Laura Evelia Torres Velázquez

The adaptor protein Lnk, and the closely related proteins APS and SH2B, form a subfamily of SH2 domain-containing proteins implicated in growth factor, cytokine, and immunoreceptor signaling. To elucidate the physiological function of Lnk, we derived Lnk-deficient mice. Lnk(-/-) mice are viable, but display marked changes in the hematopoietic compartment, including splenomegaly and abnormal lymphoid and myeloid homeostasis. The in vitro proliferative capacity and absolute numbers of hematopoietic progenitors from Lnk(-/-) mice are greatly increased, in part due to hypersensitivity to several cytokines. Moreover, an increased synergy between stem cell factor and either interleukin (IL)-3 or IL-7 was observed in Lnk(-/-) cells. Furthermore, Lnk inactivation causes abnormal modulation of IL-3 and stem cell factor-mediated signaling pathways. Consistent with these results, we also show that Lnk is highly expressed in multipotent cells and committed precursors in the erythroid, megakaryocyte, and myeloid lineages. These data implicate Lnk as playing an important role in hematopoiesis and in the regulation of growth factor and cytokine receptor-mediated signaling.

Tyk2 and JAK1, members of the Janus kinase (JAK) family of protein tyrosine kinases, are required for interferon-α/β binding and signaling. Both enzymes are associated with the interferon-α/β receptor, and upon ligand binding, they undergo tyrosine phosphorylation and catalytic activation in an interdependent manner. To identify residues involved in Tyk2 regulation and to understand the basis of the interdependence of Tyk2 and JAK1, six mutated versions of Tyk2 bearing single or multiple point mutations in the tyrosine kinase domain were studied in a cell line lacking endogenous Tyk2. The Y1054F/Y1055F substitutions in the putative activation loop prevented ligand-dependent activation of Tyk2, without abolishing its catalytic potential. The K930R mutation in the ATP binding site generated a kinase-negative protein, which however, still became phosphorylated upon interferon-α treatment. The Y1054F/Y1055F substitutions in this kinase-negative Tyk2 abolished the induced phosphorylation. These results indicate that Tyk2 is activated by phosphorylation on Tyr-1054 and/or Tyr-1055 and that this phosphorylation requires another kinase, most likely JAK1. While the Tyk2 forms mutated on Tyr-1054 and Tyr-1055 or on Lys-930 allowed some inducible gene expression, the combination of the three point mutations totally abolished signaling. Tyk2 and JAK1, members of the Janus kinase (JAK) family of protein tyrosine kinases, are required for interferon-α/β binding and signaling. Both enzymes are associated with the interferon-α/β receptor, and upon ligand binding, they undergo tyrosine phosphorylation and catalytic activation in an interdependent manner. To identify residues involved in Tyk2 regulation and to understand the basis of the interdependence of Tyk2 and JAK1, six mutated versions of Tyk2 bearing single or multiple point mutations in the tyrosine kinase domain were studied in a cell line lacking endogenous Tyk2. The Y1054F/Y1055F substitutions in the putative activation loop prevented ligand-dependent activation of Tyk2, without abolishing its catalytic potential. The K930R mutation in the ATP binding site generated a kinase-negative protein, which however, still became phosphorylated upon interferon-α treatment. The Y1054F/Y1055F substitutions in this kinase-negative Tyk2 abolished the induced phosphorylation. These results indicate that Tyk2 is activated by phosphorylation on Tyr-1054 and/or Tyr-1055 and that this phosphorylation requires another kinase, most likely JAK1. While the Tyk2 forms mutated on Tyr-1054 and Tyr-1055 or on Lys-930 allowed some inducible gene expression, the combination of the three point mutations totally abolished signaling.

tyk2 belongs to the JAK family of nonreceptor protein tyrosine kinases recently found implicated in signaling through a large number of cytokine receptors. These proteins are characterized by a large amino-terminal region and two tandemly arranged kinase domains, a kinase-like and a tyrosine kinase domain. Genetic and biochemical evidence supports the requirement for tyk2 in interferon-alpha/beta binding and signaling. To study the role of the distinct domains of tyk2, constructs lacking one or both kinase domains were stably transfected in recipient cells lacking the endogenous protein. Removal of either or both kinase domains resulted in loss of the in vitro kinase activity. The mutant form truncated of the tyrosine kinase domain was found to reconstitute binding of interferon-alpha 8 and partial signaling. While no contribution of this protein toward interferon-beta binding was evident, increased signaling could be measured. The mutant form lacking both kinase domains did not exhibit any detectable activity. Altogether, these results show that a sequential deletion of domains engenders a sequential loss of function and that the different domains of tyk2 have distinct functions, all essential for full interferon-alpha and -beta binding and signaling.