As anyone whose car has ever broken down knows, working out the mechanical defect that is responsible can be rather tricky. Similarly, elucidating the basis for the phenotypes that result from gene-targeting experiments can also prove to be difficult. Mice that are deficient for T-cell protein tyrosine phosphatase (Tcptp) have severe defects in the haematopoietic compartment — including splenomegaly, lymphadenopathy, and defects in lymphocyte proliferation and haematopoiesis — but the pathways that are disrupted in these mice have not been identified. Simoncic and colleagues now report in Current Biology that the Janus-family tyrosine kinases Jak1 and Jak3 are physiological substrates for Tcptp, and that defects in cytokine signalling are to blame for the defective immune homeostasis that is seen in Tcptp−/− mice.

The authors used an in vivo substrate-trapping approach to identify substrates of Tcptp. They constructed wildtype and substrate-trapping mutants of Tcptp (which bind, but cannot dephosphorylate, substrates); these were transfected into cells and, after cytokine stimulation, immunoprecipitation and anti-phosphotyrosine immunoblotting were performed. Using this approach, they showed that tyrosine phosphorylated Jak1 and Jak3 are substrates for Tcptp downstream of interleukin-2 (IL-2), and that Jak1 is a substrate for Tcptp downstream of interferon-γ (IFN-γ). They also showed that the interaction between Tcptp and Jak1/Jak3 is mediated by the catalytic domain of Tcptp.

Cytokine signalling results in the activation of Jaks and subsequent tyrosine phosphorylation of signal transducer and activator of transcription (Stat) proteins, which then dimerize and translocate to the nucleus to activate target genes. Simoncic and co-workers investigated the effect of overexpression and loss of function of Tcptp on Stat phosphorylation. When IL-2-treated T cells were transfected with wildtype Tcptp, Stat5 phosphorylation was impaired, whereas in Tcptp−/− T cells, Stat5 was hyperphosphorylated. To investigate the role of Tcptp in additional downstream events, the authors assessed the effect of IFN-γ treatment on Tcptp−/− T cells and macrophages. IFN-γ treatment resulted in an increased level of Stat1 phosphorylation in Tcptp−/− T cells, and an increased level of Jak1 tyrosine phosphorylation and increased expression of inducible nitric oxide synthase (iNOS; a primary target of IFN-γ signalling) in Tcptp−/− macrophages, compared with wildtype cells.

These results indicate that Tcptp has a negative regulatory role in cytokine signalling through the dephosphorylation of Jak1 and Jak3, a role that correlates with the phenotype of Tcptp−/− animals.