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

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 orthologs 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 localized 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 signaling 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.

Deficiency of X-Linked Protein Kinase Nrk during Pregnancy Triggers Breast Tumor in Mice.

The onset and/or growth of breast tumor are controlled, at least in part, by estrogen. Therefore, to prevent the development of breast tumor, estrogen-dependent proliferation of mammary epithelial cells during pregnancy needs to be suppressed once the mammary gland is fully developed to enable lactation. However, the underlying molecular mechanisms remain unknown. Nrk is an X-linked protein serine/threonine kinase in the germinal center kinase family. Herein, we demonstrate a frequent occurrence of breast tumors in homozygous and heterozygous Nrk mutant mice that have experienced pregnancy/parturition. The tumors never developed in the mutant mice without a history of pregnancy/parturition. They exhibited histopathological features of noninvasive tubular adenocarcinoma, and expressed estrogen receptor α. At late gestation when estrogen receptor α expression was significantly reduced in the wild-type mammary gland, grossly normal mammary glands in the pregnant Nrk mutant mice occasionally contained hyperplastic foci continuously expressing the receptor. Consistently, Nrk expression was induced in the wild-type mammary gland at this period of pregnancy. On the other hand, the pregnant Nrk mutant mice also showed elevated blood estrogen levels at late gestation. We suggest that Nrk suppresses the excessive proliferation of mammary epithelial cells during pregnancy, and the impairment of this regulatory system leads to frequent occurrence of breast tumor in Nrk mutant mice.

Ubiquitin-interacting motifs confer full catalytic activity, but not ubiquitin chain substrate specificity, to deubiquitinating enzyme USP37.

Ubiquitin-specific proteases (USPs) consist of a family of deubiquitinating enzymes with more than 50 members in humans. Three of them, including USP37, contain ubiquitin-interacting motifs (UIMs), an ∼20-amino acid α-helical stretch that binds to ubiquitin. However, the roles of the UIMs in these USP enzymes remain unknown. USP37 has three UIMs, designated here as UIMs 1, 2, and 3 from the N-terminal side, between the Cys and His boxes comprising the catalytic core. Here, we examined the role of the UIMs in USP37 using its mutants that harbor mutations in the UIMs. The nuclear localization of USP37 was not affected by the UIM mutations. However, mutations in UIM2 or UIM3, but not UIM1, resulted in a significant decrease in USP37 binding to ubiquitinated proteins in the cell. In vitro, a region of USP37 harboring the three UIMs also bound to both Lys(48)-linked and Lys(63)-linked ubiquitin chains in a UIM2- and UIM3-dependent manner. The level of USP37 ubiquitination was also reduced by mutations in UIM2 or UIM3, suggesting their role in ubiquitination of USP37 itself. Finally, mutants lacking functional UIM2 or UIM3 exhibited a reduced isopeptidase activity toward ubiquitinated proteins in the cell and both Lys(48)-linked and Lys(63)-linked ubiquitin chains. These results suggested that the UIMs in USP37 contribute to the full enzymatic activity, but not ubiquitin chain substrate specificity, of USP37 possibly by holding the ubiquitin chain substrate in the proximity of the catalytic core.

Nrk, an X-linked protein kinase in the germinal center kinase family, is required for placental development and fetoplacental induction of labor.

The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.

Nik-related kinase is targeted for proteasomal degradation by the chaperone-dependent ubiquitin ligase CHIP.

Nik-related kinase (Nrk) is a member of the germinal center kinase IV family and suppresses Akt signaling. In vivo, Nrk prevents placental hyperplasia and breast cancer formation. Here, we show that Nrk is regulated by the chaperone-dependent ubiquitin ligase carboxyl terminus of heat-shock protein (Hsp)70-interacting protein (CHIP). Immunoprecipitation and liquid chromatography-tandem mass spectrometry analysis reveal that Nrk preferentially interacts with CHIP and Hsp70/90 family proteins. Nrk protein levels are decreased by CHIP overexpression and increased by siRNA-mediated CHIP knockdown. Our results indicate that Nrk is ubiquitinated by CHIP in a chaperone-dependent manner, resulting in its proteasomal degradation. CHIP targets a fraction of Nrk molecules that have lost the ability to regulate Akt signaling. We conclude that CHIP plays an important role in regulating Nrk protein levels.

Proteomic analysis of Nrk gene-disrupted placental tissue cells explains physiological significance of NRK.

OBJECTIVE: NRK is a unique X chromosome-linked protein kinase expressed predominantly in placenta. The gene knockout causes placental overgrowth and delayed labor of Nrk-null fetuses from dams in mouse. To clarify unknown mechanisms behind the Nrk-null phenotypes, protein expression profiles were analyzed in the Nrk-null placenta using a high-performance two-dimensional electrophoresis methodology. RESULTS: Among around 1800 spots detected, we characterized a dozen protein spots whose expression levels were significantly altered in the Nrk-null placenta compared to wild-type. Analyzing these data sets is expected to reflect the difference physiologically in the presence or absence of NRK, facilitating the development of therapeutic strategies.

Functional characterization of Kunitz domains in hepatocyte growth factor activator inhibitor type 1.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor identified as a strong inhibitor of hepatocyte growth factor (HGF) activator and matriptase. HAI-1 is first produced in a membrane-integrated form with two Kunitz domains in its extracellular region, and subsequent ectodomain shedding releases two major secreted forms, one with a single Kunitz domain and one with two Kunitz domains. To determine the roles of the Kunitz domains in the inhibitory activity of HAI-1 against serine proteases, we constructed various HAI-1 mutant proteins and examined their inhibitory activity against HGF activator and trypsin. The N-terminal Kunitz domain (Kunitz I) had potent inhibitory activity against both HGF activator and trypsin, whereas the C-terminal Kunitz domain (Kunitz II) had only very weak inhibitory activity against HGF activator, although its potency against trypsin was equivalent to that of Kunitz I. These results indicate that Kunitz I is the functional domain of HAI-1 for inhibiting the HGF-converting activity of HGF activator. Furthermore, the presence of two Kunitz domains affected the inhibitory activity of HAI-1 against HGF activator, and it showed a similar, but not additive, level of inhibitory activity against trypsin when compared with that of the individual Kunitz domains. These results suggest that serine protease binding sites of Kunitz I and Kunitz II are located close to each other and that proteolytic processing to generate HAI-1 with only one Kunitz domain regulates the activity of HAI-1.