Yash Chhabra

Mechanism of Activation of Protein Kinase JAK2 by the Growth Hormone Receptor

Introduction Class I cytokines regulate key processes such as growth, lactation, hematopoiesis, and immune function and contribute to oncogenesis. Although the extracellular domain structures of their receptors are well characterized, little is known about how the receptors activate their associated JAK (Janus kinase) protein kinases. We provide a mechanistic description for this process, focusing on the growth hormone (GH) receptor and its associated JAK2. Receptor-JAK2 activation process. (Top) Cartoons of the GH receptor basal state (state 1, left) and the active state (state 2, right) with (Bottom) transmembrane helix alignments for these states derived by modeling. GHR, GH receptor. Rationale We tested whether the receptor exists as a dimer in the inactive state by homo-FRET [fluorescence resonance energy transfer (FRET) between the proteins labeled with the same fluorophore] and other means. Then, to define receptor movements resulting from activation, we attached FRET reporters to the receptor below the cell membrane and correlated their movement with receptor activation, measured as increased cell proliferation. We controlled the position of the transmembrane helices with leucine zippers and mutagenesis, and we again monitored FRET and receptor activation. We used cysteine cross-linking data to define the faces of the transmembrane helices in contact in the basal state and verified this with molecular dynamics, which allowed us to model the activation process. We also used FRET reporters to monitor the movement of JAK2, and we matched this with molecular dynamics docking of the crystal structures of the kinase and its pseudokinase domains to derive a model for activation, which we then verified experimentally. Results We found that the GH receptor exists predominantly as a dimer in vivo, held together by its transmembrane helices. These helices are parallel in the basal state, and binding of the hormone converts them into a left-hand crossover state that induces separation of helices at the lower transmembrane boundary (hence, Box1 separation). This movement is triggered by increased proximity of the juxtamembrane sequences, a consequence of locking together of the lower module of the extracellular domain on hormone binding. This movement is triggered by increased proximity of the juxtamembrane sequences , a Both this locking and the helix state transition require rotation of the receptors, but the key outcome is separation of the Box1 sequences. Because these sequences are bound to the JAK2 FERM (4.1, ezrin, radixin, moesin) domains, this separation results in removal of the pseudokinase inhibitory domain of one JAK2, which is blocking the kinase domain of the other JAK2, and vice versa. This brings the two kinase domains into productive apposition, triggering JAK2 activation. We verified this mechanism by kinase-pseudokinase domain swap, by changes in JAK2 FRET signal on activation, by showing association of pseudokinase-kinase domain pairs, and by docking of the crystal structures. An animation of our complete model of GH receptor activation is provided at http://web-services.imb.uq.edu.au/waters/hgh.html. Conclusion The proposed mechanism will be useful in understanding the many actions of GH, which include altered growth, metabolism, and bone turnover. We expect that it may extend to other members of this important receptor family. The mechanism provides a molecular basis for understanding the oncogenic JAK2 mutations responsible for polycythemia vera and certain other hematologic disorders and may thus be of value in the design of small-molecule inhibitors of clinical applicability. Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors. A molecular mechanism for transmembrane signaling by the growth hormone receptor is elucidated. [Also see Perspective by Wells and Kossiakoff] The Hormones Message The receptor for growth hormone is a well-studied representative of a family of cytokine receptors through which binding of hormone molecules at the cell surface is converted into a biochemical signal within the cell. Brooks et al. (10.1126/science.1249783; see the Perspective by Wells and Kossiakoff) used a combination of crystal structures, biophysical measurements, cell biology experiments with modified receptors, and molecular dynamics and modeling to decipher how the receptor actually transmits the information that a hormone molecule is bound. The results suggest that the receptors exist in inactive dimeric complexes in which two associated JAK2 protein kinase molecules interact in an inhibitory manner. Binding of growth hormone causes a structural change in the receptor that results in movement of the receptor intracellular domains apart from one another. This relieves the inhibition of the JAK2 molecules and allows them to activate one another, thus initiating the cellular response to the hormone.

