Abstract
T cell receptor (TCR) phosphorylation requires the kinase Lck and phosphatase CD45. CD45 activates Lck by dephosphorylating an inhibitory tyrosine of Lck to relieve autoinhibition. However, CD45 also dephosphorylates the TCR, and the spatial exclusion of CD45 from TCR clustering in the plasma membrane appears to attenuate this negative effect of CD45. To further investigate the role of CD45 in signal initiation, we reconstituted membrane TCR clusters in vitro on supported lipid bilayers. Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of TCR clusters, but only when Lck co-clustered with TCR. We found that clustered Lck autophosphorylated the inhibitory tyrosine and thus could be activated by CD45, whereas diffusive Lck molecules did not. In the TCR-Lck clusters and at low CD45 density, we speculate that the effect of Lck activation may overcome dephosphorylation of TCR, resulting in a net positive regulation. The CD45 density in physiological TCR clusters is also low because of the exclusion of CD45. Thus, we propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important not only for modulating the negative role of CD45 but also for creating conditions in which CD45 has a positive role in signal initiation.
Highlights
The roles of protein clustering in T cell signaling are poorly understood
Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of T cell receptor (TCR) clusters, but only when Lck co-clustered with TCR
We propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important for modulating the negative role of CD45 and for creating conditions in which CD45 has a positive role in signal initiation
Summary
The roles of protein clustering in T cell signaling are poorly understood. Results: Lck clustering recreates conditions in which a phosphatase has positive and negative roles in signal initiation. CD45 dephosphorylates Tyr(P)505 and increases the catalytic activity of Lck, and Lck activation by CD45 is thought to be indispensable for T cell signaling and development [1] Another regulatory mechanism is clustering of TCR and other proteins in the plasma membrane. CD45 phosphatase activity is decreased and the balance of biochemical reactions is altered in the clusters [13,14,15] These multiple regulatory mechanisms are coupled, resulting in diverse phenomena observed in T cell signaling. The spatial exclusion of CD45 from TCR clusters in plasma membranes likely play a role in reducing TCR dephosphorylation, highlighting the negative regulatory role of CD45 in T cell signaling [13,14,15]. We investigated how such effects might underlie the dual positive/negative regulatory functions of CD45 in TCR phosphorylation
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