KITN822K and KITV560G mislocalize to EL in leukemia cells

To investigate the sub-cellular localization of endogenous KIT, we performed confocal immunofluorescence microscopic analyses in pt18 (mouse mast cell line, KIT wild-type (WT)), Kasumi-1 (human AML, KITWT/N822K), SKNO-1 (human AML, KITN822K/N822K), and HMC-1.1 (human MCL, KITWT/V560G) (Fig. 1a). As previously described [24], most KITWT localized to the PM in pt18 (Fig. 1b, left). In contrast, KIT accumulated in vesicular structures in Kasumi-1, SKNO-1, and HMC-1.1 (Fig. 1b). We then performed co-staining assays to identify these structures. In Kasumi-1, KIT was co-localized with TFR (transferrin receptor, endosome marker) and LAMP1 (lysosome marker) rather than with calnexin (ER marker) or giantin (Golgi marker) (Fig. 2a). Similarly, in HMC-1.1, KIT in vesicular structures was colocalized with TFR and cathepsin D (lysosome marker) (Fig. 2b; Additional file 1: Figure S1A). By calculating Pearson’s R correlation coefficients (Pearson’s R) for KIT versus organelle markers, the intensity from KIT-TFR was significantly greater than that from KIT-calnexin, −giantin, and -LAMP1 in Kasumi-1 (Fig. 2c, left graph), suggesting that KIT mainly localizes to endosomes. The quantification showed that in HMC-1.1, KIT was colocalized with TFR to a similar extent as with cathepsin D (Fig. 2c, right graph). In both types of cells, KIT was colocalized with EL markers rather than with ER/Golgi markers. We previously showed that in MCL and GISTs, KIT mutants are normally complex-glycosylated in the Golgi [24, 26]. To test for the KIT glycosylation state in Kasumi-1 and HMC-1.1, we treated KIT with endoglycosidase H, which digests immature high-mannose forms of KIT but not mature complex-glycosylated forms. Figure 2d shows that most KIT in these leukemia cells was in a complex-glycosylated form, similar to normal KIT [24, 28, 29]. KIT shifted to a non-glycosylated form following the complete digestion of N-linked glycans by peptide-N-glycosidase F. In SKNO-1, as with Kasumi-1 and HMC-1.1, KIT was complex-glycosylated (Additional file 1: Figure S1B). Pearson’s R quantification from immunofluorescence images showed that in SKNO-1, KIT localized to endosomes to a similar extent as to lysosomes and it was found in EL rather than on ER/Golgi (Additional file 1: Figure S1C & D). These results suggest that complex-glycosylated KIT accumulates in EL in leukemia cells but not in the early secretory compartments.

Fig. 1 N822K- or V560G-mutated KIT mis-localizes to vesicular structures in leukemia cells. a Schematic representations of wild-type KIT (KITWT) and constitutively active KIT mutants (KITN822K and KITV560G) showing the extracellular domain (ECD), the transmembrane domain (TM), the kinase domain, the lysine mutation at 822 (K in red), and the glycine mutation at 560 (V560G). b Kasumi-1, SKNO-1, HMC-1.1, or pt18 cells were immunostained with anti-KIT. Bars, 10 μm. Note that KITWT localized to the PM, whereas KIT mutants accumulated on vesicular compartments Full size image

Fig. 2 KIT mutants localize to EL but not to the PM in leukemia cells. a & b Kasumi-1 (a) or HMC-1.1 cells (b) were double-stained with anti-KIT (green) plus the indicated antibody (red). Insets show magnified images. Bars, 10 μm. c Pearson’s R correlation coefficients were calculated by analyzing the intensity of KIT vs. organelle markers. Results are means ± s.d. (n = 12~22). *P < 0.05, ***P < 0.001. NS, not significant. Calnexin (ER marker); giantin (Golgi marker); GM130 (Golgi marker); TFR (endosome marker); LAMP1 (lysosome marker); cathepsin D (cathD, lysosome marker). d Lysates from Kasumi-1 (left) or HMC-1.1 cells (right) were treated with peptide N-glycosidase F (PNGase F) or endoglycosidase H (endo H) then immunoblotted with anti-KIT. CG, complex-glycosylated form; HM, high mannose form; DG, deglycosylated form. Note that most KIT was present in a complex-glycosylated form in these leukemia cells Full size image

