Fyn specifically Regulates the activity of red cell glucose-6-phosphate-dehydrogenase

Abstract

Fyn is a tyrosine kinase belonging to the Src family (Src-Family-Kinase, SFK), ubiquitously expressed. Previously, we report that Fyn is important in stress erythropoiesis. Here, we show that in red cells Fyn specifically stimulates G6PD activity, resulting in a 3-fold increase enzyme catalytic activity (k cat ) by phosphorylating tyrosine (Tyr)-401. We found Tyr-401 on G6PD as functional target of Fyn in normal human red blood cells (RBC), being undetectable in G6PD deficient RBCs (G6PD-Mediterranean and G6PD-Genova). Indeed, Tyr-401 is located to a region of the G6PD molecule critical for the formation of the enzymatically active dimer. Amino acid replacements in this region are mostly associated with a chronic hemolysis phenotype. Using mutagenesis approach, we demonstrated that the phosphorylation status of Tyr401 modulates the interaction of G6PD with G6P and stabilizes G6PD in a catalytically more efficient conformation. RBCs from Fyn -/− mice are defective in G6PD activity, resulting in increased susceptibility to primaquine-induced intravascular hemolysis. This negatively affected the recycling of reduced Prx2 in response to oxidative stress, indicating that defective G6PD phosphorylation impairs defense against oxidation. In human RBCs, we confirm the involvement of the thioredoxin/Prx2 system in the increase vulnerability of G6PD deficient RBCs to oxidation. In conclusion, our data suggest that Fyn is an oxidative radical sensor, and that Fyn-mediated Tyr-401 phosphorylation, by increasing G6PD activity, plays an important role in the physiology of RBCs. Failure of G6PD activation by this mechanism may be a major limiting factor in the ability of G6PD deficient RBCs to withstand oxidative stress.

Tyrosine Phosphorylation Modulates Peroxiredoxin-2 Activity in Normal and Diseased Red Cells

“…On the other hand, the Prx2 phosphorylation by Lyn and Fgr was negligible (lanes 3 and 5). These results support the notion that the substrate specificity of SFKs can widely vary, as further highlighted recently [ 36 , 37 , 38 ]. That the incorporation of 32 P labels had occurred on tyrosine residues was further confirmed by Western blot analysis of Prx2 with anti-phospho-tyrosine antibodies ( Figure 5 b).…”

“…The oxidative dependent compartmentalization of Prx2 was further supported by the observation that dithiotretol (DTT), a known thiol group donor, prevented the membrane association of Tyr-phosphorylated Prx2 ( Figure 4 b) [ 15 , 21 ]. Noteworthily, Prx2 was associated with the membrane as both a monomer and dimer in diamide-treated red cells, and DTT again prevented Prx2 dimer formation, as previously reported by us [ 13 , 36 ] ( Figure S3b ). Taken together, our data suggest that Prx2 is Tyr-phosphorylated by Syk in response to oxidation, binding to the membrane of diamide-treated red cells.…”

“…recently [36][37][38]. That the incorporation of 32 P labels had occurred on tyrosine residues was further confirmed by Western blot analysis of Prx2 with anti-phospho-tyrosine antibodies (Figure 5b).…”

“…We found that Syk specifically phosphorylates Prx2 at Tyr-193, resulting in increased Prx2 activity. This is extremely interesting, since Prx2 is the third most abundant protein in red cells, playing a key role in the antioxidant system and as an atypical chaperone in normal and diseased erythrocytes [ 17 , 36 , 40 ].…”

Enzymatic Changes in Red Blood Cells of Diamond-Blackfan Anemia

“…An increase in GSH-Px activity is supported by the upregulation of GSH levels. In the cross-analysis reported in the present study, we noticed that the protein level of G6PD did not increase significantly, suggesting that the discrepancy between activity and protein levels was attributable to the oxidation/reduction states of G6PD (Luzzatto 1967;Taniguchi et al 2016), the glycosylation states of G6PD (Rao et al 2015), the post-translational deacetylated state (Wang et al 2014), and the stimulation caused by the phosphorylation of G6PD (Matte et al 2020).…”