Differential Use of Myristoyl Groups on Neuronal Calcium Sensor Proteins as a Determinant of Spatio-temporal Aspects of Ca2+ Signal Transduction

O'Callaghan, Dermott W.; Ivings, Lenka; Weiss, Jamie L.; Ashby, Michael C.; Tepikin, Alexei V.; Burgoyne, Robert D.

doi:10.1074/jbc.m111750200

Cited by 133 publications

(168 citation statements)

References 47 publications

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“…In some cases, the membrane association conferred by these moieties is dynamically regulated by Ca 2þ binding when a sequestered mysristoyl chain becomes solvent-exposed following a Ca 2þ -driven shift in conformation as originally described for recoverin (Ames et al 1997). VSNL proteins are also cytosolic at resting [Ca 2þ ] i but localize to the plasma membrane or Golgi complex upon Ca 2þ elevation (O'Callaghan et al 2002;Spilker et al 2002;O'Callaghan et al 2003b). Each of the NCS proteins displays distinct subcellular localizations, which are in part determined by additional interactions with specific phosphoinositides mediated by basic aminoterminal residues immediately proximal to the site of acylation (O'Callaghan et al 2003a;O'Callaghan et al 2005).…”

Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 94%

“…It displays a high Ca 2þ -binding affinity and is able to respond to small fluctuations in [Ca 2þ ] i . NCS-1 is amino-terminally myristoylated and is constitutively associated with membranes including plasma and Golgi membranes (O'Callaghan et al 2002), although in some cell lines, NCS-1 has been found to be partially cytosolic (de Barry et al 2006) and it is able to rapidly exchange between membrane and cytosolic pools (Handley et al 2010). In contrast to all other NCS family members, NCS-1 is not neuron specific and is expressed in neuroendocrine cells (McFerran et al 1998) and at low levels in several nonneuronal cell types.…”

Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 99%

“…Upon binding of Ca 2þ , the amino-terminal domain comprising EF-1 and EF-2 rotates through 458 relative to the carboxy-terminal domain driving extrusion of its buried myristoyl group to permit association with membranes and revealing a hydrophobic surface, which can mediate interaction with the target protein rhodopsin kinase (Ames et al 2006). The residues involved in the interaction of the myristoyl group with the hydrophobic pocket are also conserved in the other members of the NCS family, however not all of the other family members display this Ca 2þ /myristoyl switch (O'Callaghan et al 2002;Stephen et al 2007). NCS-1 and KChIP1 expose a similar hydrophobic surface upon Ca 2þ -binding, which could be similarly important for target interactions (Bourne et al 2001;Scannevin et al 2004;Zhou et al 2004b;Pioletti et al 2006).…”

Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 99%

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The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

McCue

Haynes²,

Burgoyne

2010

Cold Spring Harbor Perspectives in Biology

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Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 94%

Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 99%

Section: Calcium Sensor Proteins In Neuronal Functionmentioning

confidence: 99%

See 1 more Smart Citation

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

McCue

Haynes²,

Burgoyne

2010

Cold Spring Harbor Perspectives in Biology

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“…X-rhod-1 was imaged using the Leica TCS-SP-MP microscope with excitation at 543 nm and light collection at 600 -650 nm. X-rhod-1 was used in the study of PC12 cells rather than Fura Red (22), since the latter dye could not be successfully loaded into PC12 cells, and other dyes were not compatible with measurements in EYFP-expressing cells. The X-rhod-1 fluorescence was diffusely and uniformly distributed, suggesting cytosolic localization of the probe.…”

Section: Methodsmentioning

confidence: 99%

Calcium-binding Protein 1 Is an Inhibitor of Agonist-evoked, Inositol 1,4,5-Trisphosphate-mediated Calcium Signaling

Haynes

Tepikin

Burgoyne

2004

Journal of Biological Chemistry

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117

133

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Calcium is a ubiquitous intracellular second messenger in all vertebrate cells (1). Intracellular resting free Ca 2ϩ concentration ([Ca 2ϩ ] i ) is acutely maintained at around 100 nM via a variety of mechanisms, and even small local rises above this base line are able to elicit dramatic cellular responses. Rises in [Ca 2ϩ ] i are generated through two primary routes (2). First, many physiological agonists activate Ca 2ϩ channels that mediate the direct entry of extracellular Ca 2ϩ . Second, [Ca 2ϩ ] i can be elevated through release of sequestered Ca 2ϩ from intracellular stores (3), most notably the endoplasmic reticulum (ER). 1 Ca 2ϩ release from intracellular stores is controlled predominantly by two related Ca 2ϩ channel families, the inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) and the ryanodine receptor (RyR). InsP 3 Rs couple to extracellular stimuli that drive the activation of phospholipase C and subsequent liberation of the soluble second messenger InsP 3. Activation of ER-localized InsP 3 Rs by InsP 3 in turn elicits Ca 2ϩ release into the cytosol. InsP 3 R activity can be further modulated by direct binding of Ca 2ϩ itself, and this regulation explains a form of calciuminduced calcium release that is responsible for the establishment of complex regenerative Ca 2ϩ signals within cells (2). In neurons, such Ca 2ϩ signals have been intimately linked to processes as divergent as neurodegeneration, neurite outgrowth, alterations in neuronal gene expression, and neurotransmitter release (4 -10). Factors affecting the generation and propagation of intracellular Ca 2ϩ signals therefore represent potential critical integrators of neuronal responses to complex and varied stimuli.Although Ca 2ϩ is capable of eliciting direct cellular responses such as the aforementioned gating of InsP 3 Rs, many of its effects are registered indirectly through specific Ca 2ϩ -binding proteins. Calmodulin (CaM) is a universal Ca 2ϩ -binding protein, homologues of which are present in all eukaryotes (11), and a large number of Ca 2ϩ driven alterations in cell physiology can be mapped to its Ca 2ϩ sensing activity. CaM coordinates Ca 2ϩ ions through specialized EF-hand domains, and a large number of related proteins have now been characterized. In neurons, these include the neuronal calcium sensor (NCS) family of Ca 2ϩ -binding proteins (12). More recently, a group of CaM-like proteins, termed caldendrins (13, 14) or calciumbinding proteins (CaBPs) (15) have been identified that are vertebrate-specific and display a predominantly neuronal/retinal pattern of expression (14 -16). It has since been reported that members of the CaBP family are able to interact with and modulate the activity of InsP 3 Rs (17). Uniquely, it has been suggested that CaBPs may be capable of activating the InsP 3 R independently of the natural ligand ‡ To whom all correspondence should be addressed.

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“…N-terminal myristoylation of NCS-1 is thought to be important for protein structure, cellular localization [14], and function [15]. Myristoylation mediates structural changes in NCS-1 that increase its stability and affect the unfolding pathway of the protein [16].…”

Section: Introductionmentioning

confidence: 99%

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

Cotiis

Woll

Fox

et al. 2008

Protein Expression and Purification

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We have developed a protocol to produce large quantities of high purity myristoylated and nonmyristoylated Neuronal Calcium Sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6X His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100 -200 µM). With optimized growth conditions and a high capacity metal affinity purification scheme, > 50 mg of homogenous myristoylated NCS-1 is obtained from 1 L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 EF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.

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Differential Use of Myristoyl Groups on Neuronal Calcium Sensor Proteins as a Determinant of Spatio-temporal Aspects of Ca2+ Signal Transduction

Cited by 133 publications

References 47 publications

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

Calcium-binding Protein 1 Is an Inhibitor of Agonist-evoked, Inositol 1,4,5-Trisphosphate-mediated Calcium Signaling

Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli

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