Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTKsubstrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substratedocking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 ‫؍‬ 21 M) Csk phosphorylation of Src but only moderately inhibited (IC50 ‫؍‬ 422 M) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.

GPRC5A is a G-protein-coupled receptor expressed in lung tissue but repressed in most human lung cancers.

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Protein tyrosine kinases (PTKs) are important regulators of mammalian cell function and their own activities are tightly regulated. Underlying their tight regulation, all PTKs contain multiple regulatory domains in addition to a catalytic domain. C-terminal Src kinase (Csk) contains a catalytic domain and a regulatory region, consisting of an SH3 and an SH2 domain. In this study, we probed the communication between the regulatory and catalytic domains of Csk. First, kinetic characterization of SH3 and SH2 domain deletion mutants demonstrated that the SH3 and SH2 domains were crucial in maintaining the full activity of Csk, but were not directly involved in Csk recognition of its physiological substrate, Src. Second, highly conserved Trp188, corresponding to a key residue in domain-domain communication in other PTKs, was found to be important for maintaining the active structure of Csk by the presence of the regulatory region, but not required for Csk activation triggered by a phosphopeptide binding to the SH2 domain. Third, structural alignment indicated that the presence of the regulatory domains modulated the conformation of multiple substructures in the catalytic domain, some directly and others remotely. Mutagenic and kinetic studies supported this assignment. This report extended previous studies of Csk domain-domain communication, and provided a foundation for further detailed investigation of this communication.

GFP-TR3 cotransfected with RXRα was exported out of the nucleus in response to 9-cis retinoic acid. Moreover, specific reduction of RXRα levels caused by anti-sense RXRα abolished TR3 nuclear export. In contrast, specific knockdown of TR3 by antisense-TR3 or TR3-siRNA did not affect RXRα shuttling. These results indicate that RXRα is responsible for TR3 nucleocytoplasmic translocation, which is facilitated by the RXRα ligand 9-cis retinoic acid. In addition, mitochondrial TR3, but not RXRα, was critical for apoptosis, as TR3 mutants that were distributed in the mitochondria induced apoptosis in the presence or absence of 9-cis retinoic acid. These data reveal a novel aspect of RXRα function, in which it acts as a carrier for nucleocytoplasmic translocation of orphan receptors.

The imaging characteristics of PCCCL are similar to those of CHCC and could be useful for differentiating these from other liver tumors (such as hemangioma and hepatic metastases). PCCCLs are more prone than CHCCs to form pseudo capsules.

AIM:To investigate the roles of PLCγ2 and PKCα in TPAinduced apoptosis of gastric cancer cells.

Tripodal peptide analogues were designed based on the phosphotyrosine binding pocket of the Src SH2 domain and assayed for their ability to bind to fluorescein-labeled phosphopeptides. Fluorescence polarization assays showed that a number of amphipathic linear peptide analogues (LPAs), such as LPA4, bind to fluorescein-labeled GpYEEI (F-GpYEEI). LPA4 was evaluated for potential application in cellular delivery of phosphopeptides. Fluorescence microimaging cellular uptake studies with fluorescein-attached LPA4 (F-LPA4) alone or the mixture of LPA4 and FGpYEEI in BT-20 cells showed dramatic increase of the fluorescence intensity in cytosol of cells, indicating that LPA4 can function as a delivery tool of F-GpYEEI across the cell membrane. Fluorescent flow cytometry studies showed the cellular uptake of F-LPA4 in an energy-independent pathway, and confirmed the cellular uptake of F-GpYEEI in the presence of LPA4. These studies suggest that amphipathic tripodal peptide analogues, such as LPA4, can be used for cellular delivery of phosphopeptides.