Roles of Tyrosine Residues 845, 892 and 922 in Constitutive Activation of Murine FLT3 Kinase Domain Mutant

Roles of tyrosine residues 845, 892 and 922 in constitutive activation of murine FLT3 kinase domain mutant

Jun Ishiko1, Masao Mizuki*,1, Itaru Matsumura1, Hirohiko Shibayama1, Hiroyuki Sugahara1, Glen Scholz2, Hubert Serve3 and Yuzuru Kanakura1

1Department of Hematology and Oncology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan; 2Department of Medicine, University of Melbourne, The Royal Melbourne Hospital, Parkville 3050, Australia; 3Department of Medicine, Hematology and Oncology, University of Mu¨nster, Albert-Schweitzer-Strasse 33, Mu¨nster 48129, Germany

FLT3 tyrosine kinase domain (TKD)mutations are cies (Reilly, 2002). Among them, two types of consti- detected in B7% of acute myeloid leukemia patients, tutively active FLT3 mutations are frequently identified and suggested to correlate with poor prognosis and confer in leukemic blasts from the patients with acute myeloid resistance to FLT3 inhibitors. To explore activation leukemia (AML): one is internal tandem duplication mechanism of FLT3 TKD mutation, we analysed critical (ITD) in the juxtamembrane domain and the other is tyrosine residues for the constitutive activation and missense mutations in the activation loop of the second downstream signaling of the mutant by generating a tyrosine kinase domain (TKD) (Nakao et al., 1996; series of single Tyr-Phe substitution mutant of all 22 Yokota et al., 1997; Abu-Duhier et al., 2001; Yamamoto cytoplasmic tyrosine residues of murine FLT3 et al., 2001). The ITD mutation is found in about TKD-mutant (mFLT3Asp838Val). Tyr845Phe, Tyr892Phe B20% of AML patients, and associated with poor and Tyr922Phe substitutions suppressed the phosphoryla- prognosis (Kiyoi et al., 1999; Abu-Duhier et al., 2000; tion of mFLT3Asp838Val itself, the activation of Erk1/2, Schnittger et al., 2002; Thiede et al., 2002). The TKD STAT3 and STAT5, and the factor-independent cell mutation is detected in B7% of AML patients. proliferation and survival. In contrast, these three Although the clinical relevance of TKD mutation has Tyr-Phe mutations partially suppressed but maintained not been confirmed, some reports suggested the possible the ligand-dependent activation and anti-apoptotic activity association with poor prognosis in younger patients with of wild-type FLT3, suggesting that these tyrosine residues intermediate risk (Yamamoto et al., 2001; Thiede et al., were more critical for the constitutive activation and 2002; Moreno et al., 2003). TKD mutations are mainly signaling of mFLT3Asp838Val. These three Tyr-Phe muta- caused by the missense mutations at Asp835 (Asp at codon tions also inhibited the constitutive activation of other 835) of human FLT3 (Abu-Duhier et al., 2001; Yamamoto FLT3 mutants bearing internal tandem duplication, et al., 2001). The Asp residue lies near the highly conserved Asp838Tyr or Ile839del. The suppression of mFLT3Asp838- Asp-Gly-Phesequenceoftyrosinekinasefamily,and Val activation and signaling by these substitutions was occupies the equivalent position in other members of partially recovered by shifting the culture temperature RTKs such as c-Kit and PDGFRa. TKD mutation of from 37 to 331C, or by the introduction of Cdc37 and c-Kit has been reported to cluster at the corresponding Hsp90. Taken together, Tyr845, Tyr892 and Tyr922 are the Asp816 residue, and are associated with mastocytosis or critical residues in mFLT3Asp838Val activation, possibly AML (Furitsu et al., 1993; Longley et al., 1996; Worobec through stabilizing the active conformation of et al., 1998; Care et al., 2003). Furthermore, constitutively mFLT3Asp838Val. activating mutation at the Asp residue of kinase domain of Oncogene (2005) 24, 8144–8153. doi:10.1038/sj.onc.1208957; PDGFRa has recently been identified in several cases of publshed online 1 August 2005 gastrointestinal stromal tumor (Heinrich et al., 2003; Hirota et al., 2003). Keywords: FLT3; activation loop; receptor tyrosine The clinical significance of these TKD mutations is kinase; leukemia; Hsp90; Cdc37 characterized by the resistance to the tyrosine kinase inhibitors. TKD mutations of c-Kit and PDGFRa showed resistance to imatinib, which can inhibit the Introduction wild-type (WT) and the juxtamembrane mutants of these kinases (Ma et al., 2002; Ueda et al., 2002; Constitutive active mutations of type III receptor Heinrich et al., 2003; Hirota et al., 2003). FLT3 TKD tyrosine kinases (RTK) such as FLT3, c-Kit and mutation also confers the resistance to some kind of PDGFR are major causes of hematological malignan- tyrosine kinase inhibitors (Grundler et al., 2003). Thus, TKD mutations confer resistance to tyrosine kinase *Correspondence: M Mizuki; E-mail: [email protected] inhibitors in different kinds of tyrosine kinases. Received 7 January 2005; revised 17 June 2005; accepted 23 June 2005; We have previously found that Phe substitution of publshed online 1 August 2005 Tyr719 (Tyr719Phe) in c-Kit TKD-mutant, c-KitAsp814Val, Critical tyrosine residues in FLT3 mutation J Ishiko et al 8145 not only abolished the binding of PI3 kinase p85 subunit cell lysates were immunoprecipitated with anti-mFLT3 but also completely suppressed the receptor phosphory- antibody and subjected to Western blotting with lation and ligand-independent proliferation (Hashimoto anti-phosphotyrosine antibody and anti-FLT3 antibody et al., 2003). These results suggested that specific (Figure 2). All Tyr-Phe mutants of mFLT3Asp838Val as tyrosine residue may regulate the constitutive activation well as mFLT3Asp838Val were composed of 140-kDa in the c-Kit TKD-mutant. Since not only the affected (immature) and 160-kDa (mature) form, and expressed residue of FLT3 TKD-mutant and c-Kit TKD-mutant at comparable level. mFLT3Asp838Val showed distinct is conserved but also cytoplasmic tyrosine residues are receptor phosphorylation without ligand stimulation. almost conserved in both receptors, we examined The substitution of phenylalanine for tyrosine at whether specific tyrosine residues exist in FLT3 codon 796, 845, 892 or 922 (Tyr796Phe, Tyr845Phe, TKD-mutant, which regulate its constitutive activation. Tyr892Phe, Tyr922Phe, respectively) considerably impaired the phosphorylations of mFLT3Asp838Val, both of mature and immature form, whereas other Tyr-Phe mutations did not. Results For further study, we introduced Tyr796Phe, Tyr845Phe, Tyr892Phe and Tyr922Phe mutants of Effects of substitution of phenylalanine for tyrosine on the mFLT3Asp838Val stably into Ba/F3 cells. All transfected constitutive activation of mFLT3Asp838Val cells sufficiently expressed mutated mFLT3 on cell Murine FLT3 (mFLT3) has 22 cytoplasmic tyrosine surface (Figure 3a). We obtained cell lysates of these residues, which correspond to respective tyrosine resi- transfected cells and assessed the phosphorylation of dues of human FLT3 (Figure 1). At first, we intended to these receptors by anti-phosphotyrosine antibody estimate as to which cytoplasmic tyrosine residues are (Figure 3b). Tyr845Phe, Tyr892Phe and Tyr922Phe Asp838Val important for the activation of mFLT3Asp838Val. Expres- impaired the phosphorylation of mFLT3 in sion vectors pcDNA3 carrying 22 types of Tyr-Phe Ba/F3 cells, but Tyr796Phe did not. The extra phos- mutations of mFLT3Asp838Val were transiently transfected photyrosine bands in Tyr892Phe lanes were not mFLT3 to Plat-E cells. Without FLT3 ligand stimulation, the bands judging from the molecular weight. We also analysed the activation of FLT3 by immunoblotting with anti-phospho-FLT3 monoclonal antibody (Figure 3c). This monoclonal antibody detects FLT3 only when phosphorylated at Tyr592 of mFLT3. Consistent with the results by anti-phophotyrosine antibody, Tyr845Phe, Tyr892Phe and Tyr922Phe impaired the autophosphorylation of Tyr592 of mFLT3. Tyr892Phe and Tyr922Phe appeared to inhibit the phosphorylation more extensively than Tyr845Phe, as was indicated by the quantified phosphorylation rates. mFLT3Asp838Val

