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Daniel R. Storm - One of the best experts on this subject based on the ideXlab platform.
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Neuromodulin (GAP43): A Neuronal Protein Kinase C Substrate Is Also Present in 0-2A Glial Cell Lineage. Characterization of Neuromodulin in Secondary Cultures of Oligodendrocytes and Comparison with the Neuronal Antigen
2013Co-Authors: Jeanchristophe Deloulme, Daniel R. Storm, Thierry Janet, Monique Sensenbrenner, Jacques BaudierAbstract:pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of Neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellateshaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that Neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of Neuromodulin i
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The Prooncoprotein EWS Binds Calmodulin and Is Phosphorylated by Protein Kinase C through an IQ Domain
The Journal of biological chemistry, 1997Co-Authors: Jeanchristophe Deloulme, Lisa Prichard, Olivier Delattre, Daniel R. StormAbstract:Abstract A growing family of proteins is regulated by protein kinase C and calmodulin through IQ domains, a regulatory motif originally identified in Neuromodulin (Alexander, K. A., Wakim, B. T., Doyle, G. S., Walsh, K. A., and Storm, D. R. (1988) J. Biol. Chem. 263, 7544–7549). Here we report that EWS, a nuclear RNA-binding prooncoprotein, contains an IQ domain, is phosphorylated by protein kinase C, and interacts with calmodulin. Interestingly, PKC phosphorylation of EWS inhibits its binding to RNA homopolymers, and conversely, RNA binding to EWS interferes with PKC phosphorylation. Several other RNA-binding proteins, including TLS/FUS and PSF, co-purify with EWS. PKC phosphorylation of these proteins also inhibits their binding to RNAin vitro. These data suggest that PKC may regulate interactions of EWS and other RNA-binding proteins with their RNA targets and that IQ domains may provide a regulatory link between Ca2+ signal transduction pathways and RNA processing.
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Dynamic palmitoylation of Neuromodulin (GAP-43) in cultured rat cerebellar neurons and mouse N1E-115 cells
Neuroscience Letters, 1997Co-Authors: Lauren P. Baker, Daniel R. StormAbstract:We conducted pulse-chase and metabolic labeling experiments to determine directly whether palmitoylation of Neuromodulin in neurons is dynamic, and if acylation is regulated. The rates of turnover of Neuromodulin protein and associated palmitoyl groups were quantified using cultured cerebellar granule neurons and the neuronal cell line N1E-115. The half-life of [3H]palmitate bound to Neuromodulin was approximately 5 h, whereas the half-life of the [35S]methionine-labeled Neuromodulin was greater than 50 h. Metabolic and pulse-chase labeling experiments were carried out in the presence of various activators of cellular signaling pathways. Our data indicate that dynamic acylation and deacylation of Neuromodulin in neurons are constitutive and are not regulated by G protein activation or other signals that control growth cone dynamics.
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Analysis of the palmitoylation and membrane targeting domain of Neuromodulin (GAP-43) by site-specific mutagenesis.
Biochemistry, 1993Co-Authors: Yuechueng Liu, Daniel A. Fisher, Daniel R. StormAbstract:Neuromodulin (GAP-43) is a neurospecific calmodulin binding protein which is implicated in neuronal growth and regeneration. It is concentrated in neuronal growth cones and associates with membranes through the palmitoylation of the N-terminal peptide MLCCMRRTK at Cys-3 and Cys-4. In the present study, we have identified critical amino acid residues required for palmitoylation and membrane association of Neuromodulin in vivo. Several Neuromodulin constructs with point mutations were tested for membrane association and palmitoylation. Wild-type Neuromodulin expressed in COS-7 cells incorporated [3H]palmitic acid, whereas a mutant in which both Cys-3 and Cys-4 were substituted with glycine was not palmitoylated in vivo. Mutant proteins in which either Cys-3 or Cys-4 was substituted with leucine incorporated 75% and 25% of [3H]palmitic acid, respectively, compared to wild-type Neuromodulin. The relative distribution of mutant Neuromodulins expressed in COS-7 cells was quantitated by immunoblot analysis of the membrane and cytosolic fractions. There was a general correlation between membrane association of mutant Neuromodulins and the extent to which they were palmitoylated in vivo. Additional point mutations in the acylation domain of Neuromodulin indicated that a short hydrophobic amino acid sequence N-terminal to Cys-4 may be required for optimal palmitoylation and membrane association. We conclude that Cys-4 is critical for the palmitoylation and membrane association of Neuromodulin.
