Feedback inhibition of calcineurin and Ras by a dual inhibitory protein Carabin

Vol 445 | 25 January 2007 | doi:10.1038/nature05476

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Feedback inhibition of calcineurin and Ras by a dual inhibitory protein Carabin

Fan Pan1, Luo Sun1{, David B. Kardian4, Katharine A. Whartenby4, Drew M. Pardoll4 & Jun O. Liu1,2,3

Feedback regulation of adaptive immunity is a fundamental mechanism for controlling the overall output of different signal transduction pathways, including that mediated by the T-cell antigen receptor (TCR)1. Calcineurin24 and Ras57 are known to have essential functions during T-cell activation. However, how the calcineurin signalling pathway is terminated in the process is still largely unknown. Although several endogenous inhibitors of calcineurin have been reported813, none fulfils the criteria of a feedback inhibitor, as their expression is not responsive to TCR signalling. Here we identify an endogenous inhibitor of calcineurin, named Carabin, which also inhibits the Ras signalling pathway through its intrinsic Ras GTPase-activating protein (GAP) activity. Expression of Carabin is upregulated on TCR signalling in a manner that is sensitive to inhibitors of calcineurin, indicating that Carabin constitutes part of a negative regulatory loop for the intracellular TCR signalling pathway. Knockdown of Carabin by short interfering RNA led to a significant enhancement of interleukin-2 production by antigen-specific T cells in vitro and in vivo. Thus, Carabin is a negative feedback inhibitor of the calcineurin signalling pathway that also mediates crosstalk between calcineurin and Ras.

To identify new calcineurin-binding proteins we performed a yeast two-hybrid screen of a human T-cell complementary DNA library, using a truncated and catalytically inactive calcineurin mutant as bait8. One interacting protein identified was Cabin1, which has been extensively characterized1,1416. The other calcineurin-binding protein was named Carabin on the basis of its ability to interact with both calcineurin and Ras. Carabin is unrelated to Cabin1. Human Carabin (GenBank accession number NP_940919) consists of 446 amino acid residues and is 88% identical and 91% similar in sequence to the mouse orthologue (GenBank accession number BAC30605; Fig. 1a). Carabin contains a putative Ras/Rab GAP domain at its amino terminus (residues 89294) and a carboxy-terminal domain (residues 406446) that mediates its interaction with calcineurin. Northern blot analysis revealed that Carabin is most abundant in spleen and peripheral blood leukocytes (Supplementary Fig. 1).

The interaction between Carabin and calcineurin was confirmed both in vitro and in vivo. A...