Figure 2 Effects of Tyr-Phe mutation on the constitutive activation of mFLT3Asp838Val in Plat-E cells. Various Tyr-Phe mutants of mFLT3Asp838Val were immunoprecipitated with anti- mFLT3 antibody from the lysates of transiently transfected Plat-E cells, and subjected to immunoblotting with anti-phosphotyrosine Figure 1 Structure of murine FLT3 (mFLT3) and list of antibody (upper lanes) and anti-mFLT3 (lower lanes). Mobility of cytoplasmic tyrosine residues. Substitution of valine (Val) for the mature (160 kDa) and immature (140 kDa) forms of mFLT3 is aspartic acid (Asp) at codon 838 (Asp838Val) results in constitutive indicated by the arrows. All samples were cultured without FLT3 activation of mFLT3. mFLT3 has 22 cytoplasmic tyrosine residues. ligand. The results are shown as the representative of three Each tyrosine residue of mFLT3Asp838Val at codon from 567 to 972 independent experiments. ‘Asp838Val’ represents mFLT3Asp838Val was changed to phenylalanine. Tyrosine residues of human FLT3, without Tyr-Phe mutation. ‘Tyr567Phe’ represents Tyr567-Phe which correspond to those of mFLT3, are listed on the right panel mutant of mFLT3Asp838Val, and so on