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Palmitylation of Neuromodulin (GAP-43) is not required for phosphorylation by protein kinase C.
The Journal of biological chemistry, 1992Co-Authors: Edwin R. Chapman, Roger P. Estep, Daniel R. StormAbstract:Abstract Neuromodulin (also designated GAP-43, B-50, and F-1) is a prominent protein kinase C substrate attached to the membranes of neuronal growth cones during development and to presynaptic membranes in discrete subsets of adult synapses. In this study, we have examined the relationship between the attachment of Neuromodulin to membranes and its phosphorylation by protein kinase C. To address this issue, we have compared wild-type and mutant Neuromodulins expressed in cells that normally lack the protein. Wild-type Neuromodulin expressed in Chinese hamster ovary cells was associated with membranes, incorporated [3H]palmitic acid, and was phosphorylated in response to phorbol ester treatment. Substitution of serine 41, the in vitro protein kinase C site, abolished the phorbol ester response, indicating that serine 41 serves as the sole protein kinase C phosphorylation site in vivo. Substitution of the putative fatty acylation sites, cysteines 3 and 4, abolished membrane association as well as [3H]palmitic acid labeling of Neuromodulin. Fatty acylation therefore appears to serve as the mechanism for anchoring Neuromodulin to membranes. Surprisingly, the soluble cysteine substitution mutant was phosphorylated by protein kinase C at a rate indistinguishable from that of the wild-type protein. Therefore, membrane association may not be required for the phosphorylation of Neuromodulin by protein kinase C.
Jeanchristophe Deloulme - One of the best experts on this subject based on the ideXlab platform.
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Neuromodulin (GAP43): A Neuronal Protein Kinase C Substrate Is Also Present in 0-2A Glial Cell Lineage. Characterization of Neuromodulin in Secondary Cultures of Oligodendrocytes and Comparison with the Neuronal Antigen
2013Co-Authors: Jeanchristophe Deloulme, Daniel R. Storm, Thierry Janet, Monique Sensenbrenner, Jacques BaudierAbstract:pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of Neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellateshaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that Neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of Neuromodulin i
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The Prooncoprotein EWS Binds Calmodulin and Is Phosphorylated by Protein Kinase C through an IQ Domain
The Journal of biological chemistry, 1997Co-Authors: Jeanchristophe Deloulme, Lisa Prichard, Olivier Delattre, Daniel R. StormAbstract:Abstract A growing family of proteins is regulated by protein kinase C and calmodulin through IQ domains, a regulatory motif originally identified in Neuromodulin (Alexander, K. A., Wakim, B. T., Doyle, G. S., Walsh, K. A., and Storm, D. R. (1988) J. Biol. Chem. 263, 7544–7549). Here we report that EWS, a nuclear RNA-binding prooncoprotein, contains an IQ domain, is phosphorylated by protein kinase C, and interacts with calmodulin. Interestingly, PKC phosphorylation of EWS inhibits its binding to RNA homopolymers, and conversely, RNA binding to EWS interferes with PKC phosphorylation. Several other RNA-binding proteins, including TLS/FUS and PSF, co-purify with EWS. PKC phosphorylation of these proteins also inhibits their binding to RNAin vitro. These data suggest that PKC may regulate interactions of EWS and other RNA-binding proteins with their RNA targets and that IQ domains may provide a regulatory link between Ca2+ signal transduction pathways and RNA processing.
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Noradrenergic, but not Adrenergic Chromaffin Cells in the Adrenal Gland Express Neuromodulin (GAP-43).
The European journal of neuroscience, 1992Co-Authors: Nancy J. Grant, Jeanchristophe Deloulme, Fabienne König, Dominique Aunis, Keith LangleyAbstract:Neuroendocrine chromaffin cells of the adrenal gland express certain molecular markers either transiently during development or permanently. In the present study, the expression of Neuromodulin (GAP-43), a neuronal protein often associated with neurite outgrowth, was examined in adult adrenals. Neuromodulin was detected by Western blot analysis in extracts of both rat adrenals and cultured bovine chromaffin cells, and was localized in situ in a subpopulation of chromaffin cells, as well as in nerve fibres and Schwann cells. The use of anti-tyrosine hydroxylase or anti-phenylethanolamine N-methyltransferase antibodies in combination with anti-Neuromodulin antibodies in double immunofluorescent labelling of cryostat sections of rat glands demonstrated that Neuromodulin is expressed by noradrenergic, and not by adrenergic chromaffin cells. The results provide further evidence that Neuromodulin is not limited to neurons; it is also expressed in a subpopulation of neuroendocrine chromaffin cells. Neuromodulin may play a role in the development of the adrenal medulla or in the specific regulation of noradrenalin secretion from chromaffin cells.