KIT mutants autonomously migrate from the PM to EL through endocytosis in a manner dependent on their kinase activity

Next, we examined the role of KIT kinase activity on cell proliferation, growth signals, and KIT localization. As previously reported [36,37,38,39], Kasumi-1 and HMC-1.1 proliferate autonomously, and KIT tyrosine kinase inhibitors (TKIs), such as imatinib and PKC412, suppressed cell proliferation in a dose-dependent manner (Fig. 3a). Immunoblotting showed that phosphorylation of KIT, AKT, ERK, and STAT5 occurred in the absence of TKIs, and PKC412 and imatinib reduced these phosphorylations (Fig. 3b), confirming that KIT activates AKT, ERK, and STAT5 in Kasumi-1 and HMC-1.1. Next, we investigated the localization of KIT in TKI-treated cells by immunofluorescence and flow cytometry. In Kasumi-1 cells treated with TKIs, KIT localized more in PM (outside ER staining, Fig. 3c & d) and less in endosomes (Fig. 3e & f). Similar results were seen with HMC-1.1 and SKNO-1 (Additional file 1: Figure S1E). Collectively, in these leukemia cells, newly synthesized KIT in the ER moves to the PM along the secretory pathway and subsequently traffics to EL through kinase activity-dependent endocytosis. In addition to our previous findings that KITD816V and KIT⊿560–578 in the PM are increased by TKI treatment [24, 26, 40], these results suggest that retention in the PM by TKIs is a ubiquitous feature of KIT mutants.

Fig. 3 KIT migrates to EL through endocytosis in a manner dependent on their kinase activity. a Kasumi-1 or HMC-1.1 cells were cultured for 24 h in the presence of KIT kinase inhibitors (imatinib, open circles; PKC412, closed circles). The graphs show the levels of [3H] thymidine deoxyribonucleotide (TdR) incorporation into cells (counts per minute, c.p.m., growth index) at the indicated inhibitor concentrations. Results are means ± s.d. (n = 3). b Kasumi-1 or HMC-1.1 cells were treated for 4 h with 1 μM PKC412 or 1 μM imatinib. Lysates were immunoblotted for KIT, phospho-KIT Y703 (pKITY703), AKT, pAKT, STAT5, pSTAT5, ERK, and pERK. c-f Kasumi-1 cells were treated for 12 h with 1 μM PKC412 (PKC) or 1 μM imatinib (IMA). c Cells were immunostained with anti-KIT (green) and anti-calnexin (ER marker, red). Confocal immunofluorescence images are shown. Insets show magnified images of the PM region. Bars, 10 μm. d Cell surface KIT levels determined by flow cytometry are shown. Non-permeabilized cells were stained with anti-KIT extracellular domain antibody. Green histogram, with KIT inhibitor treatment; white histogram, no KIT inhibitor; gray histogram, no anti-KIT antibody control. e Cells were immunostained with anti-KIT (green) and anti-TFR (endosome marker, red). Confocal immunofluorescence images are shown. Bars, 10 μm. f Pearson’s R correlation coefficients were calculated by analyzing the intensity of KIT vs. TFR. Results are means ± s.d. (n = 22~29). ***P < 0.001. Note that these inhibitors lowered KIT in vesicular structures and increased KIT in the PM Full size image

Autophosphorylation of KITN822K and KITV560G predominantly occurs on the Golgi in leukemia cells