Oncogene Critical tyrosine residues in FLT3 mutation J Ishiko et al 8146

Figure 3 Effects of Tyr796Phe, Tyr892Phe, Tyr845Phe and Tyr922Phe on the constitutive activation of mFLT3Asp838Val in Ba/F3 cells. (a) Surface expression of mFLT3 mutants on Ba/F3 cell lines. Comparable expressions of mutant mFLT3 on cell Figure 4 Effects of Tyr-Phe mutation on the downstream signal surface were confirmed by flow cytometry. (b) mFLT3 was transductions in Ba/F3 cells. (a) Effects on the activation of Erk1/2, immunoprecipitated with anti-mFLT3 antibody from lysates of STAT3 and STAT5. The lysates of Ba/F3 cells expressing Ba/F3 cells stably expressing Tyr-Phe mutants of mFLT3Asp838Val Tyr-Phe mutants of mFLT3Asp838Val were subjected to immuno- with or without FLT3 ligand (FL), and immunoblotted with blotting with anti-phospho-Erk1/2 antibody and anti-Erk1/2 anti- anti-phosphotyrosine antibody (upper lanes) and with anti-mFLT3 body (upper panel), anti-phospho-Stat3 and anti-Stat3 (middle (lower lanes). (c) The immunoprecipitates that was similarly panel), anti-phospho-Stat5 and anti-Stat5 (bottom panel). These prepared as (b) were also subjected to immunoblotting with anti- results are representative of three independent experiments. (b) The phospho-FLT3 (Tyr591) monoclonal antibody (upper lanes) and expressions of Pim-2 were measured by real-time RT–PCR. The with anti-mFLT3 (lower lanes). This monoclonal antibody against relative expression was calculated as the ratio of the expression phospho-FLT3 detects FLT3 only when phosphorylated at compared with that of mock-transfected Ba/F3 cells without IL-3. tyrosine at codon 592 of mFLT3. The relative phosphorylation The results are shown as mean7s.d. of two independent rate of each FLT3 mutant was assessed by chemical image analyzer experiments and expressed as the ratio of the phosphorylation rate of each mutant compared with that of mFLT3Asp838Val. These results are representative of three independent experiments antibody. The expressions of these downstream signal transduction molecules were confirmed by the immunoblotting with anti-Erk1/2 antibody, anti-STAT3 anti- showed constitutive activation and ligand stimulation body and anti-STAT5 antibody (Figure 4a). did not augment the activation, which suggested the FLT3Asp838Val and Tyr796Phe mutant of mFLT3Asp838Val different activation mechanism from WT receptor. The showed constitutive activation of Erk1/2, STAT3 and lack of ligand-dependent increase of activation in other STAT5, the levels of which were slightly weaker or mutant receptors may reflect the unique activation comparable with that of the activation by interleukin-3 mechanism of FLT3Asp838Val. These results indicated that (IL-3). Tyr845Phe, Tyr892Phe and Tyr922Phe signifi- these Tyr-Phe substitutions impaired the kinase cantly impaired the activation of Erk1/2, STAT3 and activity of mFLT3Asp838Val itself. STAT5, the levels of which were much weaker than that of the activation by IL-3. The expression of Pim-2, which is the target gene of STAT3/5, was also Activation of downstream signal transduction molecules examined by real-time RT–PCR (Figure 4b). Ba/F3 Next, we examined the downstream signal transductions cells expressing mFLT3Asp838Val or Tyr796Phe mutant of of Tyr-Phe mutants of mFLT3Asp838Val. Total cell mFLT3Asp838Val showed about 12 to 17 times expression lysates of these transfected Ba/F3 cells were immuno- of Pim-2 with or without FLT3 ligand compared with blotted with anti-phospho-Erk1/2 antibody, anti-phos- that of mock-transfected Ba/F3 cells, suggesting pho-STAT3 antibody and anti-phospho-STAT5 that mFLT3Asp838Val or Tyr796Phe mutant induced

Oncogene Critical tyrosine residues in FLT3 mutation J Ishiko et al 8147 comparable activation of STAT3 and STAT5. In Ba/F3 cells proliferated in response to IL-3 at the Tyr845Phe, Tyr892Phe and Tyr922Phe mutants of similar level, that is to say, these clones had no intrinsic mFLT3Asp838Val, the expression of Pim-2 was signiﬁcantly differences in proliferative activity. Different from reduced compared to that of mFLT3Asp838Val or Tyr892Phe and Tyr922Phe mutants, Tyr845Phe mutant Tyr796Phe mutant of mFLT3Asp838Val. The suppressed retained the proliferative activity possibly due to the expression of Pim-2 in these mutants was not induced by residual kinase activity, which was shown in Figure 3. FLT3 ligand stimulation either. These results were We also examined apoptosis of these transfected Ba/F3 consistent with the impaired activation of STAT3 and cells after IL-3 depletion. In DNA histograms, the sub- STAT5 in Tyr845Phe, Tyr892Phe and Tyr922Phe G1 fractions corresponded to apoptotic cells mutants of FLT3Asp838Val. (Figure 5b). Only a small population of Ba/F3 cells expressing mFLT3Asp838Val showed apoptosis in the absence of IL-3. In contrast, IL-3 deprivation led to a Cell proliferation and cell survival striking increase in apoptosis in the Ba/F3 cells Asp838Val To examine the effects of Tyr-Phe substitutions on expressing Tyr892Phe and Tyr922Phe of mFLT3 , proliferative activity, the cell proliferation of transfected indicating that Tyr892Phe and Tyr922Phe impaired the Asp838Val Ba/F3 cells was measured by 3-(4, 5-Dimethyl-2- survival function of mFLT3 . thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay with various concentrations of FLT3 Effects of substitution of phenylalanine for tyrosine on ligand or IL-3 (Figure 5a). The Ba/F3 cells expressing WT and other constitutive active mutants of FLT3 Asp838Val Asp838Val mFLT3 and Tyr796Phe of mFLT3 caused - ligand-independent proliferation. The proliferation of To examine the effects of Tyr Phe mutations on Asp838Val ligand-induced phosphorylation of wild-type mFLT3 the Ba/F3 cells expressing Tyr845Phe of mFLT3 WT wasreducedby60%comparedtothatofmFLT3Asp838Val, (mFLT3 ), we constructed Tyr845Phe, Tyr892Phe and and the proliferations of the Ba/F3 cells expressing Tyr892Phe and Tyr922Phe of mFLT3Asp838Val were completely abolished, whereas all of these transfected

