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JAK2 activation by growth hormone and other cytokines.

Waters MJ, Brooks AJ - Biochem. J. (2015)

Bottom Line: Binding of the bivalent ligand reorientates and rotates the receptor subunits, resulting in a transition from a form with parallel TMDs to one where the TMDs separate at the point of entry into the cytoplasm.This results in phosphorylation and activation of STATs and other signalling pathways linked to this receptor which then regulate postnatal growth, metabolism and stem cell activation.We believe that this model will apply to most if not all members of the class I cytokine receptor family, and will be useful in the design of small antagonists and agonists of therapeutic value.

View Article: PubMed Central - PubMed

Affiliation: *Institute for Molecular Bioscience, The University of Queensland Institute, QLD 4072, Australia.

ABSTRACT
Growth hormone (GH) and structurally related cytokines regulate a great number of physiological and pathological processes. They do this by coupling their single transmembrane domain (TMD) receptors to cytoplasmic tyrosine kinases, either as homodimers or heterodimers. Recent studies have revealed that many of these receptors exist as constitutive dimers rather than being dimerized as a consequence of ligand binding, which has necessitated a new paradigm for describing their activation process. In the present study, we describe a model for activation of the tyrosine kinase Janus kinase 2 (JAK2) by the GH receptor homodimer based on biochemical data and molecular dynamics simulations. Binding of the bivalent ligand reorientates and rotates the receptor subunits, resulting in a transition from a form with parallel TMDs to one where the TMDs separate at the point of entry into the cytoplasm. This movement slides the pseudokinase inhibitory domain of one JAK kinase away from the kinase domain of the other JAK within the receptor dimer-JAK complex, allowing the two kinase domains to interact and trans-activate. This results in phosphorylation and activation of STATs and other signalling pathways linked to this receptor which then regulate postnatal growth, metabolism and stem cell activation. We believe that this model will apply to most if not all members of the class I cytokine receptor family, and will be useful in the design of small antagonists and agonists of therapeutic value.

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Related in: MedlinePlus

Model of JAK2 activation for the GH receptorIn the inactive state (top), two JAK2 molecules, each bound to a GH receptor intracellular domain, interact so that the kinase domain of one JAK2 is inhibited by the pseudokinase domain of the other JAK2. GH binding causes separation of the ICD Box1 motifs and leads to separation of the pseudokinase–kinase trans-interaction and results in kinase–kinase trans-interaction and activation (bottom).
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Figure 5: Model of JAK2 activation for the GH receptorIn the inactive state (top), two JAK2 molecules, each bound to a GH receptor intracellular domain, interact so that the kinase domain of one JAK2 is inhibited by the pseudokinase domain of the other JAK2. GH binding causes separation of the ICD Box1 motifs and leads to separation of the pseudokinase–kinase trans-interaction and results in kinase–kinase trans-interaction and activation (bottom).

Mentions: Our group sought in vivo support for the JAK2 trans-inhibition model initially by introducing FRET reporters in place of the PK domain or at the C-terminus, where the kinase domain is located. The prediction was that receptor activation would result in a decrease in FRET for the PK domain located reporter, but an increase for the kinase domain located reporter. Using the wild-type GH receptor and the EED to KKR charge reversal receptor construct either separately or together, it was possible to ensure that essentially all of the receptors would be in inactive State 1 (when wild-type or KKR mutant were expressed alone) or consist of a subset of active State 2 receptors (when co-expressed). A decrease in FRET for the PK position reporter was indeed observed when transiently transfected HEK293T cells expressed JAK2 FRET reporters with both wild-type and KKR mutant receptors together (giving constitutive activation) compared with cells with expression of a single receptor construct alone. Conversely, similar analysis using the kinase position reporter showed an increase in FRET demonstrating that activation results in an increase in distance between the JAK2 PK domains while bringing the kinase domains in closer proximity, as predicted. Further in vivo evidence for the trans-model was obtained by reversing the positions of the kinase and PK domains in the JAK2 expression construct, then co-transfecting this construct with a construct for wild-type JAK2 (along with the GH receptor and STAT5) into JAK-deficient γ2A fibrosarcoma cells. The JAK2 trans-model (Figure 5) predicts constitutive activity with this co-transfection, since the combination would remove the PK domain from the other kinase domain and appose the kinase domains. This was indeed evident in multiple experiments, but only in the presence of the receptor, consistent with the trans-activation mechanism [44].


