Abstract
Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase–Akt (PI3K–Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek–Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)’s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.
Highlights
In cancer tissue from gastrointestinal stromal tumor (GIST) patients, Kit(mut) accumulates on the Golgi apparatus To investigate the sub-cellular localization of Kit, we performed confocal immunofluorescence microscopic analyses of cancer tissue from GIST patients
Since the perinuclear region may connect to the Golgi apparatus,[28,29,30,31,32,33,35] we stained with a Golgi marker GM130 to see if Kit and PDGFRα were located there
In imatinib-resistant GISTs with a secondary Kit mutation, Kit localized at the Golgi (Figure 1g, Supplementary Figure S1b, and Table 1)
Summary
A cell-surface receptor for stem cell factor, belongs to the typeIII receptor tyrosine kinase (RTK) family that includes plateletderived growth factor receptor α/β (PDGFRα/β), Flt[3] and Fms.[1,2,3] Kit is expressed on interstitial cells of Cajal (ICC), mast cells, hematopoietic cells, germ cells and melanocytes.[4,5,6]Kit is composed of the amino-terminal extracellular portion that binds stem cell factor, a transmembrane domain, and the carboxyterminal intracellular tyrosine kinase domain.[4,7] The binding of stem cell factor autophosphorylates Kit on specific tyrosine residues, for example, Tyr[568], Tyr[570], Tyr[703] and Tyr721.4,8,9 Kit binds to other cytoplasmic proteins, and this complex phosphorylates other proteins.[3,4,8] This activates the PI3K–Akt pathway, the Ras–Mek–Erk cascade and Src kinases, which regulate gene expression and cytoskeletal structures, resulting in cell proliferation and survival.[7,8,9,10]In many gastrointestinal stromal tumors (GISTs) (~85%) and mastocytomas, Kit has gain-of-function mutations, causing ligandindependent auto-activation of the receptor[11,12,13,14] A cell-surface receptor for stem cell factor, belongs to the type. III receptor tyrosine kinase (RTK) family that includes plateletderived growth factor receptor α/β (PDGFRα/β), Flt[3] and Fms.[1,2,3] Kit is expressed on interstitial cells of Cajal (ICC), mast cells, hematopoietic cells, germ cells and melanocytes.[4,5,6]. In many gastrointestinal stromal tumors (GISTs) (~85%) and mastocytomas, Kit has gain-of-function mutations, causing ligandindependent auto-activation of the receptor[11,12,13,14]
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