Figure 6 Effects of Tyr-Phe mutation on mFLT3WT and other types of constitutive active mFLT3 mutants. (a) Various Tyr-Phe mutants of mFLT3WT were transiently transfected into Plat-E cell with or without FLT3 ligand, immunoprecipitated with anti- mFLT3 antibody, and immunoblotted with anti-phosphotyrosine antibody (upper lane) and anti-mFLT3 antibody (lower lane). (b) Cell viability of Ba/F3 cell expressing Tyr-Phe mutants of mFLT3WT. Ba/F3 cells were cultured without IL-3 at 371C with or without FLT3 ligand (100 ng/ml). Cells were collected at indicated times and cell viability was determined by trypan blue dye Figure 5 Cell proliferation and survival of the transfected Ba/F3 exclusion test. The results are shown as mean7s.d. of three cells. (a) Cell proliferation of Ba/F3 cells stably expressing independent experiments. (c) Tyr-Phe mutants of various types of Tyr-Phe mutants of mFLT3Asp838Val. Cells were cultured with constitutive active mFLT3, such as ITD, Asp838Tyr or Ile839Del, various concentrations of FLT3 ligand (FL) or IL-3 for 48 h, and were transiently transfected into Plat-E cell as (a). Cell lysates were subjected to MTT assay. (b) Cell survival of transfected Ba/F3 immunoprecipitated with anti-mFLT3 antibody and subjected to cells. Cells were cultured without IL-3 for each indicated time, immunoblotting with anti-phosphotyrosine antibody (upper panel) stained with propidium iodide and subjected to FACS analysis. and anti-mFLT3 antibody (lower panel). These results are These results are representative of three independent experiments representative of three independent experiments

Oncogene Critical tyrosine residues in FLT3 mutation J Ishiko et al 8148 Tyr922Phe mutants of mFLT3WT. These mFLT3WT Tyr892Phe and Tyr922Phe mutations (Figure 6c). These mutants were transiently transfected to Plat-E cells, ﬁndings suggested that Tyr845, Tyr892 and Tyr922 were and cell lysates were obtained. mFLT3WT with these commonly important tyrosine residues for the activation Tyr-Phe mutants showed ligand-dependent phosphor- of various oncogenic FLT3 mutations. ylation, the level of which was weaker than that of mFLT3WT (Figure 6a). We also established Ba/F3 cell lines stably expressing Tyr-Phe mutant of mFLT3WT, Effects of temperature shifting on the function and - Asp838Val and examined cell viability of these transfected cells with half-life of Tyr Phe mutants of mFLT3 or without FLT3 ligand stimulation by trypan blue dye It has been reported that missense mutations in the exclusion test (Figure 6b). Although all of these kinase domain convert the tyrosine kinase into tem- transfected Ba/F3 cells could not survive without perature-sensitive mutant (Mayer et al., 1986; Carlesso FLT3 ligand, the viabilities of these cells recovered with et al., 1994). A temperature-sensitive mutant of v-Src ligand stimulation. This suggested that all Tyr-Phe has been created by two mutations; one is substitution WT mutants of mFLT3 showed anti-apoptotic activity on of Val461 for Met and the other is substitution of Pro503 ligand stimulation. These results indicated that these for Ser (Mayer et al., 1986). A temperature-sensitive Tyr-Phe substitutions partially suppressed but main- mutant of Bcr-Abl has been caused by two mutations, WT tained the activation and the function of mFLT3 which are substitutions, Arg457 to His and Tyr469 to His upon ligand binding. of c-Abl (Carlesso et al., 1994). Since Val461 of v-Src and - We next investigated the effects of Tyr Phe mutants Tyr892 of mFLT3 are located close to each other in the on the phosphorylation of other constitutive active conserved sequence of tyrosine kinase family, and Tyr469 mutants of FLT3, such as ITD, Asp838Tyr and of c-Abl corresponds to Tyr922 of mFLT3, it was ITD Asp838Tyr Ile839Del. All of mFLT3 , mFLT3 and speculated that Tyr-Phe mutants of mFLT3Asp838Val Ile839Del mFLT3 were constitutively phosphorylated, and may have a temperature-sensitive character as well. their phosphorylation was impaired with Tyr845Phe, Transfected Ba/F3 cells were cultured at 33 or 371C after the depletion of serum and IL-3 for 12 h. Western blotting analysis revealed that both phosphorylation and expression of Tyr-Phe mutants of mFLT3Asp838Val increased signiﬁcantly at 331C compared to at 371C (Figure 7a). The impaired phosphorylations of downstream

Figure 7 Effects of temperature shifting on the function and half- life of Tyr-Phe mutants of mFLT3Asp838Val.(a) Effects of temperature shifting on the expression and kinase activity of Tyr-Phe mutants. Ba/F3 cells stably expressing Tyr-Phe mutants of mFLT3Asp838Val were cultured at 33 or 371C, and then were lysed after 12 h starvation. Immunoprecipitates with anti- mFLT3 antibody were subjected to immunoblotting with anti- phosphotyrosine (top panel) and anti-mFLT3 antibodies (second panel). Total lysates were also subjected to immunoblotting with anti-phospho-Erk1/2 antibody (third panel), anti-Erk1/2 antibody (fourth panel) and anti-actin antibody (bottom panel). (b) Cell viability of Ba/F3 cells, which are stably expressing Tyr-Phe mutants of mFLT3Asp838Val. Ba/F3 cells were cultured without IL-3 at 33 or 371C. Cells were collected at indicated times and cell viability was determined by trypan blue dye exclusion test. The results are shown as mean7s.d. of three independent experiments. (c) Protein half-life of Tyr-Phe mutants. The transfected Ba/F3 cells were cultured at 371C with the protein synthesis inhibitor, CHX and lysed at the indicated times. The immunoprecipitates with anti-mFLT3 antibody from these lysates were subjected to immunoblotting with anti-mFLT3 (upper panel). Total lysates were also subjected to immunoblotting with anti-actin antibody (lower panel). We quantified the levels of mFLT3 expression at each time points after CHX treatment by the chemical image analyzer, calculated the ratio of mFLT3 amount at indicated times compared to that without CHX in each mutant and shown as relative amount. The quantified level was shown as the mean7s.d. of three independent experiments. (d) The transfected Ba/F3 cells were permeabilized, both membrane and intracellular mFLT3 were stained with anti-mFLT3 antibody at 0 h (dotted line) or 8 h (solid line) after CHX treatment, and then analysed by flow cytometry to examine the expression of mFLT3. The gray line indicates negative control, staining with normal rabbit IgG. The ratio of the mean fluorescence intensity (MFI) at 8 h to MFI at 0 h in each mutant was shown as the reduction ratio of MFI. These results are representative of three independent experiments

