Localization of Mutant Calreticulin in the Golgi Apparatus Is Required for Its Oncogenic Capacity

Recurrent somatic mutations of the calreticulin (CALR) gene, which encodes a molecular chaperone, have been previously reported in patients with JAK2- and MPL-unmutated essential thrombocythemia and primary myelofibrosis, a subcategory of myeloproliferative neoplasms (MPNs). Reports from our group, as well as others, have shown that the expression of mutant CALR results in the transformation of cells through the interaction and activation of the thrombopoietin receptor MPL. To further elucidate the molecular mechanism of mutant CALR in the development of MPNs, we studied the subcellular localization of mutant CALR in the megakaryocytic cell line UT-7/TPO, in which the oncogenic properties of mutant CALR were previously demonstrated. By immunofluorescence staining and subsequent confocal microscopic imaging, we determined that mutant CALR accumulated in the Golgi apparatus. Furthermore, we have demonstrated that mutant CALR lacking its signal to localize to the Golgi apparatus did not induce cytokine-independent growth. Consistent with this, the mislocalized mutant CALR lost its capacity to interact with MPL. In a reciprocal experiment, tethering mutant CALR to the cellular compartment by attaching localization signals from unrelated proteins rescued the capacity of mutant CALR in terms of MPL-binding and induction of cytokine-independent growth. These results imply that localization of mutant CALR in the Golgi apparatus is required for its oncogenic capacity.