JAK-STAT Signaling, Cytokine Receptors and Polycythemia Vera

Cytokine receptors lack intrinsic catalytic activity and transmit their signals by one or several of the four known Janus tyrosine kinases (JAKs). As they are appended to receptors' cytosolic domains during intracellular traffic, JAKs become transiently activated upon receptor activation by ligand. JAKs phosphorylate downstream proteins, like the receptors themselves, signal transducers and activators of transcription (STAT) proteins and a variety of other signaling proteins.

For a number of years we have been studying the mechanisms by which cytokine receptors such as the erythropoietin receptor (EpoR) interact and activate JAKs (1). In addition to its crucial role in signal transduction, JAK2 plays the role of a chaperone that promotes traffic (2). We showed that JAK2 also promotes traffic of the thrombopoietin receptor (TpoR), but in this case the expression of JAK2 equally protects the receptor from degradation and stimulates its recycling (3). For enhancing traffic and promoting stability JAKs do not need their catalytic domain or the kinase activity.

The work on TpoR traffic triggered our interest in the cause of Polycythemia Vera (PV), which is characterized by excessive production of mature red cells. Closely related to PV are Essential Thrombocythemia (ET) and Idiopathic Myelofibrosis (IMF), characterized by excessive platelet production and myelofibrosis, respectively, with all three progressing to acute leukemia. Dr Jerry Spivak had reported that one hallmark of PV is the reduction of the cell surface and intracellular levels of mature TpoR. In our hands, JAK2 promoted the very process that was found to be defective in PV and IMF (3). We hypothesized that JAK2 or a JAK2-binding protein may be involved in these diseases. In the frame of a collaboration on hematopoiesis with Prof. William Vainchenker at the Institut Gustave Roussy in Paris, our groups planned to attempt expression cloning of such a JAK2 partner involved in PV and IMF, as Dr Vainchenker had demonstrated that Epo-independent erythroid differentiation in PV required JAK2. Sequencing of JAK2 in PV patients by the Vainchenker lab, showed that the majority of patients harbor a unique V617F mutation in the pseudokinase domain of JAK2 (4). Jointly we showed that murine and human JAK2 V617F mutants are constitutively active kinases and that the wild type JAK2 inhibits signaling by the mutant, thus explaining the strong proliferation advantage of homozygous blood progenitors (4). The mutation alters a physiologic inhibition exerted by the pseudokinase domain on the kinase domain and is found in >85% of PV patients and in 50% of ET and IMF (5,6).

Questions we are trying to answer include which effects does JAK2 V617F exert on signaling by Epo, Tpo and G-CSF, how does it affect receptor traffic, which other factors explain why this mutant apparently can induce the three related disease phenotypes, and what is the structural basis for JAK activation by this mutation. Point mutations in other JAKs may be involved in different forms of cancers, as we have just shown that the homologous V to F mutation can also activate other JAKs. Cells transformed by JAK2 V617F or by oncogenic receptor mutants exhibit constitutive activation of STAT5 and STAT3 proteins. Using chromatin immunoprecipitation, we are sequencing promoters bound by constitutively active STAT5 in transformed hematopoietic cells (7), or in blood progenitors that harbor the JAK2 V617F mutant. Here at the branch we are collaborating with Jean-Christophe Renauld on the oncogenic signaling by overexpressed/mutated JAKs and on traffic by cytokine receptors coupled with JAKs. Nicolas van Baren and Pierre van der Bruggen in the group of Prof. Thierry Boon had the idea to test whether the JAK2 V617F mutant may be a tumor antigen. A vaccine against JAK2 V617F may eliminate the malignant hematopoietic stem cells and restore hematopoiesis from non-mutated stem cells.

A large effort aims at determining the structural basis of cytokine receptor dimerization and activation, as no structure is available for the cytokine receptor cytosolic domains. A flexible region and a helix-cap precede the transmembrane (TM) domain (8) of the EpoR, while downstream, the TM is continued by a rigid α-helix (9). We found that the EpoR can be rendered constitutively active by mutating one extracellular or either of the first two predicted transmembrane (TM) residues to cysteine (8). Many other cysteine mutants lead to disulfide bond formation, but only these three activated the receptor, showing that close apposition of TM domains by disulfide bonding in a certain configuration leads to receptor activation.

However, we needed a way to determine which residues are actually in the interface of the active EpoR dimer configuration. To answer this question we established a system where the receptors' extracellular domains are replaced by short dimeric coiled-coils. These can impose their register on the downstream α-helical TM residues, which, in turn, transmit their orientation to the cytosolic domains. All seven possible dimeric orientations were individually engineered for EpoR or for TpoR, by varying the coiled-coil-TM junction. In this way we identified one active conformation of the EpoR dimer (1). Surprisingly, for the TpoR several conformations induce cell proliferation, while different TpoR orientations were required to induce cell-to-cell adhesion, stem cell renewal or megakaryocyte differentiation. Distinct signaling molecules can be apportioned to different dimeric conformations of EpoR and TpoR. This approach may help disentangle the role of the many/redundant signaling molecules recruited to receptor cytosolic domains.