Oncogene Critical tyrosine residues in FLT3 mutation J Ishiko et al 8149 signal transduction molecule, Erk1/2, also recovered at 331C compared to at 371C in these transfected Ba/F3 cells, as shown by the intensity of p-Erk in Figure 7a. We also examined the cell viability of these transfected Ba/F3 cells at 33 and 371C by trypan blue dye exclusion test (Figure 7b). While the viability of Ba/F3 cells expressing mFLT3Asp838Val was the same between at 33 and 371C, the decreased viability of Ba/F3 cells transfected with Tyr845Phe, Tyr892Phe and Tyr922Phe mutants of mFLT3Asp838Val were improved to some extent by shifting the culture temperature from 37 to 331C. These results indicated that the Tyr-Phe mutants had the temperature-sensitive character, and suggested the possibility that the degradation of mFLT3Asp838Val may be enhanced in Tyr-Phe mutants. To confirm this possibility, we performed the protein degradation assay of Tyr-Phe mutants. The transfected Ba/F3 cells were cultured at 371C with the inhibitor of protein synthesis, cycloheximide (CHX), and were lysed at indicated hours after treatment with CHX. The immunoprecipitates with anti-mFLT3 antibody from these lysates were subjected to immunoblotting with anti-mFLT3 antibody (Figure 7c). We quantified both mature and immature bands of mFLT3 mutants by chemical image analyzer, and calculated the ratios of mFLT3 expression at the indicated times compared to mFLT3 expression without CHX in each mutants (mFLT3 expression at the indicated time/mFLT3 expression at time 0). The expressions of Tyr-Phe mutants of mFLT3Asp838Val declined more rapidly than the expression of mFLT3Asp838Val. We also examined the intracellular and membrane expression of mFLT3 mutants by FACS analysis after permeabilization (Figure 7d). This analysis - confirmed that the expressions of Tyr Phe mutants of Figure 8 Effects of coexpression of Hsp90 and Cdc37 on the mFLT3Asp838Val after CHX treatment were significantly activation and cell proliferation of Tyr-Phe mutants of reduced compared to that of mFLT3Asp838Val. These mFLT3Asp838Val.(a) Effects of coexpression of Hsp90 and Cdc37. results indicated that the mutations of Tyr845Phe, Tyr-Phe mutants of mFLT3Asp838Val, Cdc37 and Hsp90 were Asp838Val transiently cotransfected into Plat-E cells. The cells were grown at Tyr892Phe and Tyr922Phe caused mFLT3 un- 371C and lysed, immunoprecipitates with anti-mFLT3 antibody stable and easy to degrade, which reflected the and subjected to immunoblotting with anti-phosphotyrosine anti- temperature-sensitive character. body (upper panel), anti-mFLT3 antibody (lower panel). The phosphorylation rates indicate the ratio of phosphorylation level of each mFLT3 mutants quantified by chemical image analyzer Effects of chaperone proteins, Hsp90 and Cdc37, on the compared to that of mFLT3Asp838Val without Hsp90 and Cdc37. The activation of Tyr-Phe mutants of mFLT3Asp838Val values of fold increase indicate the ratio of phosphorylation rate with chaperones to without chaperones in each mFLT3 mutants. The temperature-sensitive character suggested that these (b) The same immunoprecipitated samples as (a) were subjected to mutations caused the conformational change, which led immunoblotting with anti-Hsp90 and anti-Cdc37 antibody (upper panels). Total lysates were also immunoblotted with anti-Hsp90 to the impaired kinase activity and easy degradation. and anti-Cdc37 antibody to confirm the expression of both Since the conformation and the activity of various chaperones (lower panels). (c) Total lysates of Ba/F3 cells stably kinases are regulated by chaperone proteins such as expressing Tyr-Phe mutants of mFLT3Asp838Val, Hsp90 and Cdc37 Hsp90 and Cdc37, we examined whether the suppressed were also subjected to immunoblotting with anti-phospho-Erk1/2 - Asp838Val and anti-Erk1/2 antibody. (d) Cell proliferation of Ba/F3 cells kinase activity of Tyr Phe mutants of mFLT3 stably expressing Tyr-Phe mutants of mFLT3Asp838Val, and/or could be rescued by Hsp90 and Cdc37. We chaperone proteins (Hsp90 and Cdc37). Cells were cultured co-transfected chaperone proteins, Hsp90 and Cdc37, without IL-3 or FLT3 ligand at 371C for 48 h, and subjected to with mFLT3 mutants to Plat-E cells at 371C, and MTT assay. The results are shown as mean7s.d. of three analysed the activation of mFLT3. The co-expression of independent experiments. P-values are also indicated Hsp90 and Cdc37 augmented the kinase activity of Tyr845Phe, Tyr892Phe and Tyr922Phe mutants about overexpression of these mutants. Hsp90 and Cdc37 were 1.30–1.64-fold as shown in fold increase (Figure 8a). In detected in FLT3 immunoprecipitates (Figure 8b), this experiment, mFLT3 of Tyr845Phe and Tyr922Phe which indicated that Hsp90 and Cdc37 could bind to were phosphorylated even without co-expression of and serve as chaperone proteins for FLT3Asp838Val and chaperones. We considered that this was due to the Tyr-Phe mutants. These results suggested that two

