Addition of ubiquitin or ubiquitin chains to target proteins leads to their mono-or polyubiquitination, respectively. Whereas polyubiquitination targets proteins for degradation, monoubiquitination is thought to regulate receptor internalization and endosomal sorting. Cbl proteins are major ubiquitin ligases that promote ligand-dependent polyubiquitination and degradation of receptor tyrosine kinases. They also recruit CIN85-endophilin in the complex with activated receptors, thus controlling receptor endocytosis. Here we show that the adaptor protein CIN85 and its homologue CMS are monoubiquitinated by Cbl͞Cbl-b after epidermal growth factor (EGF) stimulation. Monoubiquitination of CIN85 required direct interactions between CIN85 and Cbl, the intact RING finger domain of Cbl and a ubiquitin acceptor site present in the carboxyl terminus of CIN85. Cbl-b and monoubiquitinated CIN85 are found in the complex with polyubiquitinated EGF receptors during prolonged EGF stimulation and are degraded together in the lysosome. Dominant interfering forms of CIN85, which have been shown previously to delay EGF receptor degradation, were also impaired in their monoubiquitination. Thus, our data demonstrate that Cbl͞Cbl-b can mediate polyubiquitination of cargo as well as monoubiquitination of CIN85 to control endosomal sorting and degradation of receptor tyrosine kinases.

The cDNA of a Cat'-binding protein regucalcin was cloned from a rat liver cDNA library which was constructed in jlZAPI1 by immunoscreening. Positive clones were obtained from which spanned the region of interest, and they gave a sequence of 1.7 kb by sequencing with the dideoxynucleotide method. Analysis of the sequence of the cloned cDNA showed that the cDNA encoded the complete amino acid sequence of the regucalcin molecule. Regucalcin was composed of 299 amino acid residues and its molecular weight was estimated to be 33,388 Da. The hydropathy profile of regucalcin showed a highly hydrophilic character. The nucleotide and amino acid sequences of regucalcin did not have statistically significant homology, as compared with the registered sequences which are found in the EMBL and GenBank databases containing several other Ca'+-binding proteins (calmodulin, calbindin-D28k and S-100s). The regucalcin molecule did not contain the EF-hand motif as a Ca2'-binding domain. The present study demonstrates that regucalcin is a unique Ca*'-binding protein in the liver of rats.

The distribution and expression of mRNA ene.oding the Ca2"-binding protein regucalcin in rats were investigated by Northern blot analys~. Li~ver regucalein eDNA (0.6 kb) was used as a prob~. The analyses of total RNAs extracted from various tissues of rat indicated that r~gu~tlcin mRlqA was mainly prc~ent in liver but only slightly in kidney with a size of 1.8 kb. The expression level decr~d with increasing age (3, 10 and 25 wc~ks). A single intra~riton~i administration of calcium chloride (15 mg Ca/100 g body weight) induced a remarkable increase in regucalcin mRNA in liver; the level was about 200% of control at 30 rain after the administration. Sub~quently, the expression level began to decrease with time and was about 40% of control level at 120 rain after the administration. The il~emas~ in regueaicin mRNA levels at 30 rain after calcium administration was dos@dependent. These observations show that the expression of regueal¢in mRNA is specific in liver of various tissues, and that it is regulated by Ca :" administration. Regu~l¢in may have a role as regulatory protein for calcium homeostasis in liver cells.

Protein tyrosine kinase Pyk2 and multifunctional adaptor protein Cbl are implicated in the regulation of the cytoskeleton in several cell types. We report that Pyk2 and Cbl form a signaling complex that is translocated to lipid rafts and is enriched in growth cones of differentiating PC12 cells following growth factor stimulation. We found that Pyk2 and Cbl interacted with the adaptor protein ArgBP2, which also bound to flotillin-1, a component of lipid raft microdomains. These interactions contributed to recruitment of the Pyk2/Cbl complex to lipid raft compartments. In addition, Pyk2, Cbl and ArgBP2 were found co-localized with actin in axons and growth cones of differentiated PC12 cells. Moreover, co-expression of Pyk2, ArgBP2 and Cbl facilitated growth factor-induced formation of lamellipodia at the tip of neurites. Formation of these growth cone lamellipodia was dependent on intact lipid rafts and the Cbl-associated effectors Crk and phosphatidylinositol 3 (PI 3)-kinase. Our results indicate that recruitment of Pyk2/Cbl complexes to lipid rafts participates in growth factor-induced regulation of the actin cytoskeleton in growing neurites.

BackgroundPolybrominated diphenyl ethers (PBDEs) have been used as flame retardants and are becoming a ubiquitous environmental contaminant. Adverse effects in the developing brain are of great health concern.ObjectiveWe investigated the effect of PBDEs/hydroxylated PBDEs (OH-PBDEs) on thyroid hormone (TH) receptor (TR)-mediated transcription and on TH-induced dendrite arborization of cerebellar Purkinje cells.MethodsWe examined the effect of PBDEs/OH-PBDEs on TR action using a transient transfection-based reporter gene assay. TR–cofactor binding was studied by the mammalian two-hybrid assay, and TR–DNA [TH response element (TRE)] binding was examined by the liquid chemiluminescent DNA pull-down assay. Chimeric receptors generated from TR and glucocorticoid receptor (GR) were used to identify the functional domain of TR responsible for PBDE action. The change in dendrite arborization of the Purkinje cell in primary culture of newborn rat cerebellum was also examined.ResultsSeveral PBDE congeners suppressed TR-mediated transcription. The magnitude of suppression correlated with that of TR–TRE dissociation. PBDEs suppressed transcription of chimeric receptors containing the TR DNA binding domain (TR-DBD). We observed no such suppression with chimeras containing GR-DBD. In the cerebellar culture, PBDE significantly suppressed TH-induced Purkinje cell dendrite arborization.ConclusionsSeveral PBDE congeners may disrupt the TH system by partial dissociation of TR from TRE acting through TR-DBD and, consequently, may disrupt normal brain development.