Enzymatic Characterization of Wild-Type and Mutant Janus Kinase 1

Janus kinases (JAKs) are found constitutively associated with cytokine receptors and are present in an inactive state prior to cytokine exposure. Activating mutations of JAKs are causative for a number of leukemias, lymphomas, and myeloproliferative diseases. In particular, the JAK2V617F mutant is found in most human cases of polycythemia vera, a disease characterized by over-production of erythrocytes. The V617F mutation is found in the pseudokinase domain of JAK2 and it leads to cytokine-independent activation of the kinase, as does the orthologous mutation in other JAK-family members. The mechanism whereby this mutation hyperactivates these kinases is not well understood, primarily due to the fact that the full-length JAK proteins are difficult to produce for structural and kinetic studies. Here we have overcome this limitation to perform a series of enzymatic analyses on full-length JAK1 and its constitutively active mutant form (JAK1V658F). Consistent with previous studies, we show that the presence of the pseudokinase domain leads to a dramatic decrease in enzymatic activity with no further decrease from the presence of the FERM or SH2 domains. However, we find that the mutant kinase, in vitro, is indistinguishable from the wild-type enzyme in every measurable parameter tested: KM (ATP), KM (substrate), kcat, receptor binding, thermal stability, activation rate, dephosphorylation rate, and inhibitor affinity. These results show that the V658F mutation does not enhance the intrinsic enzymatic activity of JAK. Rather this data is more consistent with a model in which there are cellular processes and interactions that prevent JAK from being activated in the absence of cytokine and it is these constraints that are affected by disease-causing mutations

SOCS3 binds specific receptor-JAK complexes to control cytokine signaling by direct kinase inhibition

The inhibitory protein SOCS3 plays a key part in the immune and hematopoietic systems by regulating signaling induced by specific cytokines. SOCS3 functions by inhibiting the catalytic activity of Janus kinases (JAKs) that initiate signaling within the cell. We determined the crystal structure of a ternary complex between mouse SOCS3, JAK2 (kinase domain) and a fragment of the interleukin-6 receptor β-chain. The structure shows that SOCS3 binds JAK2 and receptor simultaneously, using two opposing surfaces. While the phosphotyrosine-binding groove on the SOCS3 SH2 domain is occupied by receptor, JAK2 binds in a phosphoindependent manner to a noncanonical surface. The kinase-inhibitory region of SOCS3 occludes the substrate-binding groove on JAK2, and biochemical studies show that it blocks substrate association. These studies reveal that SOCS3 targets specific JAK-cytokine receptor pairs and explains the mechanism and specificity of SOCS action

The molecular basis of JAK/STAT inhibition by SOCS1

The SOCS family of proteins are negative-feedback inhibitors of signalling induced by cytokines that act via the JAK/STAT pathway. SOCS proteins can act as ubiquitin ligases by recruiting Cullin5 to ubiquitinate signalling components; however, SOCS1, the most potent member of the family, can also inhibit JAK directly. Here we determine the structural basis of both these modes of inhibition. Due to alterations within the SOCS box domain, SOCS1 has a compromised ability to recruit Cullin5; however, it is a direct, potent and selective inhibitor of JAK catalytic activity. The kinase inhibitory region of SOCS1 targets the substrate binding groove of JAK with high specificity and thereby blocks any subsequent phosphorylation. SOCS1 is a potent inhibitor of the interferon gamma (IFNγ) pathway, however, it does not bind the IFNγ receptor, making its mode-of-action distinct from SOCS3. These findings reveal the mechanism used by SOCS1 to inhibit signalling by inflammatory cytokines

Epitaxie par jets moleculaire d'heterostructures GaAs/compose metallique/GaAs

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