We next examined the site of KIT activation in leukemia cells. To determine the signal platform for KIT, we immuno-stained for phospho-tyrosine residues in the kinase domain which would indicate KIT activation [7,8,9, 26, 27]. In Kasumi-1 cells, phospho-KIT Y703 (pKIT [Y703]) was clearly detected (Fig. 4a), although pKIT [Y721], [Y730], and [Y936] were undetectable by our immunofluorescence staining (data not shown). Interestingly, compared with KIT localization to EL, pKIT [Y703] was restricted to the perinuclear region in Kasumi-1 (Fig. 4a, top panels, arrowheads). Similar to KIT in Kasumi-1, KIT in HMC-1.1 was found in the perinuclear compartment (Fig. 4b). Perinuclear KIT autophosphorylation was colocalized with GM130 (Golgi) rather than with PDI (ER), TFR (endosomes), or LAMP1 (lysosomes) (Fig. 4c; Additional file 1: Figure S2A). Similar results were obtained with SKNO-1 (Additional file 1: Figure S2B & C). These results suggest that in leukemia cells, activation of KITN822K and KITV560G occurs predominantly on the Golgi although KIT itself is found mainly in EL.

Fig. 4 Autophosphorylation of KITN822K and KITV560G occurs preferentially on the Golgi in leukemia cells. a & b Kasumi-1 (a) or HMC-1.1 cells (b) were immunostained for KIT (green), phospho-KIT Y703 (pKITY703, red or green) together with GM130 (Golgi marker, blue), PDI (protein disulfide isomerase, ER marker, red), TFR (endosome marker, red), or LAMP1 (lysosome marker, red). Insets show magnified images. Bars, 10 μm. c Pearson’s R correlation coefficients were calculated by analyzing the intensity of pKITY703 vs. organelle markers. Results are means ± s.d. (n = 12~21). ***P < 0.001. Note that pKITY703 was colocalized with GM130 rather than with PDI, TFR, or LAMP1 both in Kasumi-1 and HMC-1.1 cells Full size image

KITN822K and KITV560G mainly activate downstream molecules on the Golgi in leukemia cells

We then examined whether KIT activated downstream molecules on the Golgi in leukemia cells. To resolve this question, we used inhibitors of intracellular trafficking, such as brefeldin A (BFA), 2-methylcoprophilinamide (M-COPA) (inhibitors of ER export to the Golgi) [27, 34, 35, 41], monensin (an inhibitor of intra-Golgi trafficking) [26, 42], and bafilomycin A1 (BafA1, an inhibitor of endosome-to-lysosome trafficking) [24, 43]. In Kasumi-1 and HMC-1.1, treatment with BFA or M-COPA significantly increased colocalization of KIT with an ER marker, calnexin (Fig. 5a & Additional file 1: Figure S3A), confirming that the treatment inhibited ER export of KIT. Immunoblotting showed that KIT shifted to a lower molecular weight in BFA- or M-COPA-treated cells because of a defect in full glycosylation on the Golgi apparatus (Fig. 5b & c, top panels). KIT on the ER was dephosphorylated and unable to activate downstream molecules (Fig. 5b & c). Previous studies showed that a major target of BFA/M-COPA is Golgi-specific BFA-resistance guanine nucleotide exchange factor 1 (GBF1) that plays a role in the secretory pathway through activation of ADP ribosylation factor 1 (ARF1) [34, 44, 45]. Interestingly, knockdown (KD) of ARF1 and GBF1 with siRNAs did not cause a defect in full glycosylation of KIT or inhibition of signaling (Additional file 1: Figure S3B). Since BFA and M-COPA bind not only to the ARF1-GBF1 complex but also to other complexes [44, 45], the blockers affect KIT trafficking in a manner independent of ARF1-GBF1 inhibition in the leukemia cells used in this study. Further study will be required for understanding how the inhibitors block KIT trafficking from the ER.

