CALR Type 1-Like Mutations Increase Endoplasmic Reticulum Free Ca2+ and Induce ERK1/2 Activation Independent of Thrombopoietin Receptor Activation

Abstract

Calreticulin is a multifunctional protein found in the endoplasmic reticulum lumen that is important for calcium homeostasis and glycoprotein folding. Mutations in exon 9 of the CALR gene are the second most common genetic cause of myeloproliferative neoplasms. CALR-mutated megakaryocyte proliferation in myeloproliferative neoplasms involves cytokine-independent constitutive activation of JAK/STAT signaling caused by binding of mutant calreticulin to the thrombopoietin receptor. However, whether the partial or complete removal of wildtype calreticulin from the endoplasmic reticulum has additional effects on megakaryocyte biology is not clear. To explore the impact of calreticulin mutations independent of thrombopoietin receptor signaling we generated type 1-like CALR mutations in K-562 cells, which do not express the thrombopoietin receptor. We confirmed that the loss of endoplasmic reticulum-retention KDEL motif causes the majority of mutant calreticulin to be secreted from cells. The CALR mutated cells have higher endoplasmic reticulum free Ca2+ but basal cytosolic Ca2+ is unchanged. Cells in which the KDEL endoplasmic reticulum retention motif was lost from all CALR alleles had increased ERp57 expression however the unfolded protein response was not induced. The calreticulin mutated cells also showed elevated basal phosphorylation of ERK1/2. Overall, these results suggest that the phenotype of type 1 CALR mutated myeloproliferative neoplasms is not solely due to cytokine independent activation of the thrombopoietin receptor by the mutant calreticulin, and that increased endoplasmic reticulum Ca2+ and/or basal ERK1/2 activation may contribute to the abnormal megakaryocyte proliferation characteristic of CALR mutant myeloproliferative neoplasms.