Xin-Yuan Fu

CHIP Mediates Degradation of Smad Proteins and Potentially Regulates Smad-Induced Transcription

ABSTRACT Transforming growth factor beta (TGF-β)/bone morphogenetic protein (BMP) family ligands interact with specific membrane receptor complexes that have serine/threonine kinase activities. The receptor phosphorylation and activation induced by the ligands leads to phosphorylation of the Smad proteins, which translocate to the nucleus, controlling gene expression. Thus, regulation of Smad proteins is a key step in TGF-β/BMP-induced signal transduction. Here we report a novel mechanism of the regulation of SMAD-mediated signaling, by which the Smad1 protein level is controlled through expression of the CHIP protein. CHIP is a U-box-dependent E3 ubiquitin ligase, previously identified as a cochaperon protein. However, we have isolated CHIP as a Smad-interacting protein in a yeast two-hybrid screen using Smad1 as bait. Furthermore we have shown CHIP-Smad interaction using the 35S-labeled CHIP protein, which can interact with glutathione S-transferase (GST)-Smad1 and GST-Smad4 in an in vitro protein-binding assay. The CHIP-Smad interaction has been confirmed in vivo in mammalian cells through coimmunoprecipitation. Interestingly, we demonstrate that the coexpression of Smad1 and Smad4 with the CHIP protein results in the degradation of the Smad proteins through a ubiquitin-mediated process. Consistent with the observation that CHIP induces Smad1 degradation, we further show that the expression of CHIP can inhibit the transcriptional activities of the Smad1/Smad4 complex induced by BMP signals. Intriguingly, pBS/U6/CHIPi, which diminishes CHIP expression, significantly enhanced Smad1/Smad4- or BMPRIB(QD)-induced gene transcription. These results suggest that CHIP can interact with the Smad1/Smad4 proteins and block BMP signal transduction through the ubiquitin-mediated degradation of Smad proteins.

Varp is a Rab21 guanine nucleotide exchange factor and regulates endosome dynamics.

The small GTPases Rab5 and Rab21 are closely related, and play essential roles in endocytic trafficking. Rab5 is regulated by VPS9-domain-containing guanine nucleotide exchange factors. Here, we describe a new VPS9-domain protein with ankyrin repeats, the VPS9-ankyrin-repeat protein (Varp). Varp interacts preferentially with GDP-bound Rab21 and has a much stronger guanine nucleotide exchange activity towards Rab21 than Rab5. Furthermore, RNAi-mediated depletion of endogenous Varp significantly disrupts the activity of Rab21 in HeLa cells. Ectopically expressed Varp mainly localizes to early endosomes and causes enlargement of early endosomes and giant late endosomes. Both the VPS9 domain and ankyrin-repeats are required for the endosomal localization and the activity of Varp in vivo. These results suggest that Varp is a potential Rab21 guanine nucleotide exchange factor and might regulate endosome dynamics in vivo.

Point Mutation at Single Tyrosine Residue of Novel Oncogene NOK Abrogates Tumorigenesis in Nude Mice

Receptor protein-tyrosine kinases (RPTKs) are tightly regulated during normal cellular processes including cell growth, differentiation, and metabolism. Recently, a RPTK-like molecule named novel oncogene with kinase-domain (NOK) has been cloned and characterized. Overexpression of NOK caused severe cellular transformation as well as tumorigenesis and metastasis in nude mice. In the current study, we generated two tyrosine-->phenylalanine (Y-->F) point mutations (Y327F and Y356F) within the endodomain of NOK that are well conserved in many RPTK subfamilies and are the potential tyrosine phosphorylation sites important for major intracellular signaling. Using BaF3 cells stably expressing the ectodomain of mouse erythropoietin receptor, and the transmembrane and endodomain of NOK (BaF3-E/N), we were able to show that point mutations at either Y327 or Y356 dramatically blocked cellular transformation by NOK as examined by colony formation and cellular DNA synthesis. In addition, tumorigenesis induced by BaF3-E/N was completely abrogated upon the introduction of either single mutation. Importantly, signaling studies revealed that the activation of extracellular signal-regulated kinase was inhibited by Y356F and was significantly reduced by Y327F. Both mutations significantly impaired Akt phosphorylation. Interestingly, both mutations did not affect the kinase activity of NOK. Moreover, apoptotic analysis revealed that both mutations accelerated cell death by activating caspase-3-mediated pathways. Thus, our study shows that these potential tyrosine phosphorylation sites may play critical roles in NOK-mediated tumorigenesis both in vitro and in vivo.