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The interactions of the brain-specific calmodulin-binding protein kinase C substrate, Neuromodulin (GAP 43), with membrane phospholipids.
The Journal of biological chemistry, 1991Co-Authors: D Houbre, Jeanchristophe Deloulme, Guy Duportail, Jacques BaudierAbstract:The interaction of the brain-specific calmodulin-binding protein kinase C (PKC) substrate, Neuromodulin (GAP 43), with membrane phospholipids was studied. Specific binding of Neuromodulin to negatively charged phospholipids through electrostatic interactions was demonstrated. Comparison of the binding of Neuromodulin to acidic phospholipids with that of neurogranin, a newly characterized calmodulin-binding PKC substrate (Baudier J., Deloulme, J. C., Van Dorsselaer, A., Black, D., and Mathes H. (1991) J. Biol. Chem. 266, 229-237) suggested that the conserved basic amino acid sequence which characterizes the two proteins and which corresponds to the PKC phosphorylation and calmodulin binding domain also serves as phospholipid binding site. In the absence of calmodulin, binding of Neuromodulin to phosphatidylserine at low concentration parallels its phosphorylation by PKC, suggesting that formation of a ternary complex between Neuromodulin, phosphatidylserine, and PKC is required for optimum Neuromodulin phosphorylation. In the presence of calmodulin, the binding of Neuromodulin to phosphatidylserine is inhibited, resulting in total inhibition of Neuromodulin phosphorylation. Our results suggest that, in vivo, phosphorylation of Neuromodulin may not only depend on protein kinase C (PKC)1 activation but also on the accessibility of the Neuromodulin phosphorylation domain to activated membrane-bound PKC that could regulated by CaM.
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purification and characterization of a brain specific protein kinase c substrate neurogranin p17 identification of a consensus amino acid sequence between neurogranin and Neuromodulin gap43 that corresponds to the protein kinase c phosphorylation sit
Journal of Biological Chemistry, 1991Co-Authors: Jacques Baudier, Jeanchristophe Deloulme, Alain Dorsselaer, D. Black, Hans W D MatthesAbstract:Abstract Neurogranin, formerly designated p17 (Baudier, J., Bronner, C., Kligman, D., and Cole, R. D.) (1989) J. Biol. Chem. 264, 1824-1828), a brain-specific in vitro substrate for protein kinase C (PKC), has been purified to homogeneity from bovine forebrain. The purified protein has a molecular mass of 7837.1 +/- 0.5 Da, determined by electrospray mass spectrometry. In the absence of reducing agent, dimers and higher oligomers accumulated. On sodium dodecyl sulfate-polyacrylamide gels the protein monomer migrated abnormally with an apparent molecular mass of 15,000-19,000 Da, depending on the percentage of polyacrylamide. The native protein is blocked at its amino terminus. The majority of the primary amino acid sequence was determined following proteolytic and chemical fragmentation. A comparison of the amino acid sequence of neurogranin with that of the brain-specific PKC substrate Neuromodulin, revealed a strikingly conserved amino acid sequence AA(X)KIQA-SFRGH(X)(X)RKK(X)K. The two proteins are not related over the rest of their sequences. Neurogranin was shown to be phosphorylated in hippocampal slices incubated with 32Pi and phorbol esters stimulated neurogranin phosphorylation, suggesting that neurogranin is likely to be an in vivo substrate for PKC. In vitro phosphorylation of neurogranin by PKC produced a shift of the isoelectric point of the protein (pI 5.6) to a more acidic value (pI 5.4). Tryptic digestion of the phosphorylated protein yielded a single phosphopeptide having the sequence IQASFR, where the serine residue is the phosphorylated amino acid. This phosphopeptide is part of the conserved sequence shared with Neuromodulin and also corresponds to the PKC phosphorylation site on Neuromodulin (Apel, E. D., Byford, M. F., Au, D., Walsh, K. A., and Storm, D. R. (1990) Biochemistry 29, 2330-2335). Evidence was obtained suggesting that neurogranin binds to calmodulin in the absence of Ca2+, a feature that also characterizes Neuromodulin. We propose that the amino acid sequence shared by neurogranin and Neuromodulin reflects a functional relationship between these two proteins and that the consensus sequence represents a conserved PKC phosphorylation site and a calmodulin binding domain that characterizes a class of brain-specific PKC substrates.