Role of the growth hormone- IGF-1 axis in cancer

A substantial body of evidence supports a role for the growth hormone (GH)–IGF-1 axis in cancer incidence and progression. This includes epidemiological evidence relating elevated plasma IGF-1 to cancer incidence as well as a lack of cancers in GH/IGF-1 deficiency. Rodent models lacking GH or its receptor are strikingly resistant to the induction of a wide range of cancers, and treatment with the GH antagonist pegvisomant slows tumor progression. While GH receptor expression is elevated in many cancers, autocrine GH is present in several types, and overexpression of autocrine GH can induce cell transformation. While the mechanism of autocrine action is not clear, it does involve both STAT5 and STAT3 activation, and probably nuclear translocation of the GH receptor. Development of a more potent GH receptor antagonist or secretion inhibitor is warranted for cancer therapy.

NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF

While invasion and metastasis of tumour cells are the principle factor responsible for cancer related deaths, the mechanisms governing the process remain poorly defined. Moreover, phenotypic divergence of sub-populations of tumour cells is known to underpin alternative behaviors linked to tumour progression such as proliferation, survival and invasion. In the context of melanoma, heterogeneity between two transcription factors, BRN2 and MITF, has been associated with phenotypic switching between predominantly invasive and proliferative behaviors respectively. Epigenetic changes, in response to external cues, have been proposed to underpin this process, however the mechanism by which the phenotypic switch occurs is unclear. Here we report the identification of the NFIB transcription factor as a novel downstream effector of BRN2 function in melanoma cells linked to the migratory and invasive characteristics of these cells. Furthermore, the function of NFIB appears to drive an invasive phenotype through an epigenetic mechanism achieved via the upregulation of the polycomb group protein EZH2. A notable target of NFIB mediated up-regulation of EZH2 is decreased MITF expression, which further promotes a less proliferative, more invasive phenotype. Together our data reveal that NFIB has the ability to promote dynamic changes in the chromatin state of melanoma cells to facilitate migration, invasion and metastasis.

A new mechanism for growth hormone receptor activation of JAK2, and implications for related cytokine receptors

The growth hormone receptor was the first cytokine receptor to be cloned and crystallized, and provides a valuable exemplar for activation of its cognate kinase, JAK2. We review progress in understanding its activation mechanism, in particular the molecular movements made by this constitutively dimerized receptor in response to ligand binding, and how these lead to a separation of JAK-binding Box1 motifs. Such a separation leads to removal of the pseudokinase inhibitory domain from the kinase domain of a partner JAK2 bound to the receptor, and vice versa, leading to apposition of the kinase domains and transactivation. This may be a general mechanism for class I cytokine receptor action.

BRN2, a POUerful driver of melanoma phenotype switching and metastasis

The POU domain family of transcription factors play a central role in embryogenesis and are highly expressed in neural crest cells and the developing brain. BRN2 is a class III POU domain protein that is a key mediator of neuroendocrine and melanocytic development and differentiation. While BRN2 is a central regulator in numerous developmental programs, it has also emerged as a major player in the biology of tumourigenesis. In melanoma, BRN2 has been implicated as one of the master regulators of the acquisition of invasive behaviour within the phenotype switching model of progression. As a mediator of melanoma cell phenotype switching, it coordinates the transition to a dedifferentiated, slow cycling and highly motile cell type. Its inverse expression relationship with MITF is believed to mediate tumour progression and metastasis within this model. Recent evidence has now outlined a potential epigenetic switching mechanism in melanoma cells driven by BRN2 expression that induces melanoma cell invasion. We summarize the role of BRN2 in tumour cell dissemination and metastasis in melanoma, while also examining it as a potential metastatic regulator in other tumour models.

A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation

Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial–mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer.

