Regulation of the Jak2 Tyrosine Kinase by Its Pseudokinase Domain

Abstract

Activation of Jak tyrosine kinases through hematopoietic cytokine receptors occurs as a consequence of ligand-induced aggregation of receptor-associated Jaks and their subsequent autophosphorylation. Jak kinases consist of a C-terminal tyrosine kinase domain, a pseudokinase domain of unknown function, and Jak homology (JH) domains 3 to 7, implicated in receptor-Jak interaction. We analyzed the functional roles of the different protein domains in activation of Jak2. Deletion analysis of Jak2 showed that the pseudokinase domain but not JH domains 3 to 7 negatively regulated the catalytic activity of Jak2 as well as Jak2-mediated activation of Stat5. Phosphorylation of Stat5 by wild-type Jak2 was dependent on the SH2 domain of Stat5; however, this requirement was lost upon deletion of the pseudokinase domain of Jak2. Investigation of the mechanisms of the pseudokinase domain-mediated inhibition of Jak2 suggested that this regulation did not involve protein tyrosine phosphatases. Instead, analysis of interactions between the tyrosine kinase domain and Jak2 suggested that the pseudokinase domain interacted with the kinase domain. Furthermore, coexpression of the pseudokinase domain inhibited the activity of the single tyrosine kinase domain. Finally, deletion of the pseudokinase domain of Jak2 deregulated signal transduction through the gamma interferon receptor by significantly increasing ligand-independent activation of Stat transcription factors. These results indicate that the pseudokinase domain negatively regulates the activity of Jak2, probably through an interaction with the kinase domain, and this regulation is required to keep Jak2 inactive in the absence of ligand stimulation. Furthermore, the pseudokinase domain may have a role in regulation of Jak2-substrate interactions.

Rac1 and a GTPase-activating protein, MgcRacGAP, are required for nuclear translocation of STAT transcription factors

“…To construct baculovirus vectors, the cDNAs encoding STAT5A, MgcRac-GAP, V12Rac1, L61Rac1, N17Rac1, importin αs, importin β1, Ran, and NTF2 with the C-terminal Flag epitope tag, and a kinase domain of JAK2 (JH1; Saharinen et al, 2000) were subcloned into pBacPAK (BD Biosciences). The resulting constructs were used to obtain recombinant baculoviruses by cotransfection with Bsu36 I-digested BacPAK viral DNA (BD Biosciences) into Sf-9 cells according to the manufacturer's protocol.…”

Loss of the JAK2 intramolecular auto‐inhibition mechanism is predicted by structural modelling of a novel exon 12 insertion mutation in a case of idiopathic erythrocytosis

“…JAK2 is maintained in a low activity state through various auto-regulatory mechanisms, preventing the proper orientation for catalysis of the activation loop (A-loop) in the kinase domain (JH1; Huse & Kuriyan, 2002). Previous studies confirmed the regulatory role of the catalytically inactive JH2 pseudo-kinase domain, including exons 12-19, on modulating basal enzymatic activity (Luo et al, 1997;Chen et al, 2000;Saharinen et al, 2000Saharinen et al, , 2003Saharinen & Silvennoinen, 2002). The crystallographic structure of JAK2 is not yet available, but computer-based homology models and extensive experimental data on mutant JAK2 indicate that the JH2 domain negatively regulates the activity of JAK2 by intramolecular interactions with the adjacent C-terminal JH1 domain (Saharinen et al, 2000(Saharinen et al, , 2003Lindauer et al, 2001;Saharinen & Silvennoinen, 2002).…”

The role of Janus kinases in haemopoiesis and haematological malignancy

“…The JH2 domain is thought to play a significant role in the regulation of JAK catalytic activity. Deletion of the JH2 domain, or of subregions within it, have been shown to result in an increased basal activity of JAK2 and JAK3 (Saharinen et al , 2000); however, such deletions can also abolish the response to cytokine stimulation of JAKs indicating that, although in the absence of ligand JAK2 is autoinhibited by an intramolecular interaction between JH2 and JH1 domains, induction of maximal activation requires an intact JH2 domain (Saharinen & Silvennoinen, 2002; Saharinen et al , 2003). The crystal structures of the isolated catalytic domains of both JAK2 and JAK3 have been recently reported, in both instances in complex with a kinase inhibitor (Boggon et al , 2005; Lucet et al , 2006).…”