Figures

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Figure 1. Expression of JAK2 in Ba/F3 cells at 5 fold higher than endogenous levels leads to 4-5 fold increase in the cell surface levels of HA-tagged TpoR, as shown by immunofluorescence with anti-HA antibodies on stably transduced and permeabilized cells (B). JAK2 increases the total cellular levels of TpoR protein, especially of the mature (upper arrow) form of the receptor, as shown by Western blotting with anti-HA antibodies (C). JAK2 stabilizes the mature form of the TpoR (upper arrow) and not the immature EndoH-sensitive form of the receptor (lower arrow).

Figure 1

Figure 2. Janus kinases contain an NH2-terminal FERM-like domain, an SH2 domain and then a pseudokinase and a tyrosine kinase domain. There are four known JAKs, JAK1, JAK2, JAK3 and Tyk2. Based on homology between JAKs, JAK-homology domains have been identified (JH1-JH7). The NH2-terminus of JAKs is important for attachment to cytokine receptors' cytosolic domains and for the "chaperone" effect of JAKs on cytokine receptor traffic. The pseudokinase (JH2) domain is specific to the JAK family and has been suggested to inhibit the kinase domain, and to be required for activation by cytokine receptors. The V617F mutation found in myeloproliferative diseases in JAK2 is located in the pseudokinase domain and leads to constitutive activation of JAK2.

Figure 1

Figure 3. (A) Epo binding to the extracellular domain of the EpoR imposes a dimeric conformation that is compatible with activation of JAK2, phosphorylation of the receptor cytosolic tyrosine residues and downstream signaling. (B) To functionally probe the orientation requirements of the EpoR dimer we replaced the extracellular domains by a short (30 amino acid residues) dimeric coiled coil. We have fused the coiled coil to the TM-cytosolic domains of EpoR in all seven possible orientations, as the coiled coil will impose its interface and register to the downstream TM helices. Shown are one inactive dimeric orientation, where no JAK2 activation occurs, and an active fusion protein, which signals via the JAK2-STAT5 pathway. TM residues L241, L244 and cytosolic juxtamembrane residue W258 are in the interface of the active dimeric conformation, as determined by the register of the coiled-coil and by cysteine mutagenesis and cross-linking.

References

  1. Seubert, N., Royer, Y., Staerk, J., Kubatzky, K. F., Moucadel, V., Krishnakumar, S., Smith, S. O., Constantinescu, S. N. Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer. Mol Cell 12, 1239-50 (2003).
  2. Huang, L. J., Constantinescu, S. N., Lodish, H. F. The N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor. Mol Cell 8, 1327-38. (2001).
  3. Royer, Y., Staerk, J., Costuleanu, M., Courtoy, P. J., Constantinescu, S. N. Janus kinases affect thrombopoietin receptor cell surface localization and stability. J Biol Chem 280, 27251-61 (2005).
  4. James, C., Ugo, V., Le Couedic, J. P., Staerk, J., Delhommeau, F., Lacout, C., Garcon, L., Raslova, H., Berger, R., Bennaceur-Griscelli, A., Villeval, J. L., Constantinescu, S. N. et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434, 1144-8 (2005).
  5. Vainchenker, W., Constantinescu, S. N. A unique activating mutation in JAK2 is at the origin of Polycythemia Vera and allows a new classification of myeloproliferative diseases. Hematology (Am Soc Hematol Educ Program) In press, (2005).
  6. Zhao, Z. J., Krantz, S. B., Vainchenker, W., Casadevall, N., Constantinescu, S. N. Role of tyrosine kinases and phosphatases in Polycythemia Vera. Semin Hematol In press, (2005).
  7. Moucadel, V., Constantinescu, S. N. Differential STAT5 signaling by ligand-dependent and constitutively active cytokine receptors. J Biol Chem 280, 13364-73 (2005).
  8. Kubatzky, K. F., Liu, W., Goldgraben, K., Simmerling, C., Smith, S. O., Constantinescu, S. N. Structural requirements of the extracellular to transmembrane domain junction for erythropoietin receptor function. J Biol Chem 280, 14844-54 (2005).
  9. Constantinescu, S. N., Huang, L. J., Nam, H., Lodish, H. F. The erythropoietin receptor cytosolic juxtamembrane domain contains an essential, precisely oriented, hydrophobic motif. Mol Cell 7, 377-85. (2001).

Pictures of the group

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Left to right - postdoc alumni Virginie Moucadel, PhD student Yohan Royer, Visiting Scientist Marcel Costuleanu (Univ. of Medicine Iasi, Romania), Stefan Constantinescu, PhD student Judith Staerk, technician Yan Yin, postdoc alumni Katharina Kubatzky (presently a faculty at University of Freiburg, http://www.pharmakologie.uni-freiburg.de/i/staff.htm),

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Left to right - postdoc Carmen Diaconu (permanent staff at the Stefan S. Nicolau Institute of Virology, Bucharest, Romania), PhD student Alexandra Dusa, administrative assistant Julie Klein and postdoc Michael Girardot.

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