Oncogene Critical tyrosine residues in FLT3 mutation J Ishiko et al 8150 chaperone proteins, Hsp90 and Cdc37, could support these mutations partially inhibited but retained the the activity of Tyr-Phe mutants of mFLT3Asp838Val phosphorylation and the anti-apoptotic function of under the condition of high expression of mFLT3Asp838Val. mFLT3WT by ligand stimulation. Consistent with our To investigate the effects of chaperone proteins on results about Tyr922Phe, Beslu et al. (1996) reported downstream signaling and cell function, we constructed that Tyr922Phe did not inhibit the kinase activity of Ba/F3 cell lines stably expressing Hsp90 and Cdc37, in mFLT3WT. Then, it was suggested that Tyr892 and Tyr922 addition to mFLT3 mutants. Stable expression of Hsp90 had more critical roles in the constitutive activation of and Cdc37 showed the recovery of Erk1/2 activation mFLT3Asp838Val. In addition, Tyr845, Tyr892 and Tyr922 and cell proliferation in the cell line expressing mutations inhibited the kinase activation of other Tyr845Phe mutant (Figure 8c and d). In contrast, mFLT3 mutations, corresponding to human FLT3 Hsp90 and Cdc37 could not rescue the Erk activation mutations such as ITD, Tyr835Phe and Ile836del, which and the proliferation substantially in Tyr892Phe and have been detected in patients with AML. These results Tyr922Phe mutants, although the increase of prolifera- suggested that these tyrosine residues were also the tion was statistically significant. These results suggested common regulatory region of various active mutations that the effects of Hsp90 and Cdc37 were different of FLT3. between Tyr-Phe mutants, and the sufficient recovery Tyr845 is the tyrosine residue located in the activation of Tyr892Phe and Tyr922Phe mutants may require loop of mFLT3, and has been identified as the additional support by other chaperone proteins. autophosphorylation site (Yu et al., 2003). The tyrosine residues in the activation loop are conserved in all tyrosine kinase family, the phosphorylation of which has been shown to change the conformation of activation Discussion loop into active open form in the crystal structural analysis of FGFR1 and IRK (Mohammadi et al., 1996; We have previously shown that there are crucial Hubbard, 1997). The crystal structure of human FLT3 cytoplasmic tyrosine residues regulating the activation indicated that the activation loop of FLT3 has similar of murine c-Kit TKD-mutant (KITAsp814Val) (Hashimoto configuration with that of IRK, and Tyr842 of human et al., 2003). The phenylalanine substitution of Tyr719 in FLT3, corresponding to Tyr845 of mFLT3, adopted an the kinase insert of KITAsp814Val impaired not only the orientation identical to its equivalent tyrosine of binding of p85 subunit of PI3-kinase but also the activation loop in IRK (Tyr1142) (Griffith et al., 2004). activation of the receptor itself, resulting in the complete These reports suggested that the phosphorylation of inhibition of KITAsp814Val-mediated growth (Hashimoto Tyr845 may regulate the activation of WT FLT3, and et al., 2003). These results suggested that the constitutive possess a similar role in the activation of TKD-mutant. activation of KITAsp814Val depend on the specific tyrosine Concerning the significance of Tyr892 and Tyr922,ithas residues. With the advent of the similar approach, we been reported that there are temperature-sensitive have found that Tyr845, Tyr892 and Tyr922 critically mutants of v-Src and Bcr-Abl, which has a mutation regulate the activation of mFLT3 TKD-mutant. in the similar region. Tyr892 of mFLT3 is located in the Although c-Kit and FLT3 have 22 cytoplasmic tyrosine sequence of Gly-X-X-Pro-Tyr892-Pro, which is the residues and two-third of these tyrosine residues are conserved sequence among tyrosine kinase family. The conserved, the most critical tyrosine residues were corresponding sequence of v-Src is Gly-Arg-Val461-Pro- different in both tyrosine kinases, which suggested the Tyr-Pro, and the substitution of Met for Val461 in this specific regulation of kinase activity in each RTK. We region together with another Pro503Ser mutation made have confirmed the inhibition of mFLT3Asp838Val activa- v-Src into temperature-sensitive mutant (Mayer et al., tion by these three tyrosine residues in different cell lines 1986). The temperature-sensitive mutant of Bcr-Abl is and also in different transfection systems. Since we created by Tyr457His and Tyr469His mutations of found that the phosphorylation of Tyr592, which is the c-Abl, the latter corresponding to Tyr922 of mFLT3 autophosphorylated residue on receptor activation, was (Carlesso et al., 1994). Therefore, we addressed the clearly reduced in these mutants, the impaired tyrosine temperature-sensitive character of Tyr-Phe mutants of phosphorylation of the receptor was not simply due to mFLT3Asp838Val. Tyr845Phe, Tyr892Phe and Tyr922Phe the loss of each tyrosine residue, but to the inhibition of mutants showed the improvement in the receptor the receptor activation. Tyr845Phe, Tyr892Phe and phosphorylation, MAP kinase activation and the sur- Tyr922Phe not only inhibited the several downstream vival at 331C compared to 371C, indicating temperature- signal transductions, Ras/MAP kinase, STAT3 and sensitive character of these mutants. The half-life of these STA5 but also the ligand-independent proliferation Tyr-Phe mutants was shorter than mFLT3Asp838Val, and survival mediated by mFLT3Asp838Val. Taken to- which suggested that Tyr-Phe mutations affected the gether, these results indicated that Tyr845, Tyr892 and protein stability of the receptor. The temperature- Tyr922 critically regulated the constitutive activation and sensitive character suggested that these Tyr-Phe multiple downstream signal transduction of mutations caused the conformational change of mFLT3Asp838Val. Among these three residues, Tyr892 and mFLT3Asp838Val, which led to the impaired kinase activity Tyr922 completely abolished the activation and cell and easy degradation. We suggested that lowering function by mFLT3Asp838Val. Concerning the roles of temperature could correct the unstable deformed Tyr845, Tyr892 and Tyr922 on the activation of mFLT3WT, structure of the Tyr-Phe mutants of mFLT3Asp838Val,