CRIM1 is necessary for coronary vascular endothelial cell development and homeostasis

Endothelial cells form a critical component of the coronary vasculature, yet the factors regulating their development remain poorly defined. Here we reveal a novel role for the transmembrane protein CRIM1 in mediating cardiac endothelial cell development. In the absence of Crim1 in vivo, the coronary vasculature is malformed, the number of endothelial cells reduced, and the canonical BMP pathway dysregulated. Moreover, we reveal that CRIM1 can bind IGFs, and regulate IGF signalling within endothelial cells. Finally, loss of CRIM1 from human cardiac endothelial cells results in misregulation of endothelial genes, predicted by pathway analysis to be involved in an increased inflammatory response and cytolysis, reminiscent of endothelial cell dysfunction in cardiovascular disease pathogenesis. Collectively, these findings implicate CRIM1 in endothelial cell development and homeostasis in the coronary vasculature.

Growth hormone activated STAT5 is required for induction of beige fat in vivo

OBJECTIVE The anti-obesity actions of growth hormone (GH) led us to investigate if GH signaling is able to regulate beige/brite fat development of white adipose tissue (WAT). METHODS We studied WAT in GHR-391 mice engineered to be unable to activate STAT5 in response to GH, in mice with adipose specific deletion of GHR, in GHR-/- mice and in bGH transgenic mice. QPCR, immunoblots and immunohistochemistry were used to characterize WAT. The in vivo effects of β-3 adrenergic activation with CL-316,243 and that of FGF21 infusion were also studied. RESULTS GHR-391 mice had lower surface temperature than WT, with deficiency in β-oxidation and beiging transcripts including Ucp1. Oxidative phosphorylation complex subunit proteins were decreased dramatically in GHR-391 inguinal white adipose tissue (iWAT), but increased in bGH iWAT, as were proteins for beige/brown markers. In accord with its lack of β-3 adrenergic receptors, iWAT of GHR-391 mice did not beige in response to administration of the β-3 specific agonist CL-316,243 in contrast to WT mice. GHR-391 mice are deficient in FGF21, but unlike WT, infusion of the purified protein was without effect on extent of beiging. Finally, fat-specific deletion of the GHR replicated the loss of beiging associated transcripts. CONCLUSION In addition to promoting lipolysis, our study suggests that GH is able to promote formation of beige adipose tissue through activation of STAT5 and induction of Adrb3. This sensitizes WAT to adrenergic input, and may contribute to the anti-obesity actions of GH.

NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR

Exposure of melanocytes to ultraviolet radiation (UVR) induces the formation of UV lesions that can produce deleterious effects in genomic DNA. Encounters of replication forks with unrepaired UV lesions can lead to several complex phenomena, such as the formation of DNA double-strand breaks (DSBs). The NR4A family of nuclear receptors are transcription factors that have been associated with mediating DNA repair functions downstream of the MC1R signaling pathway in melanocytes. In particular, emerging evidence shows that upon DNA damage, the NR4A2 receptor can translocate to sites of UV lesion by mechanisms requiring post-translational modifications within the N-terminal domain and at a serine residue in the DNA-binding domain at position 337. Following this, NR4A2 aids in DNA repair by facilitating chromatin relaxation, allowing accessibility for DNA repair machinery. Using A2058 and HT144 melanoma cells engineered to stably express wild-type or mutant forms of the NR4A2 proteins, we reveal that the expression of functional NR4A2 is associated with elevated cytoprotection against UVR. Conversely, knockdown of NR4A2 expression by siRNA results in a significant loss of cell viability after UV insult. By analyzing the kinetics of the ensuing 53BP1 and RAD51 foci following UV irradiation, we also reveal that the expression of mutant NR4A2 isoforms, lacking the ability to translocate, transactivate, or undergo phosphorylation, display compromised repair capacity. Implications: These data expand the understanding of the mechanism by which the NR4A2 nuclear receptor can facilitate DNA DSB repair. Mol Cancer Res; 15(9); 1184–96. ©2017 AACR.