Calreticulin mutants as oncogenic rogue chaperones for TpoR and traffic-defective pathogenic TpoR mutants.

Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell surface in states that would not pass quality control; this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi apparatus. A number of engineered or disease-associated TpoRs such as TpoR/MPL R102P, which causes congenital thrombocytopenia, are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. In addition to requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, whereas full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects.

Differential effect of inhibitory strategies of the V617 mutant of JAK2 on cytokine receptor signaling.

BACKGROUND: Janus kinase (JAK) 2 plays pivotal roles in signaling by several cytokine receptors. The mutant JAK2 V617F is the most common molecular event associated with myeloproliferative neoplasms. Selective targeting of the mutant would be ideal for treating these pathologies by sparing essential JAK2 functions. OBJECTIVE: We characterize inhibitory strategies for JAK2 V617F and assess their effect on physiologic signaling by distinct cytokine receptors. METHODS: Through structure-guided mutagenesis, we assessed the role of key residues around F617 and used a combination of cellular and biochemical assays to measure the activity of JAKs in reconstituted cells. We also assessed the effect of several specific JAK2 V617F inhibitory mutations on receptor dimerization using the NanoBiT protein complementation approach. RESULTS: We identified a novel Janus kinase homology 2 (JH2) αC mutation, A598F, which is suggested to inhibit the aromatic stacking between F617 with F594 and F595. Like other JAK2 V617F inhibitory mutations, A598F decreased oncogenic activation and spared cytokine activation while preventing JAK2 V617F-promoted erythropoietin receptor dimerization. Surprisingly, A598F and other V617F-inhibiting mutations (F595A, E596R, and F537A) significantly impaired IFN-γ signaling. This was specific for IFN-γ because the inhibitory mutations preserved responses to ligands of a series of receptor complexes. Similarly, homologous mutations in JAK1 prevented signaling by IFN-γ. CONCLUSIONS: The JH2 αC region, which is required for JAK2 V617F hyperactivation, is crucial for relaying cytokine-induced signaling of the IFN-γ receptor. We discuss how strategies aiming to inhibit JAK2 V617F could be used for identifying inhibitors of IFN-γ signaling.

Uncoupling JAK2 V617F activation from cytokine-induced signalling by modulation of JH2 αC helix.

The mechanisms by which JAK2 is activated by the prevalent pseudokinase (JH2) V617F mutation in blood cancers remain elusive. Via structure-guided mutagenesis and transcriptional and functional assays, we identify a community of residues from the JH2 helix αC, SH2-JH2 linker and JH1 kinase domain that mediate V617F-induced activation. This circuit is broken by altering the charge of residues along the solvent-exposed face of the JH2 αC, which is predicted to interact with the SH2-JH2 linker and JH1. Mutations that remove negative charges or add positive charges, such as E596A/R, do not alter the JH2 V617F fold, as shown by the crystal structure of JH2 V617F E596A. Instead, they prevent kinase domain activation via modulation of the C-terminal residues of the SH2-JH2 linker. These results suggest strategies for selective V617F JAK2 inhibition, with preservation of wild-type function.

Germ-line JAK2 mutations in the kinase domain are responsible for hereditary thrombocytosis and are resistant to JAK2 and HSP90 inhibitors.

The main molecular basis of essential thrombocythemia and hereditary thrombocytosis is acquired, and germ-line-activating mutations affect the thrombopoietin signaling axis. We have identified 2 families with hereditary thrombocytosis presenting novel heterozygous germ-line mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, whereas the other presents 2 JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of Janus kinase 2 (JAK2) R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-thrombopoietin receptor (MPL) but not of Ba/F3-human receptor of erythropoietin cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed thrombopoietin-independent phosphorylation of signal transducer and activator of transcription 1. The JAK2 R867Q and S755R/R938Q proteins had significantly longer half-lives compared with JAK2 V617F. The longer half-lives correlated with increased binding to the heat shock protein 90 (HSP90) chaperone and with higher MPL cell-surface expression. Moreover, these mutants were less sensitive to JAK2 and HSP90 inhibitors than JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL while inducing a decreased sensitivity to clinically available JAK2 inhibitors.

Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake.

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.

JAK2 V617F constitutive activation requires JH2 residue F595: a pseudokinase domain target for specific inhibitors.

The JAK2 V617F mutation present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients renders the kinase constitutively active. In the absence of a three-dimensional structure for the full-length protein, the mechanism of activation of JAK2 V617F has remained elusive. In this study, we used functional mutagenesis to investigate the involvement of the JH2 alphaC helix in the constitutive activation of JAK2 V617F. We show that residue F595, located in the middle of the alphaC helix of JH2, is indispensable for the constitutive activity of JAK2 V617F. Mutation of F595 to Ala, Lys, Val or Ile significantly decreases the constitutive activity of JAK2 V617F, but F595W and F595Y are able to restore it, implying an aromaticity requirement at position 595. Substitution of F595 to Ala was also able to decrease the constitutive activity of two other JAK2 mutants, T875N and R683G, as well as JAK2 K539L, albeit to a lower extent. In contrast, the F595 mutants are activated by erythropoietin-bound EpoR. We also explored the relationship between the dimeric conformation of EpoR and several JAK2 mutants. Since residue F595 is crucial to the constitutive activation of JAK2 V617F but not to initiation of JAK2 activation by cytokines, we suggest that small molecules that target the region around this residue might specifically block oncogenic JAK2 and spare JAK2 wild-type.