Jacques Baudier - One of the best experts on this subject based on the ideXlab platform.
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Neuromodulin (GAP43): A Neuronal Protein Kinase C Substrate Is Also Present in 0-2A Glial Cell Lineage. Characterization of Neuromodulin in Secondary Cultures of Oligodendrocytes and Comparison with the Neuronal Antigen
2013Co-Authors: Jeanchristophe Deloulme, Daniel R. Storm, Thierry Janet, Monique Sensenbrenner, Jacques BaudierAbstract:pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of Neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellateshaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that Neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of Neuromodulin i
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The interactions of the brain-specific calmodulin-binding protein kinase C substrate, Neuromodulin (GAP 43), with membrane phospholipids.
The Journal of biological chemistry, 1991Co-Authors: D Houbre, Jeanchristophe Deloulme, Guy Duportail, Jacques BaudierAbstract:The interaction of the brain-specific calmodulin-binding protein kinase C (PKC) substrate, Neuromodulin (GAP 43), with membrane phospholipids was studied. Specific binding of Neuromodulin to negatively charged phospholipids through electrostatic interactions was demonstrated. Comparison of the binding of Neuromodulin to acidic phospholipids with that of neurogranin, a newly characterized calmodulin-binding PKC substrate (Baudier J., Deloulme, J. C., Van Dorsselaer, A., Black, D., and Mathes H. (1991) J. Biol. Chem. 266, 229-237) suggested that the conserved basic amino acid sequence which characterizes the two proteins and which corresponds to the PKC phosphorylation and calmodulin binding domain also serves as phospholipid binding site. In the absence of calmodulin, binding of Neuromodulin to phosphatidylserine at low concentration parallels its phosphorylation by PKC, suggesting that formation of a ternary complex between Neuromodulin, phosphatidylserine, and PKC is required for optimum Neuromodulin phosphorylation. In the presence of calmodulin, the binding of Neuromodulin to phosphatidylserine is inhibited, resulting in total inhibition of Neuromodulin phosphorylation. Our results suggest that, in vivo, phosphorylation of Neuromodulin may not only depend on protein kinase C (PKC)1 activation but also on the accessibility of the Neuromodulin phosphorylation domain to activated membrane-bound PKC that could regulated by CaM.
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purification and characterization of a brain specific protein kinase c substrate neurogranin p17 identification of a consensus amino acid sequence between neurogranin and Neuromodulin gap43 that corresponds to the protein kinase c phosphorylation sit
Journal of Biological Chemistry, 1991Co-Authors: Jacques Baudier, Jeanchristophe Deloulme, Alain Dorsselaer, D. Black, Hans W D MatthesAbstract:Abstract Neurogranin, formerly designated p17 (Baudier, J., Bronner, C., Kligman, D., and Cole, R. D.) (1989) J. Biol. Chem. 264, 1824-1828), a brain-specific in vitro substrate for protein kinase C (PKC), has been purified to homogeneity from bovine forebrain. The purified protein has a molecular mass of 7837.1 +/- 0.5 Da, determined by electrospray mass spectrometry. In the absence of reducing agent, dimers and higher oligomers accumulated. On sodium dodecyl sulfate-polyacrylamide gels the protein monomer migrated abnormally with an apparent molecular mass of 15,000-19,000 Da, depending on the percentage of polyacrylamide. The native protein is blocked at its amino terminus. The majority of the primary amino acid sequence was determined following proteolytic and chemical fragmentation. A comparison of the amino acid sequence of neurogranin with that of the brain-specific PKC substrate Neuromodulin, revealed a strikingly conserved amino acid sequence AA(X)KIQA-SFRGH(X)(X)RKK(X)K. The two proteins are not related over the rest of their sequences. Neurogranin was shown to be phosphorylated in hippocampal slices incubated with 32Pi and phorbol esters stimulated neurogranin phosphorylation, suggesting that neurogranin is likely to be an in vivo substrate for PKC. In vitro phosphorylation of neurogranin by PKC produced a shift of the isoelectric point of the protein (pI 5.6) to a more acidic value (pI 5.4). Tryptic digestion of the phosphorylated protein yielded a single phosphopeptide having the sequence IQASFR, where the serine residue is the phosphorylated amino acid. This phosphopeptide is part of the conserved sequence shared with Neuromodulin and also corresponds to the PKC phosphorylation site on Neuromodulin (Apel, E. D., Byford, M. F., Au, D., Walsh, K. A., and Storm, D. R. (1990) Biochemistry 29, 2330-2335). Evidence was obtained suggesting that neurogranin binds to calmodulin in the absence of Ca2+, a feature that also characterizes Neuromodulin. We propose that the amino acid sequence shared by neurogranin and Neuromodulin reflects a functional relationship between these two proteins and that the consensus sequence represents a conserved PKC phosphorylation site and a calmodulin binding domain that characterizes a class of brain-specific PKC substrates.
