Jak2 FERM domain interaction with the erythropoietin receptor regulates Jak2 kinase activity

Abstract

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement.

FIG. 1.

Structure and biological activity associated with the various tyrosine-to-phenylalanine mutations in the JH1 and JH2 domains of Jak2. (A) Structure of Jak2 tyrosine kinase, the Jak homology domain (JH), and putative tyrosine phosphorylation sites. JH1, kinase domain; JH2, pseudokinase domain; JH3, SH2-like domain; JH4 to -7, FERM domain. Numbers indicate the amino acid positions of the various tyrosine residues located in the JH1 and JH2 domains. (B) Jak2-deficient mouse embryonic fibroblasts were infected with retroviruses encoding EpoR together with retroviruses encoding HA-tagged Jak2 (WT), the indicated mutants of Jak2, or retroviruses not encoding Jak2 (murine stem cell virus [MSCV]). Forty-eight hours after infection, cells were serum starved for 16 h and then stimulated or not with Epo (5 U/ml) for 15 min before solubilization. Cell lysates were subjected to immunoprecipitation using an anti-HA or anti-STAT5 antibody. Immune complexes were analyzed by Western blotting using antibodies directed against phosphotyrosines (4G10 and pY), activating tyrosine phosphorylation of Jak2 (pY1007/Y1008), the HA epitope on Jak2 (HA), and STAT5. These experiments and the similar experiments shown in subsequent figures have been done at least three times. (C and D) Biological activity ascribed to the various Jak2 tyrosine mutants. Jak2-deficient fetal liver cells were infected with retroviruses encoding the indicated Jak2 mutants and then subjected to in vitro colony formation assays in the absence or presence of Epo (5 U/ml) (C) or Epo (5 U/ml) and IL-3 (1.8 ng/ml) (D). The benzidine-positive CFU-e and BFU-e colonies were scored at day 3 and day 8, respectively. In this and subsequent figures, the error bars indicate the standard deviation in colony numbers among the replicates.

FIG. 2.

Biological activity associated with various tyrosine-to-phenylalanine or -glutamic acid mutants of the JH2 domain of Jak2. (A) Biological activity associated with mutants of Jak2 in which the various tyrosines have been mutated for either a phenylalanine or a glutamic acid. Jak2-deficient mouse embryonic fibroblasts were infected with retroviruses encoding EpoR together with retroviruses encoding HA-tagged Jak2 (WT), the indicated mutants of Jak2, or retroviruses not encoding Jak2 (murine stem cell virus [MSCV]). Forty-eight hours after infection, cells were serum deprived for 16 to 24 h and then stimulated or not with Epo (5 U/ml) for 15 min before solubilization. Cell lysates were subjected to immunoprecipitation using an anti-HA or anti-STAT5 antibody. Immunoprecipitated proteins were then analyzed by Western blotting using antibodies directed against phosphorylated tyrosines (4G10 and pY), activating tyrosine phosphorylation of Jak2 (pY1007/Y1008), the HA epitope on Jak2 (HA), and STAT5. Note the constitutive global tyrosine phosphorylation of Jak2, activating tyrosine phosphorylation of Jak2, and the phosphorylation of STAT5 in cells expressing EpoR together with Jak2-Y613E in the absence of Epo stimulation (*). (B and C) Epo-independent formation of CFU-e from Jak2-deficient fetal liver cells expressing Jak2-V613E. Jak2-deficient fetal liver cells were infected with retroviruses encoding the indicated Jak2 mutants and then subjected to in vitro colony formation assays in the absence or presence of Epo (0.2 U/ml) (B) or Epo (3 U/ml) and IL-3 (10 ng/ml) (C). The benzidine-positive CFU-e and BFU-e colonies were scored at day 3 and day 8, respectively.

FIG. 3.

The constitutive activity of Jak2-Y613E requires the expression of the EpoR and its kinase activity but not Epo stimulation. (A) Jak2-deficient MEFs were infected with retroviruses encoding the indicated Jak2 mutants together (+) or not (−) with viruses encoding EpoR-c-Flag. Forty-eight hours after infection, Jak2-deficient fibroblasts were left untreated or incubated with Epo (5 U/ml) for 15 min before solubilization. Cell lysates were subjected to immunoprecipitation using antibodies against HA (3F10) or Stat5. Cell lysates and immune complexes were then analyzed by Western blotting using antibodies directed against phosphotyrosine (4G10 and pY), activating tyrosine phosphorylation on Jak2 or the HA epitope. Lysates were also analyzed by Western blotting using the anti-Flag antibody M2 to detect EpoR expression (Flag). MSCV, empty retrovirus; K882R, Jak2 kinase dead mutant; Y613Y, Jak2-Y613F; Y613F K882R, Jak2 bearing the Y613F and K882R mutations; Y613E, Jak2-Y613E; Y613E K882R, Jak2 bearing the Y613E and K882R mutations. (B and C) Kinase activity and phosphorylation in the activation loop are required for the biological activity of Jak2-V613E mutant. Jak2-deficient fetal liver cells were infected with retroviruses encoding the indicated Jak2 mutants and then subjected to in vitro colony formation assays in the absence or presence of Epo (0.2 U/ml) (B) or Epo (3 U/ml) and IL-3 (10 ng/ml) (C). The benzidine-positive CFU-e (C) and BFU-e (B) colonies were scored at day 3 and day 8, respectively.

