Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

Co-immunoprecipitation protocol to investigate cytokine receptor-associated proteins, e.g., Janus kinases or other associated signaling proteins.

Jak binding to cytokine receptors has been shown to be a complex and tight interaction. When studying loss-of-function or gain-of-function mutants of the Jaks or cytokine receptors it is often necessary to know if a certain mutant still associates correctly in the context of the signaling complex. The standard technique to show interaction of Jaks with cytokine receptors or other signalling molecules is Co-immunoprecipitation. Here we describe our protocol and discuss different pitfalls that can be encountered during the procedure.

Analysis of Janus tyrosine kinase phosphorylation and activation.

Activation of Janus kinases (Jaks) occurs through autophosphorylation of key tyrosine residues located primarily within their catalytic domain. Phosphorylation of these tyrosine residues facilitates access of substrates to the active site and serves as an intrinsic indicator of Jak activation. Here, we describe the methods and strategies used for analyzing Jak phosphorylation and activation. Tyrosine-phosphorylated (active) Jaks are primarily detected from cell extracts using anti-phosphotyrosine-directed Western blot analysis of Jak-specific immunoprecipitates. Additionally, receptor pull-down and in vitro kinase assays can also be utilized to measure cellular Jak catalytic activity. In addition to tyrosine phosphorylation, recent evidence indicates Jaks can be serine phosphorylated upon cytokine stimulation, however the lack of commercially available antibodies to detect these sites has hindered their analysis by Western blot. However, phosphoamino acid analysis (PAA) has been employed to monitor Jak serine and threonine phosphorylation. Over the past decade, remarkable advances have been made in our understanding of Jak function and dysfunction, however much remains to be learned about their complex regulatory mechanisms.

In vitro JAK kinase activity and inhibition assays.

The discovery that a range of myeloproliferative diseases and leukemias are associated with Janus Kinase (JAK) mutations has highlighted the importance of JAK/STAT signalling in disease and sparked a renewed interest in developing JAK inhibitors. In vitro kinase assays are the most direct and quantitative method to assess mutant forms of JAK for altered enzymatic properties as well as verifying and quantifying the affinity and efficacy of potential inhibitors. Here, we describe protocols for heterologous expression and purification of JAK kinases from insect cells, assays to determine the activity of these purified kinases, and finally inhibition assays to determine the effectiveness of potential inhibitors.

Determination of protein turnover rates in the JAK/STAT pathway using a radioactive pulse-chase approach.

The turnover rate of different protein species in a signal transduction network strongly affects the impact of the given species on the outcome of a stimulus. Whereas stable, long-lived proteins mainly account for the transmission of a signal, unstable short-lived species often comprise regulatory functions. Here, we describe a method to determine the half-lives of proteins of the JAK/STAT pathway by a pulse-chase approach in cell culture. First, radioactive labeling with (35)S-methionine is carried out to label newly synthesized proteins (pulse). Subsequently, the dynamics of the decay of these proteins is monitored in the absence of labeled amino acids over a defined time period (chase). For this purpose the protein of interest is isolated by immunoprecipitation from total cell lysates, separated on an SDS-polyacrylamide gel, and subsequently visualized by autoradiography.

Production and crystallization of recombinant JAK proteins.

JAK kinases are critical mediators in development, differentiation, and homeostasis and accordingly, have become well-validated targets for drug discovery efforts. In recent years, the integration of X-ray crystallography in kinase-focused drug discovery programs has provided a powerful rationale for chemical modification by allowing a unique glimpse of a bound inhibitor to its target. Such structural information has not only led to an improved understanding of the key drivers of potency and specificity of several JAK-specific compounds but has greatly facilitated and accelerated the design of compounds with improved pharmacokinetic properties.JAK kinases are traditionally difficult candidates to express in significant quantities, generally requiring eukaryotic expression systems, protein engineering, mutations to yield soluble, homogeneous samples suitable for crystallization studies. Here we review the key methods utilized to express, purify, and crystallize the JAK kinases and provide a detail description of the methods that we have developed to express, purify, and crystallize recombinant JAK1 and JAK2 proteins in the presence of small molecule inhibitors.