Edwin R. Chapman - One of the best experts on this subject based on the ideXlab platform.
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Palmitylation of Neuromodulin (GAP-43) is not required for phosphorylation by protein kinase C.
The Journal of biological chemistry, 1992Co-Authors: Edwin R. Chapman, Roger P. Estep, Daniel R. StormAbstract:Abstract Neuromodulin (also designated GAP-43, B-50, and F-1) is a prominent protein kinase C substrate attached to the membranes of neuronal growth cones during development and to presynaptic membranes in discrete subsets of adult synapses. In this study, we have examined the relationship between the attachment of Neuromodulin to membranes and its phosphorylation by protein kinase C. To address this issue, we have compared wild-type and mutant Neuromodulins expressed in cells that normally lack the protein. Wild-type Neuromodulin expressed in Chinese hamster ovary cells was associated with membranes, incorporated [3H]palmitic acid, and was phosphorylated in response to phorbol ester treatment. Substitution of serine 41, the in vitro protein kinase C site, abolished the phorbol ester response, indicating that serine 41 serves as the sole protein kinase C phosphorylation site in vivo. Substitution of the putative fatty acylation sites, cysteines 3 and 4, abolished membrane association as well as [3H]palmitic acid labeling of Neuromodulin. Fatty acylation therefore appears to serve as the mechanism for anchoring Neuromodulin to membranes. Surprisingly, the soluble cysteine substitution mutant was phosphorylated by protein kinase C at a rate indistinguishable from that of the wild-type protein. Therefore, membrane association may not be required for the phosphorylation of Neuromodulin by protein kinase C.
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Characterization of the calmodulin binding domain of Neuromodulin. Functional significance of serine 41 and phenylalanine 42.
The Journal of biological chemistry, 1991Co-Authors: Edwin R. Chapman, Kenneth A Alexander, Teresa A. Nicolson, Daniel R. StormAbstract:Abstract Neuromodulin (also designated P-57, GAP-43, B-50) is a major presynaptic substrate for protein kinase C. Phosphorylation of Neuromodulin decreases its affinity for calmodulin, suggesting that Neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons, releasing calmodulin locally in response to phosphorylation by protein kinase C (Alexander, K. A., Cimler, B. M., Meier, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). In the present study, we have constructed and characterized several mutant Neuromodulins to demonstrate that the amino acid sequence 39-56 is required for calmodulin binding, and that this domain contains the sole in vitro protein kinase C phosphorylation site at serine 41. We also demonstrate that the adjacent phenylalanine 42, interacts hydrophobically with calmodulin. These hydrophobic interactions may be disrupted by the introduction of negative charge at serine 41, and thereby regulate the Neuromodulin/calmodulin binding interactions. The sensitivity of the Neuromodulin/calmodulin binding interaction to negative charge at serine 41 was determined by substitution of serine 41 with an aspartate or an asparagine residue. The asparagine mutant retained its affinity for calmodulin-Sepharose while the aspartate mutant did not adsorb to calmodulin-Sepharose. We conclude that protein kinase C phosphorylation of Neuromodulin abolishes calmodulin binding by introducing negative charges within the calmodulin binding domain at a position adjacent to the phenylalanine.
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Targeting of Neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.
Neuron, 1991Co-Authors: Yuechueng Liu, Edwin R. Chapman, Daniel R. StormAbstract:Abstract Neuromodulin (GAP-43) is a membrane protein that is transported to neuronal growth cones. Zuber and co-workers have proposed that the N-terminal 10 amino acid sequence of Neuromodulin is sufficient to target proteins to growth cones. We demonstrate that a Neuromodulin(β-galactosidase fusion protein is transported to growth cones of cultured rat neurons, whereas a fusion protein containing the N-terminal 10 amino acids of Neuromodulin and (β-galactosidase is not. A mutant Neuromodulin lacking cysteines 3 and 4, the palmitylation sites required for membrane attachment, does not target β-galactosi-dase to growth cones. We conclude that membrane attachment is required for growth cone accumulation and that structural elements, in addition to the first 10 amino acids of Neuromodulin, may be required for growth cone targeting.