JAK2 activation by growth hormone and other cytokines.

Waters MJ, Brooks AJ - Biochem. J. (2015)

Model of JAK2 activation for the GH receptorIn the inactive state (top), two JAK2 molecules, each bound to a GH receptor intracellular domain, interact so that the kinase domain of one JAK2 is inhibited by the pseudokinase domain of the other JAK2. GH binding causes separation of the ICD Box1 motifs and leads to separation of the pseudokinase–kinase trans-interaction and results in kinase–kinase trans-interaction and activation (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325515&req=5

Figure 5: Model of JAK2 activation for the GH receptorIn the inactive state (top), two JAK2 molecules, each bound to a GH receptor intracellular domain, interact so that the kinase domain of one JAK2 is inhibited by the pseudokinase domain of the other JAK2. GH binding causes separation of the ICD Box1 motifs and leads to separation of the pseudokinase–kinase trans-interaction and results in kinase–kinase trans-interaction and activation (bottom).
Mentions: Our group sought in vivo support for the JAK2 trans-inhibition model initially by introducing FRET reporters in place of the PK domain or at the C-terminus, where the kinase domain is located. The prediction was that receptor activation would result in a decrease in FRET for the PK domain located reporter, but an increase for the kinase domain located reporter. Using the wild-type GH receptor and the EED to KKR charge reversal receptor construct either separately or together, it was possible to ensure that essentially all of the receptors would be in inactive State 1 (when wild-type or KKR mutant were expressed alone) or consist of a subset of active State 2 receptors (when co-expressed). A decrease in FRET for the PK position reporter was indeed observed when transiently transfected HEK293T cells expressed JAK2 FRET reporters with both wild-type and KKR mutant receptors together (giving constitutive activation) compared with cells with expression of a single receptor construct alone. Conversely, similar analysis using the kinase position reporter showed an increase in FRET demonstrating that activation results in an increase in distance between the JAK2 PK domains while bringing the kinase domains in closer proximity, as predicted. Further in vivo evidence for the trans-model was obtained by reversing the positions of the kinase and PK domains in the JAK2 expression construct, then co-transfecting this construct with a construct for wild-type JAK2 (along with the GH receptor and STAT5) into JAK-deficient γ2A fibrosarcoma cells. The JAK2 trans-model (Figure 5) predicts constitutive activity with this co-transfection, since the combination would remove the PK domain from the other kinase domain and appose the kinase domains. This was indeed evident in multiple experiments, but only in the presence of the receptor, consistent with the trans-activation mechanism [44].

Bottom Line: Binding of the bivalent ligand reorientates and rotates the receptor subunits, resulting in a transition from a form with parallel TMDs to one where the TMDs separate at the point of entry into the cytoplasm.This results in phosphorylation and activation of STATs and other signalling pathways linked to this receptor which then regulate postnatal growth, metabolism and stem cell activation.We believe that this model will apply to most if not all members of the class I cytokine receptor family, and will be useful in the design of small antagonists and agonists of therapeutic value.

View Article: PubMed Central - PubMed

Affiliation: *Institute for Molecular Bioscience, The University of Queensland Institute, QLD 4072, Australia.

ABSTRACT
Growth hormone (GH) and structurally related cytokines regulate a great number of physiological and pathological processes. They do this by coupling their single transmembrane domain (TMD) receptors to cytoplasmic tyrosine kinases, either as homodimers or heterodimers. Recent studies have revealed that many of these receptors exist as constitutive dimers rather than being dimerized as a consequence of ligand binding, which has necessitated a new paradigm for describing their activation process. In the present study, we describe a model for activation of the tyrosine kinase Janus kinase 2 (JAK2) by the GH receptor homodimer based on biochemical data and molecular dynamics simulations. Binding of the bivalent ligand reorientates and rotates the receptor subunits, resulting in a transition from a form with parallel TMDs to one where the TMDs separate at the point of entry into the cytoplasm. This movement slides the pseudokinase inhibitory domain of one JAK kinase away from the kinase domain of the other JAK within the receptor dimer-JAK complex, allowing the two kinase domains to interact and trans-activate. This results in phosphorylation and activation of STATs and other signalling pathways linked to this receptor which then regulate postnatal growth, metabolism and stem cell activation. We believe that this model will apply to most if not all members of the class I cytokine receptor family, and will be useful in the design of small antagonists and agonists of therapeutic value.

Show MeSH
Related in: MedlinePlus