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Biomedical subjects

P E Burnett

Publications and source records attributed to P E Burnett.

10 recordsLinked to original sources
PubMed2000

The calcineurin-binding protein cain is a negative regulator of synaptic vesicle endocytosis.

During neurotransmitter release, exocytosed neurotransmitter vesicles are recycled by endocytosis, which involves the assembly of a complex of endocytic proteins. Assembly of endocytic proteins into a functional complex depends on their dephosphorylation by calcineurin, a calcium-sensitive protein phosphatase and the inhibitory target of immunosuppressive drugs cyclosporin A and FK506. Cain is a recently identified protein inhibitor of calcineurin. We now provide evidence that cain is a component of the endocytic protein complex. The proline-rich region of cain forms a stable association with the SH3 domain of amphiphysin 1. Using a transferrin uptake assay, we found that overexpression of cain in HEK293 cells blocks endocytosis as potently as expression of a dominant negative dynamin 1 construct. The use of other calcineurin inhibitors such as cyclosporin A and FK506 also blocks endocytosis. Since binding of cain to amphiphysin 1 does not affect amphiphysin's interaction with other endocytic proteins, our results suggest that cain negatively regulates synaptic vesicle endocytosis by inhibiting calcineurin activity, rather than sterically interfering with the assembly of the endocytic protein complex.

Animals↗
PubMed1999

The calcineurin-dynamin 1 complex as a calcium sensor for synaptic vesicle endocytosis.

Exocytosis of synaptic vesicles is calcium-dependent, with synaptotagmin serving as the calcium sensor. Endocytosis of synaptic vesicles has also been postulated as a calcium-dependent process; however, an endocytic calcium sensor has not been found. We now report a physical association between the calcium-dependent phosphatase calcineurin and dynamin 1, a component of the synaptic endocytic machinery. The calcineurin-dynamin 1 interaction is calcium-dependent, with an EC(50) for calcium in the range of 0.1-0. 4 microM. Disruption of the calcineurin-dynamin 1 interaction inhibits clathrin-mediated endocytosis. Thus, the calcium-dependent formation of the calcineurin-dynamin 1 complex, delivered to the other endocytic coat proteins, provides a calcium-sensing mechanism that facilitates endocytosis.

Animals↗
PubMed1999

Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling.

RAFT1 (rapamycin and FKBP12 target 1; also called FRAP or mTOR) is a member of the ATM (ataxia telangiectasia mutated)-related family of proteins and functions as the in vivo mediator of the effects of the immunosuppressant rapamycin and as an important regulator of messenger RNA translation. In mammalian cells RAFT1 interacted with gephyrin, a widely expressed protein necessary for the clustering of glycine receptors at the cell membrane of neurons. RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules, including the p70 ribosomal S6 kinase and the eIF-4E binding protein, 4E-BP1. The interaction with gephyrin ascribes a function to the large amino-terminal region of an ATM-related protein and reveals a role in signal transduction for the clustering protein gephyrin.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Cain, a novel physiologic protein inhibitor of calcineurin.

Calcineurin is a widely distributed protein phosphatase regulated by calcium and calmodulin. It mediates the immunosuppressive actions of drugs such as cyclosporin and FK506, and has been implicated in a number of calcium-sensitive pathways in the nervous system, including regulation of neurotransmitter release and modulation of long-term changes in synaptic plasticity. Calcineurin associates physiologically with other proteins, including calmodulin, FKBP12 (FK506-binding protein), the ryanodine receptor, and the inositol 1,4,5-trisphosphate receptor. We now report the identification, molecular cloning, and functional characterization of a novel protein, cain (calcineurin inhibitor), that interacts with and inhibits calcineurin. The full-length cain cDNA predicts a 240-kDa protein with no significant homology to any known protein. Cain associates with calcineurin both in vitro and in vivo, leading to a non-competitive inhibition of calcineurin activity. The putative calcineurin-binding domain of cain, a 38-amino acid region defined by mutational analysis, is highly basic. Like calcineurin, cain has a prominent neuronal expression and a wide tissue distribution. Cain's expression pattern in the brain closely resembles that of calcineurin, indicating a physiologic association between the two proteins.

Amino Acid Sequence↗
PubMed1998

Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton.

p70 S6 kinase (p70(S6k)) is a mitogen-activated protein kinase that plays a central role in the control of mRNA translation. It physiologically phosphorylates the S6 protein of the 40s ribosomal subunit in response to mitogenic stimuli and is a downstream component of the rapamycin-sensitive pathway, which includes the 12-kDa FK506 binding protein and includes rapamycin and the 12-kDa FK506 binding protein target 1. Here, we report the identification of neurabin (neural tissue-specific F-actin binding protein), a neuronally enriched protein of 1,095 amino acids that contains a PDZ domain and binds p70(S6k). We demonstrate the neurabin-p70(S6k) interaction by yeast two-hybrid analysis and biochemical techniques. p70(S6k) and neurabin coimmunoprecipitate from transfected HEK293 cells. Site-directed mutagenesis of neurabin implicates its PDZ domain in the interaction with p70(S6k), and deletion of the carboxyl-terminal five amino acids of p70(S6k) abrogates the interaction. Cotransfection of neurabin in HEK293 cells activates p70(S6k) kinase activity. The mRNA of neurabin and p70(S6k) show striking colocalization in brain sections by in situ hybridization. Subcellular fractionation of rat brain demonstrates that neurabin and p70(S6k) both localize to the soluble fraction of synaptosomes. By way of its PDZ domain, the neuronal-specific neurabin may target p70(S6k) to nerve terminals.