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Mutagenesis of the calmodulin binding domain of Neuromodulin.
Progress in brain research, 1991Co-Authors: Edwin R. Chapman, Teresa A. Nicolson, Daniel R. StormAbstract:Publisher Summary This chapter focuses on biochemical properties of Neuromodulin that may be important for its function in neurons. Two interesting biochemical properties that may be important for the physiological function of Neuromodulin are its affinity for CaM and the effect of protein kinase C phosphorylation on CaM interactions. The concentration of Neuromodulin in the brain and the affinity of the protein for CaM in the absence of free calcium are sufficient to complex the majority of CaM present. Consequently, interactions between Neuromodulin and CaM as well as the regulation of CaM binding by protein kinase C phosphorylation are of considerable interest. The protein kinase C phosphorylation site of Neuromodulin is serine-41. This phosphorylation significantly reduces the affinity of Neuromodulin for CaM, suggesting that the introduction of negative charge may abolish CaM binding. The chapter addresses this issue by substituting serine-41 with an aspartate or an asparagines residue. The aspartate-41 mutant Neuromodulin did not bind to CaM-Sepharose. In contrast, the asparagine-41 mutant bound to CaM-Sepharose in a manner indistinguishable from the wild type protein.
David S. Cafiso - One of the best experts on this subject based on the ideXlab platform.
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Defining protein-protein interactions using site-directed spin-labeling: the binding of protein kinase C substrates to calmodulin
Biochemistry, 1996Co-Authors: Zhihai Qin, Stacey L. Wertz, Yoko Savino, Jaison Jacob, David S. CafisoAbstract:EPR spectroscopy was used to examine protein-protein interactions between calmodulin and spin-labeled peptides based on the protein kinase C substrate domains of the myristoylated alanine rich C kinase substrate (MARCKS) and Neuromodulin. When bound to calmodulin, the C- and N-terminal ends of a 25 residue MARCKS derived peptide exhibited large amplitude motion on the nanosecond time scale and were accessible to paramagnetic agents in aqueous solution. However, residues 5-23 were highly protected and in contact with side chains from calmodulin. These data are consistent with an alpha-helical configuration for this segment of MARCKS and with structures that have been obtained for other calmodulin-substrate complexes. For the 17 residue Neuromodulin derived peptide, which is Ca2+ independent in its binding to calmodulin, oxygen collision rates demonstrate that one helical face of this peptide interacts strongly with calmodulin. The data are consistent with an interaction of this face specifically with the C-terminal lobe of calmodulin, where this lobe is either in an "open" or "semiopen" configuration. The EPR data also indicate that the N-terminal lobe of calmodulin is in contact with the peptide, but that this lobe is not as strongly associated with the peptide target. Overall, the binding pocket for Neuromodulin appears to be less compact and more dynamic than that formed by MARCKS. This behavior has not previously been seen for calmodulin substrates, and it may play a role in the Ca2+ independent binding of this class of substrates. This work demonstrates the utility of EPR spectroscopy to define protein-protein interactions; in addition, oxygen collision frequencies obtained at buried sites appear to provide information on the conformational dynamics of proteins.
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Solution and membrane bound structure of a peptide derived from the protein kinase C substrate domain of Neuromodulin.
Biochemistry, 1996Co-Authors: Stacey L. Wertz, Yoko Savino, David S. CafisoAbstract:The solution, micelle, and membrane bound structure of a peptide based on the protein kinase C and calmodulin binding domain of Neuromodulin was studied using a combination of NMR, EPR, and circular dichroism. NMR spectroscopy on this peptide indicates that there is little secondary structure in aqueous solution or detergent micelles, but that the peptide is helical in methanol. This finding is in agreement with EPR experiments utilizing double spin-labeled derivatives of the peptide as well as circular dichroism. The membrane bound structure of this peptide was investigated with EPR by synthesizing a series of spin-labeled peptides based on the protein kinase C and calmodulin binding domain of Neuromodulin. These peptides exhibit no binding to neutral membranes containing phosphatidylcholine, but associate strongly with membranes containing negatively charged lipids such as phosphatidylserine. The depth of penetration of the spin label was estimated using continuous wave power-saturation EPR and demonstr...