Faiz Mifra, Dunstan-Harrison Caitlin, Ledgerwood Elizabeth C
Department of Biochemistry, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.
Cell Biol Int. 2025 Jun 30. doi: 10.1002/cbin.70053.
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 Ca but basal cytosolic Ca 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 Ca and/or basal ERK1/2 activation may contribute to the abnormal megakaryocyte proliferation characteristic of CALR mutant myeloproliferative neoplasms.
钙网蛋白是一种存在于内质网腔中的多功能蛋白质，对钙稳态和糖蛋白折叠很重要。CALR基因第9外显子的突变是骨髓增殖性肿瘤第二常见的遗传原因。骨髓增殖性肿瘤中CALR突变的巨核细胞增殖涉及突变型钙网蛋白与血小板生成素受体结合导致的JAK/STAT信号通路的细胞因子非依赖性组成性激活。然而，内质网中野生型钙网蛋白的部分或完全去除是否会对巨核细胞生物学产生额外影响尚不清楚。为了探索独立于血小板生成素受体信号的钙网蛋白突变的影响，我们在不表达血小板生成素受体的K-562细胞中产生了1型样CALR突变。我们证实内质网保留KDEL基序的缺失导致大多数突变型钙网蛋白从细胞中分泌。CALR突变的细胞内质网游离钙较高，但基础胞质钙不变。所有CALR等位基因均缺失KDEL内质网保留基序的细胞中ERp57表达增加，然而未诱导未折叠蛋白反应。钙网蛋白突变的细胞还显示ERK1/2的基础磷酸化升高。总体而言，这些结果表明，1型CALR突变的骨髓增殖性肿瘤的表型不仅仅是由于突变型钙网蛋白对血小板生成素受体的细胞因子非依赖性激活，内质网钙增加和/或基础ERK1/2激活可能导致CALR突变的骨髓增殖性肿瘤特征性的异常巨核细胞增殖。