Amino Acid Sequence↗
PubMed1998

RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.

The complex of rapamycin with its intracellular receptor, FKBP12, interacts with RAFT1/FRAP/mTOR, the in vivo rapamycin-sensitive target and a member of the ataxia telangiectasia mutated (ATM)-related family of kinases that share homology with the catalytic domain of phosphatidylinositol 3-kinase. The function of RAFT1 in the rapamycin-sensitive pathway and its connection to downstream components of the pathway, such as p70 S6 kinase and 4E-BP1, are poorly understood. Here, we show that RAFT1 directly phosphorylates p70(S6k), 4E-BP1, and 4E-BP2 and that serum stimulates RAFT1 kinase activity with kinetics similar to those of p70(S6k) and 4E-BP1 phosphorylation. RAFT1 phosphorylates p70(S6k) on Thr-389, a residue whose phosphorylation is rapamycin-sensitive in vivo and necessary for S6 kinase activity. RAFT1 phosphorylation of 4E-BP1 on Thr-36 and Thr-45 blocks its association with the cap-binding protein, eIF-4E, in vitro, and phosphorylation of Thr-45 seems to be the major regulator of the 4E-BP1-eIF-4E interaction in vivo. RAFT1 phosphorylates p70(S6k) much more effectively than 4E-BP1, and the phosphorylation sites on the two proteins show little homology. This raises the possibility that, in vivo, an unidentified kinase analogous to p70(S6k) is activated by RAFT1 phosphorylation and acts at the rapamycin-sensitive phosphorylation sites of 4E-BP1.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Application issues in bioanalytical method validation, sample analysis and data reporting.

Although some degree of consensus has been reached concerning the requirements for acceptable method validation, the procedures used to establish them vary significantly between laboratories. Also, issues arising from application of these requirements during validation and subsequent sample analysis need to be addressed. The purpose of this paper is to discuss application issues concerning prerequisites to method validation, and all validation criteria for evaluation of method reliability and overall performance. Other poorly addressed issues such as re-validation, cross-validation, partial sample volume, multicomponent analysis and reporting will also be discussed. Although many issues discussed are of a general nature, the scope of this presentation is primarily to address issues arising from the validation and routine application of chromatographic methods.

Animals↗
PubMed1995

Issues in evaluation of bioanalytical method selectivity and drug stability.

Evaluation of selectivity is one of the most controversial aspects of method validation and application of methods to studies. The focus of selectivity testing should address the question: Above what level will interference significantly impact on study conclusions? Four key issues will be addressed: the statistical relevance of any selectivity test performed; a criterion for significant interference; experimental methods to establish selectivity; and criteria for acceptance. To ensure that compound integrity is maintained throughout the work-up process, statistically meaningful methods of stability evaluation which are associated with specific acceptance criteria are required. Suitable methods for evaluating stability of analyte and/or solutions of analyte, in process stability, processed sample stability, long term stability and freeze-thaw stability, as well as meaningful acceptance criteria, are presented.

Animals↗
PubMed1994

Transcriptional control of the yeast PDR5 gene by the PDR3 gene product.

Saccharomyces cerevisiae cells possess the ability to simultaneously acquire resistance to an array of drugs with different cytotoxic activities. The genes involved in this acquisition are referred to as pleiotropic drug resistant (PDR) genes. Several semidominant, drug resistance-encoding PDR mutations have been found that map near the centromere on chromosome II, including PDR3-1 and PDR4-1. DNA sequencing of chromosome II identified a potential open reading frame, designated YBL03-23, that has the potential to encode a protein with strong sequence similarity to the product of the PDR1 gene, a zinc finger-containing transcription factor. Here we show that YBL03-23 is allelic with PDR3. The presence of a functional copy of either PDR1 or PDR3 is essential for drug resistance and expression of a putative membrane transporter-encoding gene, PDR5. Deletion mapping of the PDR5 promoter identified a region from -360 to -112 that is essential for expression of this gene. DNase I footprinting analysis using bacterially expressed Pdr3p showed specific recognition by this protein of at least one site in the -360/-112 interval in the PDR5 promoter. A high-copy-number plasmid carrying the PDR3 gene elevated resistance to both oligomycin and cycloheximide. Increasing the number of PDR3 gene copies in a delta pdr5 strain increased oligomycin resistance but was not able to correct the cycloheximide hypersensitivity that results from loss of PDR5. These data are consistent with the notion that PDR3 acts to increase cycloheximide resistance by elevating the level of PDR5 transcription, while PDR3-mediated oligomycin resistance acts through some other target gene.

ATP-Binding Cassette Transporters↗