Fig. 5 In Kasumi-1 and HMC-1.1, KIT activates downstream pathways on the Golgi apparatus. a Kasumi-1 cells were cultured for 12 h in the presence of 1 μM BFA or 1 μM M-COPA (inhibitors of ER export to the Golgi) and immunostained for KIT and calnexin (ER marker, red). Bars, 10 μm. Pearson’s R correlation coefficients were calculated by analyzing the intensity of KIT vs. calnexin. The right graph shows Pearson’s R (KIT-calnexin) for HMC-1.1 cells treated with 5 μM BFA or 1 μM M-COPA for 16 h. Results are means ± s.d. (n = 14~20). ***P < 0.001. b-d Kasumi-1 cells were treated for 12 h with 1 μM BFA, 1 μM M-COPA, or 250 nM monensin (an inhibitor of Golgi export to the PM). HMC-1.1 cells were treated with 1~5 μM BFA, 1 μM M-COPA for 16 h, or 250 nM monensin for 24 h. Lysates were immunoblotted. e Kasumi-1 cells were immunostained for phospho-AKT (pAKT, green), pERK (green), pSTAT5 (green), and GM130 (Golgi marker, blue). Bars, 10 μm. Arrowheads indicate the Golgi region. f Cells were treated with 1 μM M-COPA for 12 h (Kasumi-1) or 16 h (HMC-1.1), including 3 mM Na 3 VO 4 (a PTP inhibitor) during the last 3 h, then immunoblotted Full size image

Fig. 5d shows that inhibition of the Golgi export of KIT through blocking intra-Golgi trafficking did not suppress KIT signaling, suggesting that Golgi-localized KIT is sufficient for oncogenic signaling in Kasumi-1 and HMC-1.1. As shown in Additional file 1: Figure S3C, KIT signals remained in BafA1-treated cells, indicating that endosome-to-lysosome trafficking is unnecessary for downstream activation. Taken together, these results suggest that the Golgi apparatus serves as the platform for KIT activation in leukemia cells. To support this conclusion, we stained for phospho-AKT (pAKT), pERK, and pSTAT5. As shown in Fig. 5e, these phosphorylations were found at the Golgi region in Kasumi-1 cells. Compared with pAKT, total AKT was barely seen in the Golgi (Additional file 1: Figure S3D, upper panels). In Kasumi-1, only part of AKT may be activated by KIT. Furthermore, total ERK and STAT5 were distributed in the Golgi region (Additional file 1: Figure S3D). These results support our hypothesis that KIT activates these downstream molecules on the Golgi in leukemia cells. In HMC-1.1, AKT, ERK, STAT5, and their phospho-forms showed a diffuse distribution compared with those in Kasumi-1 (Additional file 1: Figure S3E). Since pAKT, pERK, pSTAT5, though small, were found in the Golgi region, they could be activated on the Golgi and subsequently move elsewhere.

Recently, we showed that in GISTs, KIT on the ER is dephosphorylated by protein tyrosine phosphatases (PTPs) [27]. We then considered the role of PTPs in KIT inactivation in the ER in leukemia cells. In M-COPA-pretreated Kasumi-1, a 3-h treatment with a PTP inhibitor (sodium orthovanadate, Na 3 VO 4 ) [46] restored pKIT [Y703], resulting in downstream reactivation (Fig. 5f, left), indicating that in Kasumi-1, PTPs play a role in KIT inactivation in the ER. In M-COPA-treated HMC-1.1, pKIT [Y703] and pSTAT5 were recovered by Na 3 VO 4 treatment, but AKT and ERK did not become active on PTP inhibition (Fig. 5f, right). Negative regulation of AKT and ERK may differ among cell types. Taken together, these results suggest that ER-localized KIT is inactivated by PTPs. PTP1B, Src homology 2 containing PTP-1 (SHP-1), and SHP-2 have been reported as PTPs for KIT and FLT3 RTKs [47,48,49,50]. Thus, we knocked down these PTPs and treated cells with M-COPA to investigate the key PTPs for KIT in the ER. Additional file 1: Figure S4 shows that in M-COPA-treated cells, pKIT [Y703], pAKT, and pERK were not restored by PTP1B or SHP1/2 KD, suggesting that these PTPs are not responsible for this dephosphorylation in the ER. Interestingly, PTP1B but not SHP1/2 KD partially rescued pSTAT5 in M-COPA-treated cells (Additional file 1: Figure S4A, arrows). Although we were unable to identify KIT phospho-tyrosine sites that are dephosphorylated by PTP1B in this study, PTP1B in the ER may play a role in inactivation of the KIT-STAT5 axis.