Oncogene Critical tyrosine residues in FLT3 mutation J Ishiko et al 8151 which resulted in more proper form with less degrada- between amino acids 602/603, similar with previously reported tion and sufficient kinase activity. However, lowering human FLT3-ITD (Spiekermann et al., 2003). The other two temperature itself may not be sufficient for the complete mutations were mFLT3Asp838Tyr and mFLT3Ile839Del, which respec- Asp835Tyr Ile836del reforming of the distorted structure, which corre- tively corresponded to human FLT3 and FLT3 sponded to the incomplete recovery of kinase activity detected in patients with AML. For the expression of Cdc37 and Hsp90, we used pEF-BOS vectors, which contain human and cell survival as shown in Figure 7a and b. In order CDC37 and HSP90 (Scholz et al., 2000). to support this hypothesis that these mutations affected the steric conformation, we examined whether the Cell lines suppressed kinase activity of Tyr-Phe mutants of mFLT3Asp838Val could be rescued by Hsp90 and Cdc37. The Plat-E cells, derived from human embryonic kidney cells, We have found that co-expression of chaperone 293 T cells (Morita et al., 2000), were maintained in Dulbecco’s proteins, Hsp90 and Cdc37, could partially improve modification of Eagle’s medium (Nacalai-Tesque Inc.) supple- mented with 10% fetal bovine serum (FBS; EQUITEC-BIO the activation of the Tyr-Phe mutants of mFLT3Asp838- Val Inc.). The Ba/F3 cells, murine IL3-dependent pro-B lymphoid under the condition of high expression of FLT3. This cell line, were cultured in RPMI 1640 medium with result suggested that Hsp90 and Cdc37 could correct the L-glutamine (Nacalai-Tesque) and 10% FBS. conformation of the mutant receptors, which resulted in the recovery of the kinase activity. This finding was Transfection consistent with our previous report that Hsp90 and Cdc37 could buffer the impaired activity at higher The expression vector pcDNA3 (10 mg) was transiently temperature of a temperature-sensitive mutant of con- transfected into Plat-E cells by the calcium-phosphate method. On the next day, we confirmed the mFLT3 expression by stitutive active Hck (Scholz et al., 2001). However, staining with PE anti-mouse CD135 (Flk-2/FLT-3) antibody Hsp90 and Cdc37 could not effectively rescue the (BD Biosciences) and analysing by FACScan (Becton Dick- impaired activity of Tyr892Phe and Tyr922Phe in inson). The cells were used for further analysis 2 days after Ba/F3 cells, which suggested that other chaperone transfection. proteins may be necessary. For stable gene transfer to Ba/F3 cells, we performed the In conclusion, we have identified the critical tyrosine electroporation with expression vector pGD (20 mg), and residues, which may be important to stabilize the active selected neomycin-resistant cells by culturing with 1 mg/ml structure of FLT3 TKD-mutant. Although the crystal G418 sulfate (Sigma-Aldrich). To establish Ba/F3 cell line structure of FLT3 has been revealed, it has not been stably expressing Hsp90 and Cdc37, expression vector pEF- clear how the mutations of activation loop cause the BOS (20 mg) and pMAM/BSD (1 mg, blasticidin-resistant gene) were transfected by electroporation, and selected with 10 mg/ml structural change (Griffith et al., 2004). Although Tyr892 922 blasticidin S (Calbiochem). The expression of Cdc37 and and Tyr have not been identified as the autophos- Hsp90 was confirmed by Western blotting. For each construct, WT phorylation sites in FLT3 (Yu et al., 2003), they could we selected more than three clones that had the sufficient be phosphorylated aberrantly in FLT3 active mutants. expression, and the results are shown as the representative of It is also possible that these residues may be involved in experiments with these different clones. the proper folding of FLT3 kinase domain without phosphorylation. It has been reported that the replace- Immunoprecipitation and Western blotting ment of Tyr with Phe in IRK result in the change of After the depletion of serum and factors for 12 h, cells were basal kinase activity without tyrosine phosphorylation treated with 200 ng/ml of FLT3 ligand (PeproTech Inc.) or (Li et al., 2003). More detailed structural analysis of 10 ng/ml of IL-3 (Kirin Brewery) at 371Cfor10min.Cellswere these mutants may reveal how these tyrosine residues washed twice with phosphate-buffered saline (PBS; Nacalai- function in the stabilization of protein structure of Tesque), and lysed with lysis buffer as described previously FLT3, and may facilitate the development of small (Ueda et al., 2002). For immunoprecipitation, cell lysates were molecules inhibiting oncogenic FLT3. incubated with rabbit polyclonal antibody to mFLT3 (Santa Cruz Biotechnology) and Protein G Sepharose 4 FastFlow (Amersham Bioscience). The immunoprecipitates and total lysates were resuspended in sodium dodecyl sulfate (SDS) Materials and methods sample buffer, heated, separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and subjected for Western blotting Construction of transgene as described before. We used the following antibodies. Anti- phosphotyrosine antibody (P-Tyr-100), anti-phospho-FLT3 The constructs of mFLT3Asp838Val was described previously (Tyr591) antibody, anti-phospho-Erk1/2 antibody, anti-Erk1/2 (Fenski et al., 2000). To construct various mutants of antibody, anti-phospho-STAT3 antibody, anti-STAT3 anti- mFLT3Asp838Val in which each cytoplasmic tyrosine residue body, anti-phospho-STAT5 antibody and anti-STAT5 antibody was substituted by phenylalanine, we used QuikChanget Site- were from Cell Signaling Technology. Anti-FLT3 antibody was Directed Mutagenesis Kit (Stratagene). We made 22 primers from Upstate Biotechnology, and anti-Hsp90 and anti-Cdc37 for Tyr-Phe substitution and carried out site-directed antibodies were from Santa Cruz Biotechnology. mutagenesis on the expression vector pcDNA3 and pGD carrying mFLT3Asp838Val cDNA. The mutation as well as the Apoptosis assay and cell viability assay whole sequence was confirmed by DNA sequencing. We also made three other constitutive active mutants of mFLT3 by Apoptosis in transfected cells were analysed by cellular DNA using QuikChanget. The one mutation was mFLT3-ITD, content with propidium iodide staining. Briefly, 2 Â 105 cells which had tandem insertion of Arg-Glu-Tyr-Glu-Asp-Lys were cultured without IL-3 for indicated times, and fixed with

