Johnnie M. Moore

Saint Louis University

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Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca²⁺-evoked release of catecholamine

AJP Cell Physiology (2006)

Johnnie M. Moore Jason B. Papke Anne L. Cahill Amy B. Harkins

Synaptotagmin (syt) I is a Ca2+-binding protein that is well accepted as a major sensor for Ca2+-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca2+ sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca2+ and stimulated Ca2+ influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca2+ sensor for regulated release of transmitter.

Synaptotagmin 1

Synaptotagmin I

10.1152/ajpcell.00539.2005

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Citations (32)

SCF E3-Mediated Autoubiquitination Negatively Regulates Activity of Cdc34 E2 but Plays a Nonessential Role in the Catalytic Cycle In Vitro and In Vivo

Molecular and Cellular Biology (2007)

K. Matthew Scaglione Parmil K. Bansal Andrew E. Deffenbaugh Alexi Kiss Johnnie M. Moore

One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless K0Cdc34ΔC is indistinguishable from Cdc34ΔC in ubiquitination of the prototype SCFCdc4 substrate Sic1 in vitro, and replacement of the CDC34 gene with either the K0cdc34ΔC or the cdc34ΔC allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.

10.1128/mcb.01555-06

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Citations (21)

Variability in RNA interference in neuroendocrine PC12 cell lines stably transfected with an shRNA plasmid

Journal of Neuroscience Methods (2007)

Anne L. Cahill Johnnie M. Moore Fatima I. Sabar Amy B. Harkins

10.1016/j.jneumeth.2007.07.016

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Citations (4)

Release of Ubiquitin-Charged Cdc34-SUb from the RING Domain Is Essential for Ubiquitination

Andrew E. Deffenbaugh K. Matthew Scaglione Lingxiao Zhang Johnnie M. Moore Tione Buranda

Ubiquitin-Protein Ligases

Ubiquitin-conjugating enzyme

Source

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Potential Deep-Sea Petroleum Source Beds Related to Coastal Upwelling

Springer eBooks (1983)

Jürgen Rullkötter Vassil T. Vuchev K. Hinz Edward L. Winterer Peter O. Baumgartner

10.1007/978-1-4613-3709-6_21

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Citations (15)

Release of Ubiquitin-Charged Cdc34-S∼Ub from the RING Domain Is Essential for Ubiquitination of the SCFCdc4-Bound Substrate Sic1

Cell (2003)

Andrew E. Deffenbaugh K. Matthew Scaglione Lingxiao Zhang Johnnie M. Moore Tione Buranda

Ubiquitin-conjugating enzyme

Ubiquitin-Protein Ligases

Deubiquitinating enzyme

Ring finger

10.1016/s0092-8674(03)00641-x

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Citations (80)

Stable RNA interference of synaptotagmin I in PC12 cells results in differential regulation of transmitter release

AJP Cell Physiology (2007)

William H. Roden Jason B. Papke Johnnie M. Moore Anne L. Cahill Heather Macarthur

In sympathetic neurons, it is well-established that the neurotransmitters, norepinephrine (NE), neuropeptide Y (NPY), and ATP are differentially coreleased from the same neurons. In this study, we determined whether synaptotagmin (syt) I, the primary Ca(2+) sensor for regulated release, could function as the protein that differentially regulates release of these neurotransmitters. Plasmid-based RNA interference was used to specifically and stably silence expression of syt I in a model secretory cell line. Whereas stimulated release of NPY and purines was abolished, stimulated catecholamine (CA) release was only reduced by approximately 50%. Although expression levels of tyrosine hydroxylase, the rate-limiting enzyme in the dopamine synthesis pathway, was unaffected, expression of the vesicular monoamine transporter 1 was reduced by 50%. To evaluate whether NPY and CAs are found within the same vesicles and whether syt I is found localized to each of these NPY- and CA-containing vesicles, we used immunocytochemistry to determine that syt I colocalized with large dense core vesicles, with NPY, and with CAs. Furthermore, both CAs and NPY colocalized with one another and with large dense core vesicles. Electron micrographs show that large dense core vesicles are synthesized and available for release in cells that lack syt I. These results are consistent with syt I regulating differential release of transmitters.

Synaptotagmin 1

Synaptotagmin I

Monoaminergic

10.1152/ajpcell.00482.2006

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Citations (11)