that CD53 plays a role in the regulation of B cell signaling, but CD53 function in T cells remains unknown 14 .…”