JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway

Abstract

Key Points• In polycythemia vera RBCs, JAK2V617F is able to activate an adhesion molecule through a Rap1/Akt pathway, despite the absence of EpoR.• In polycythemia vera, the abnormal adhesion of RBCs to laminin is due to the phosphorylation of Lu/BCAM by a JAK2V617F/Rap1/Akt pathway.Polycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent IntroductionPolycythemia vera (PV) is the most common myeloproliferative neoplasm. 1 It is characterized by erythropoietin-independent erythroid colony formation in vitro, which is associated with somatic gain-of-function mutations in the gene encoding the tyrosine kinase JAK2. [2][3][4][5] The most frequent mutation is a substitution from valine to phenylalanine at position 617, which renders JAK2 constitutively active, leading to uncontrolled cell proliferation in the erythroid lineage and resulting in increased red cell mass. Patients with PV exhibit increased platelet and leukocyte counts and have a high risk of thrombosis, which is considered a major cause of mortality and morbidity in this disease. 6 In a previous work, we showed that PV RBCs were abnormally adherent to endothelial cells because of the interaction between erythroid Lutheran/basal cell-adhesion molecule (Lu/BCAM) and endothelial laminin. 7 Lu/BCAM is an adhesion protein of the immunoglobulin superfamily with a large tissue distribution. 8,9 It is the unique erythroid receptor of laminin ␣5 chain, constituent of laminin 511/521 isoforms, and a major component of the extracellular matrix. 10,11 Lu/BCAM is expressed as 2 isoforms of 85 and 78 kDa, named Lu and Lu(v13), respectively. 9,10 The 2 isoforms differ only by the length of their cytoplasmic domain, which is composed of 19 a.a. for Lu(v13) and 59 a.a. for Lu. The extra 40 a.a. of Lu comprise phosphorylated serines, 12 which have been shown to play a critical role in Lu/BCAM-mediated RBC adhesion to laminin in sickle cell disease. 13,14 We showed in our previous work that PV RBC adhesion to laminin was associated with Lu/BCAM hyperphosphorylation. Lu/BCAM phosphorylation also was strongly increased in a K562 cell line coexpressing Lu and JAK2V617F, ...

Lu/BCAM‐mediated cell adhesion as biological marker of JAK2V617F activity in erythrocytes of polycythemia vera patients

“…Recently, we showed that this abnormal adhesion stems from JAK2V617F-mediated Lu/BCAM activation through a Rap1/Akt signaling pathway [5]. Blocking this pathway by JAK2 specific inhibitors led to significant decrease of PV RBC adhesion [5]. Here, we extended our findings by a quantitative analysis to determine the relationship between the rate of RBC adhesion to laminin and the JAK2V617F allele burden.…”

“…1B). These results indicated that Lu/ BCAM expression alone did not account for the variability of PV RBC adhesion, in accordance with the role of Lu/BCAM phosphorylation in the adhesion of PV RBCs to laminin [5].…”

“…Our group showed increased adhesion of PV RBCs to endothelial cells mediated by erythroid Lu/BCAM and endothelial laminin a5 chain [4]. Recently, we showed that this abnormal adhesion stems from JAK2V617F-mediated Lu/BCAM activation through a Rap1/Akt signaling pathway [5]. Blocking this pathway by JAK2 specific inhibitors led to significant decrease of PV RBC adhesion [5].…”

“…Because Lu/BCAM phosphorylation is driven by a JAK2V617F-dependent pathway in PV RBCs [5], we asked whether RBC adhesion was influenced by JAK2V617F expression level. To date the most commonly used tool for evaluating the mutational load in one individual is by measuring the mutant allele burden in blood cell DNA.…”

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging

“…13 In PV, the JAK2V617F mutation causes activation of the RAP1/Akt signaling pathway that eventually leads to Lu/BCAM activation through phosphorylation. 14,15 Upon phosphorylation, Lu/BCAM is believed to detach from the underlying spectrin network 16,17 and thus to more easily cluster upon ligation of laminina5. Although both Lu and Lu(v13) contain the cytoplasmic motif that interacts with the underlying spectrin network, 17 only the Lu cytoplasmic tail seems to contain phosphorylation sites that modulate this interaction.…”