SKNO-1 cells were similar to Kasumi-1 in phospho-regulation of KIT in intracellular compartments (Additional file 1: Figure S5A & B). However, AKT, ERK, and STAT5 were not activated by KITN822K (Additional file 1: Figure S5C). SKNO-1 requires GM-CSF for proliferation, but the cytokine did not affect the activation of KIT, AKT, ERK, or STAT5 (Additional file 1: Figure S5C, right panels). AKT and ERK were not found in specific compartments in SKNO-1 in the presence or absence of GM-CSF (Additional file 1: Figure S5D). At present, we are unable to find downstream molecules that are activated by KITN822K in SKNO-1 cells. We will investigate the role of KITN822K in SKNO-1 growth in the near future.

Lipid rafts play a key role in KIT signaling, which occurs on the Golgi apparatus

Recent studies showed that sphingomyelin-enriched membrane microdomains (lipid rafts) in the Golgi are needed for activation of an innate immunity molecule, STING [51, 52]. Formation of normal lipid microdomains is inhibited by N-hexanoyl-D-erythro-sphingosine (cer-C6) through producing short chain sphingomyelin that disrupts the lipid order [51, 53, 54]. Figure 6a shows that in Kasumi-1, cer-C6 treatment lowered the protein levels of KIT and inhibited KIT autophosphorylation and the activation of AKT, ERK, and STAT5 in a dose-dependent manner. The treatment did not decrease but rather increased KIT on the Golgi (Fig. 6b & c). These results suggest that KITN822K and KITV560G require lipid rafts for their stability and activation in the Golgi in these leukemia cells.

Fig. 6 Lipid rafts have a role in KIT signaling at the Golgi apparatus. a-c Kasumi-1 or HMC-1.1 cells were treated with 0~40 μM cer-C6 for 8 h (for inhibition of normal lipid raft formation). a Lysates were immunoblotted. b Cells treated with 40 μM cer-C6 for 8 h were immunostained for KIT (green), giantin (Golgi marker, red), or GM130 (Golgi marker, blue). Bars, 10 μm. c Pearson’s R correlation coefficients were calculated by analyzing the intensity of KIT vs. giantin (Kasumi-1) or GM130 (HMC-1.1). Results are means ± s.d. (n = 16~22). **P < 0.01. d & e GIST-T1 cells were treated with 0~10 μM cer-C6 for 10 h. d Lysates were immunoblotted. e Lysates were treated with PNGase F or endoglycosidase H then immunoblotted with anti-KIT. CG, complex-glycosylated form; HM, high mannose form; DG, deglycosylated form. f HMC-1.2 cells were treated with 0~40 μM cer-C6 for 8 h, then immunoblotted Full size image

Finally, we asked whether lipid rafts play a role in oncogenic signaling by all KIT mutants. GIST-T1 cells (KIT⊿560–578) grow in a manner dependent on KIT signaling on the Golgi, whereas HMC-1.2 (mast cell leukemia, KITV560G/D816V) requires pAKT on EL and pSTAT5 on the ER [24,25,26,27] (Additional file 1: Figure S6A & Table 1). In both cell types, TKIs increased PM distribution of KIT mutants (Additional file 1: Figure S6B), supporting our data obtained with Kasumi-1 that mutant KIT localizes to intracellular compartments in a manner dependent on its kinase activity. In GIST-T1, cer-C6 inhibited the phosphorylation of KIT and downstream molecules (Fig. 6d). Unlike KIT in leukemia cells, that in cer-C6-treated GIST-T1 assumed an immature glycosylated form (Fig. 6e), indicating that KIT is complex-glycosylated in GIST after reaching lipid rafts. Similar to the results using Kasumi-1, the treatment did not decrease but rather increased KIT on the Golgi (Additional file 1: Figure S6C). On the other hand, in HMC-1.2, cer-C6 did not have an inhibitory effect on Golgi export of KITD816V and growth signals (Fig. 6f; Additional file 1: Figure S6D). Therefore, lipid rafts play a critical role in KIT signaling that occurs on the Golgi.