N-Glycosylation of the human kappa opioid receptor enhances its stability but slows its trafficking along the biosynthesis pathway

We examined glycosylation of FLAG-hKOR expressed in CHO cells and determined its functional significance. FLAG-hKOR was resolved as a broad and diffuse 55-kDa band and a less diffuse 45-kDa band by immunoblotting, indicating that the receptor is glycosylated. Endoglycosidase H cleaved the 45-kDa band to approximately 38 kDa but did not change the 55-kDa band, demonstrating that the 45-kDa band is N-glycosylated with high-mannose or hybrid-type glycan. Peptide-N-glycosidase F digestion of solubilized hKOR or incubation of cells with tunicamycin resulted in two species of 43 and 38 kDa, suggesting that the 43-kDa band is O-glycosylated. FLAG-hKOR was reduced to lower Mr bands by neuraminidase and O-glycosidase, indicating that the hKOR contains O-linked glycan. Mutation of Asn25 or Asn39 to Gln in the N-terminal domain reduced the Mr by approximately 5 kDa, indicating that both residues were glycosylated. The double mutant hKOR-N25/39Q was resolved as a 43-kDa (mature form) and a 38-kDa (intermediate form) band. When transiently expressed, hKOR-N25/39Q had a lower expression level than the wild type. In CHO cells stably expressing the hKOR-N25/39Q, pulse-chase experiments revealed that the turnover rate constants (ke) of the intermediate and mature forms were approximately 3 times those of the wild type. In addition, the maturation rate constant (ka) of the 43-kDa form of hKOR-N25/39Q was 6 times that of the mature form of the wild type. The hKOR-N25/39Q mutant showed increased agonist-induced receptor phosphorylation, desensitization, internalization, and downregulation, without changing ligand binding affinity or receptor-G protein coupling. Thus, N-glycosylation of the hKOR plays important roles in stability and trafficking along the biosynthesis pathway of the receptor protein as well as agonist-induced receptor regulation.     

ALK-mediated Tyr95 phosphorylation of Smad4 impairs its transcription activity and the tumor suppressive activity of TGF-β

<正>Transforming growth factorβ(TGF-β) regulates various physiological processes such as cell proliferation, differentiation, morphogenesis, andt tissue homeostasis and regeneration. The deregulation of these processes leads to many types of human diseases such as cancer (Massagué,2008). TGF-βsignaling is initiated upon active TGF-βdimer     

Akt phosphorylates MstI and prevents its proteolytic activation, blocking FOXO3 phosphorylation and nuclear translocation

Oxidative stress can induce apoptosis through activation of MstI, subsequent phosphorylation of FOXO and nuclear translocation. MstI is a common component of apoptosis initiated by various stresses. MstI kinase activation requires autophosphorylation and proteolytic degradation by caspases. The role of Akt in regulating MstI activity has not been previously examined. Here, we show that MstI is a physiological substrate of Akt. Akt phosphorylation of MstI diminishes its apoptotic cleavage by caspases and prevents its kinase activity on FOXO3. MstI directly binds to Akt, which is regulated Akt kinase activity. Akt phosphorylates MstI on the Thr(387) residue and protects MstI from apoptotic cleavage in vitro and in apoptotic cells. Interestingly, Akt phosphorylation of MstI strongly inhibits its kinase activity on FOXO3. The phosphorylation mimetic mutant MST1 T387E blocks H2O2-triggered FOXO3 nuclear translocation and apoptosis. Thus, our findings support that Akt blocks MstI-triggered FOXO3 nuclear translocation by phosphorylating MstI, promoting cell survival.