FIG. 4.

Properties of Jak2-V617F, Jak2-L611S, and TEL-Jak2 mutants. (A) Schematic representation of Jak2 mutants and fusion protein TEL-Jak2. JH1, kinase domain; JH2, pseudokinase domain; JH3, SH2-like; JH4 to -7, FERM domain; TEL-Jak2, gene product obtained from the translocation of the JAK2 gene with the translocated Ets leukemia gene TEL. The TEL-Jak2 fusion protein contains the N-terminal region of TEL, which comprises the oligomerization domain and the JH1 (kinase) domain of Jak2. (B) EpoR-independent activity of Jak2-V617F, Jak2-L611S, and TEL Jak2. Jak2-deficient MEFs were infected with retroviruses encoding the indicated Jak2 cDNAs. Forty-eight hours after infection, Jak2-deficient fibroblasts were left untreated or incubated with Epo (5 U/ml) for 15 min before solubilization. Cell lysates were subjected to immunoprecipitation using antibodies against HA (3F10). Cell lysates and immune complexes were then analyzed by Western blotting using antibodies directed against phosphotyrosine (4G10 and pY), activating tyrosine phosphorylation on Jak2, and the HA epitope, activating phosphorylation of Stat5 and Stat5. MSCV, murine stem cell virus. (C and D) Biological activities of the various Jak2 mutants and TEL-Jak2. Jak2-deficient fetal liver cells were infected with retroviruses encoding the indicated Jak2 mutants and then subjected to in vitro colony formation assays in the absence or presence of Epo (0.2 U/ml) or Epo (3 U/ml) and IL-3 (10 ng/ml) (C) or Epo alone (D). The benzidine-positive CFU-e (C) and BFU-e (D) colonies were scored at day 3 and day 8, respectively.

FIG. 5.

The EpoR is required for Jak2-Y613E biological activity but not for Jak2-V617F, Jak2-L611S, and TEL-Jak2 in vitro. Exponentially growing Ba/F3 cell lines were infected with retroviruses encoding the indicated constructs, and green fluorescent protein-positive cells were sorted and selected with puromycin as described in Materials and Methods. Ba/F3 cells were then washed twice with PBS and left untreated or stimulated with IL-3 (2.5 ng/ml) and Epo (5 U/ml), and living cells were counted using a Beckman Coulter VI-Cell analyzer at the indicated times (C). For panel A, the levels of Jak2 expression were determined by Western blotting with an antiserum against Jak2 (C-20, sc-294; Santa Cruz) that detects a nonspecific band that migrates above the Jak2 band. For panel B, the cell lysates were immunoblotted using antibodies directed against Jak2 pY1007/Y1008 to assess the levels of activated Jak2. MSCV, murine stem cell virus.

FIG. 6.

EpoR is required for Jak2-Y613E biological activity but not for Jak2-V617F, Jak2-L611S, and TEL-Jak2 activities ex vivo. Wild-type (upper panel) or EpoR-deficient (lower panel) fetal liver cells were infected with retroviruses encoding the indicated Jak2 mutants and then subjected to in vitro colony formation assays in the presence of Epo (5 U/ml). The benzidine-positive CFU-e colonies were scored at day 3. MSCV, murine stem cell virus.

FIG. 7.

Proposed model for activation of Jak2 by EpoR derived from the properties of Jak2 mutants. In the unbound and inactive state, the FERM domain and the JH1 and JH2 domains of Jak2 are tightly folded together and prevent the Jak2 catalytic domain from being active (A). The first step toward activation of the kinase is the displacement of the FERM domain by its interaction with the receptor (B). The next step in activation occurs with ligand-induced receptor aggregation. In particular, as a consequence of aggregation, the probability of tyrosine phosphorylation within the active loop is greatly increased, which may be associated with changes in JH2-JH1 domain interactions. Once phosphorylation occurs within the activation loop, the kinase is fully activated (C). The active conformation of Jak2 is likely to be mimicked by the Jak2-V617F mutant, whereas the Y613E mutant failed to undergo complete conformational changes leading to its activation.