Placental Mammals Acquired Functional Sequences in NRK for Regulating the CK2–PTEN–AKT Pathway and Placental Cell Proliferation

Abstract

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologues across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: (1) mouse NRK is localised at the plasma membrane via the CNH domain, where the CIR inhibits CK2. (2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN, and (3) leads to the inhibition of AKT signalling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.

A possible function of <i>Nik-related kinase</i> in the labyrinth layer of delayed delivery mouse placentas

“…NRK exists just below the plasma membrane of TGCs. This cell localization is consistent with a previous study showing that mouse NRK is localized to the plasma membrane via the citron homology domain [ 16 ]. In the human placenta, NRK was also expressed in the Syn-T of the villi ( Figs.…”

“…Overgrowth of the spongiotrophoblast layer in Nrk KO mice is presumed to be due to the enhancement of the AKT pathway [ 16 , 23 ]. NRK is localized inside the cell membrane and inhibits casein kinase 2, a negative regulator of phosphatase and tensin homolog (PTEN) [ 16 ].…”

“…Overgrowth of the spongiotrophoblast layer in Nrk KO mice is presumed to be due to the enhancement of the AKT pathway [ 16 , 23 ]. NRK is localized inside the cell membrane and inhibits casein kinase 2, a negative regulator of phosphatase and tensin homolog (PTEN) [ 16 ]. NRK might indirectly activate PTEN through this mechanism, thereby suppressing the AKT pathway and cell hyperproliferation [ 16 , 23 ].…”

“…NRK is localized inside the cell membrane and inhibits casein kinase 2, a negative regulator of phosphatase and tensin homolog (PTEN) [ 16 ]. NRK might indirectly activate PTEN through this mechanism, thereby suppressing the AKT pathway and cell hyperproliferation [ 16 , 23 ]. The labyrinth layer weight of the Nrk KO mice was higher than that of the WT mice ( Fig.…”

“…Placental signals might be responsible for the delayed delivery of Nrk KO mice, as WT dams with transplanted Nrk KO blastocysts exhibited delayed delivery [ 15 ]. Furthermore, a recent study has suggested that Nrk has undergone rapid evolution unique to eutherians and is strongly associated with placental development [ 16 , 17 ]. Previous studies have reported that Nrk is expressed in the spongiotrophoblast layer, and Nrk KO placentas exhibit an overgrowth of this layer [ 15 ].…”