An unexpected 2-histidine phosphoesterase activity of suppressor of T-cell receptor signaling protein 1 contributes to the suppression of cell signaling.

The suppressor of T-cell receptor (TCR) signaling (Sts) proteins Sts-1 and Sts-2 suppress receptor-mediated signaling pathways in various immune cells, including the TCR pathway in T cells and the Dectin-1 signaling pathway in phagocytes. As multidomain enzymes, they contain an N-terminal ubiquitin-association domain, a central Src homology 3 domain, and a C-terminal histidine phosphatase domain. Recently, a 2-histidine (2H) phosphoesterase motif was identified within the N-terminal portion of Sts. The 2H phosphoesterase motif defines an evolutionarily ancient protein domain present in several enzymes that hydrolyze cyclic phosphate bonds on different substrates, including cyclic nucleotides. It is characterized by two invariant histidine residues that play a critical role in catalytic activity. Consistent with its assignment as a phosphoesterase, we demonstrate here that the Sts-1 2H phosphoesterase domain displays catalytic, saturable phosphodiesterase activity toward the dinucleotide 2',3'-cyclic NADP. The enzyme exhibited a high degree of substrate specificity and selectively generated the 3'-nucleotide as the sole product. Sts-1 also had phosphodiesterase catalytic activity toward a 5-mer RNA oligonucleotide containing a 2',3'-cyclic phosphate group at its 3' terminus. To investigate the functional significance of Sts-1 2H phosphoesterase activity, we generated His-to-Ala variants and examined their ability to negatively regulate cellular signaling pathways. Substitution of either conserved histidine compromised the ability of Sts-1 to suppress signaling pathways downstream of both the TCR and the Dectin-1 receptor. Our results identify a heretofore unknown cellular enzyme activity associated with Sts-1 and indicate that this catalytic activity is linked to specific cell-signaling outcomes.

Structural and Functional Characterization of the Histidine Phosphatase Domains of Human Sts-1 and Sts-2.

The suppressor of T cell signaling (Sts) proteins, Sts-1 and Sts-2, are homologous phosphatases that negatively regulate signaling pathways downstream of the T cell receptor. Functional inactivation of Sts-1 and Sts-2 in a murine model leads to resistance to systemic infection by the opportunistic pathogen, Candida albicans. This suggests that modulation of the host immune response by inhibiting Sts function may be a viable strategy for treating these deadly fungal pathogen infections. To better understand the molecular determinants of function and structure, we characterized the structure and steady-state kinetics of the histidine phosphatase domains of human Sts-1 (Sts-1HP) and Sts-2 (Sts-2HP). We determined the X-ray crystal structures of unliganded Sts-1HP and Sts-1HP in complex with sulfate to 2.5 and 1.9 Å, respectively, and the structure of Sts-2HP with sulfate to 2.4 Å. The steady-state kinetic analysis shows, as expected, that Sts-1HP has a phosphatase activity significantly higher than that of Sts-2HP and that the human and mouse proteins behave similarly. In addition, comparison of the phosphatase activity of full-length Sts-1 protein to Sts-1HP reveals similar kinetics, indicating that Sts-1HP is a functional surrogate for the native protein. We also tested known phosphatase inhibitors and determined that the SHP-1 inhibitor, PHPS1, is a potent inhibitor of Sts-1 (Ki = 1.05 ± 0.15 µM). Finally, we demonstrated that human Sts-1 has robust phosphatase activity against the substrate, Zap-70, in a cell-based assay. Collectively, these data suggest that the human Sts proteins are druggable targets and provide a structural basis for future drug development efforts.