Oncogene Critical tyrosine residues in FLT3 mutation J Ishiko et al 8152 70% ethanol. Subsequently, cells were stained with propidium MTT assay iodide (100 mg/ml), NP-40 (0.01%), and RNase (10 mg/ml) for To investigate cell proliferation, we used an MTT rapid 10 min at 371C. Stained cells were subjected to FACS analysis. colorimetric assay as described before (Ueda et al., 2002). To estimate cell viability of transfected cells, we used trypan Briefly, 2 Â 104 transfected Ba/F3 cells were cultured in the blue dye exclusion test. After incubation for 1 min with 0.4% indicated conditions for 72 h at 371C. MTT (Sigma-Aldrich) trypan blue (Wako Pure Chemical Industries), we counted was incubated in each well for final 4 h of culture. blue-stained and nonstained cells, corresponding to dead and viable cells, respectively. Quantification of protein expression and phosphorylation RNA isolation and real-time PCR The protein expression levels were quantified by analysing the Total RNA was isolated from Ba/F3 cells stably expressing intensity of each band using chemical image analyzer mFLT3Asp838Val mutants using TRIzol reagent (Invitrogen). A FluorChem (Alpha-Innotech Corp.). The phosphorylation total of 1 mg of RNA was reverse transcribed using random rate was calculated by the ratio of the intensity of phosphory- primer and Moloney murine leukemia virus reverse transcrip- lated protein to that of total protein. tase according to the manufacturer’s protocol (Clontech). The quantification of mRNA levels was carried out with the use of Protein degradation assay real-time fluorescence detection method as described before (Muller et al., 2000; Mizuki et al., 2003). Primers and probes Transfected Ba/F3 cells were cultured in RPMI 1640 supple- were designed as described below: mented with 10% FBS without IL-3 in the presence of 10 mg/ml cycloheximide (CHX; Sigma-Aldrich). After indicated hours, a murine Pim-2 total of 1.0 Â 107 cells were lysed, immunoprecipitated with 50-primer: 50-CGG-AAC-CGT-GTG-CTA-GGC-T-30 anti-mFLT3 antibody and subjected to immunoblotting with 30-primer: 50-AGC-AGC-GCA-ACC-TCA-AGT-G-30 anti-mFLT3 antibody as described above. In addition, the probe: 50-FAM-CCA-CCG-TGT-CAG-ACT-CAG-TCA- transfected Ba/F3 cells (1.0 Â 106 cells), which were treated CCT-GC-TAMRA-30 with CHX for 8 h, were fixed and permeabilized by IntraPrept murine GAPDH permeabilization reagents (Immunotech). Then, both intra- 50-primer: 50-TTG-TGC-AGT-GCC-AGC-CTC-30 cellular and membrane mFLT3 were stained with rabbit 30-primer: 50-CCA-ATA-CGG-CCA-AAT-CCG-30 anti-mFLT3 antibody, and subsequently with goat anti-rabbit probe: 50-FAM-TCC-CGT-AGA-CAA-AAT-GGT-GAA- IgG (H þ L) fluorescein-conjugated secondary antibody GGT-CGG-TAMRA-30 (Chemicon International). The expression amounts of mFLT3 were analysed by flow cytometry. The expression level of Pim-2 was calculated using standard curves generated by the serial dilutions of cDNA from IL-3- stimulated Ba/F3 cells. All samples were independently analysed Acknowledgements at least twice. The housekeeping gene GAPDH served as an This work is supported by grants from the Japanese Ministry additional control for the cDNA quality. The expression levels of of Education, Culture, Sports, Science and Technology, and Pim-2 were divided by the GAPDH expression levels, which was Japan Society for the Promotion of Science, from the Ichiro used as the relative Pim-2 expression. Kanehara Foundation, and from the NH&MRC (Australia).

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Oncogene