Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43.

Sudo, Yasuhiko; Valenzuela, Dario; Ag, Beck-Sickinger; Fishman, Mark C.; Strittmatter, Stephen M.

doi:10.1002/j.1460-2075.1992.tb05268.x

Cited by 111 publications

(72 citation statements)

References 51 publications

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“…These remain to be elucidated. Although best characterized for promoting membrane association (30), palmitoylation also affects trafficking of proteins (39), accessibility of proteins to kinases (40), and protein-protein interactions (41,42). It was reported recently that the GAP activity of RGS4 and other RGS proteins is inhibited in vitro by palmitoylation of G i␣ (43).…”

Section: Discussionmentioning

confidence: 99%

Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

Srinivasa

Bernstein

Blumer

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

139

154

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RGS4, a mammalian GTPase activating protein for G protein ␣ subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae. To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system. Deletion of the N-terminal 33 aa of RGS4 (⌬1-33) yielded a nonfunctional protein and loss of plasma membrane localization. These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (⌬1-33). Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling. Fusion of the N-terminal 33 aa of RGS4 to green f luorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain. RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation. Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did not affect plasma membrane localization in yeast or the ability to inhibit signaling. Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast.Heterotrimeric G proteins couple receptors for hormones, neurotransmitters, and sensory signals to intracellular effector molecules, thereby eliciting cellular responses (1, 2). Guanine nucleotide exchange and hydrolysis on the G protein ␣ subunit drives the cycle of activation and deactivation of these signaling pathways. The duration of G protein-mediated responses is subject to the intrinsic GTPase rate of the G protein ␣ subunit, but is also modulated by extrinsic factors. A recently appreciated form of regulation has come from the discovery that members of a protein family called regulators of G protein signaling, or RGS proteins, stimulate the rate of GTP hydrolysis by G protein ␣ subunits (3-5). RGS proteins are found in species ranging from yeast to mammals and constitute a family of at least 20 mammalian proteins (6-8).All RGS family members share sequence similarity that extends over approximately 130 aa, separated in some cases by insertions of varying length (9-11). This conserved RGS domain is sufficient to stimulate GTPase activity of G protein ␣ subunits in vitro (12)(13)(14). Expression of the RGS homology domain of RET-RGS1 or RGS4 yields a recombinant protein that is a functional GAP (GTPase-activating protein). In the crystal structure of RGS4 bound to AlF 4 Ϫ -activated G i␣1 , only the core domain is visible (15). The RGS homology domain binds to the switch regions of G i␣1 and appears to catalyze GTP hydrolysis by stabilizing the switch regions of the G protein in a conformation that favors the transition state of the reactants (14, 15). Sequences outside the RGS homology domain exhibit considerable diversity among family members.Although rapid progress has been made in dissecting the biochemical mechanism by which RGS proteins regulate G protein a...

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Section: Discussionmentioning

confidence: 99%

Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

Srinivasa

Bernstein

Blumer

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

139

154

View full text Add to dashboard Cite

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“…Thus, the presence of FIMs in proteins such as GAP-43 and paralemmin that regulate process outgrowth strongly suggest functional relevance. Previous studies indicate that the first 10 amino acids of GAP-43 are sufficient to induce filopodia and to stimulate G o (Strittmatter et al, 1990;Sudo et al, 1992;Arni et al, 1998). Surprisingly, synthetic di-palmitoylated peptides, which include the N-terminal sequences of GAP-43 abolished G o activity (Strittmatter et al, 1994a).…”

Section: Possible Mechanisms For Induction Of Process Outgrowth By Fimsmentioning

confidence: 95%

Regulation of Dendritic Branching and Filopodia Formation in Hippocampal Neurons by Specific Acylated Protein Motifs

Gauthier-Campbell

Bredt

Murphy

et al. 2004

MBoC

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Although neuronal axons and dendrites with their associated filopodia and spines exhibit a profound cell polarity, the mechanism by which they develop is largely unknown. Here, we demonstrate that specific palmitoylated protein motifs, characterized by two adjacent cysteines and nearby basic residues, are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and the branching of dendrites and axons in neurons. Such motifs are present at the N-terminus of GAP-43 and the C-terminus of paralemmin, two neuronal proteins implicated in cytoskeletal organization and filopodial outgrowth. Filopodia induction is blocked by mutations of the palmitoylated sites or by treatment with 2-bromopalmitate, an agent that inhibits protein palmitoylation. Moreover, overexpression of a constitutively active form of ARF6, a GTPase that regulates membrane cycling and dendritic branching reversed the effects of the acylated protein motifs. Filopodia induction by the specific palmitoylated motifs was also reduced upon overexpression of a dominant negative form of the GTPase cdc42. These results demonstrate that select dually lipidated protein motifs trigger changes in the development and growth of neuronal processes. INTRODUCTIONNeurons possess an elaborate plasma membrane architecture that includes axons, dendrites, and synaptic sites (Da Silva and Dotti, 2002). Modulation of the plasma membrane shape and composition regulate processes outgrowth, axonal development, dendritic branching, and the construction of synapses (Jontes and Smith, 2000). These changes are regulated by interactions between the constituents of the plasma membrane, the exo/endocytic vesicles, and the cytoskeleton (Wood and Martin, 2002). In nonneuronal cells, differential modulation of membrane flow can result in the formation of processes such as microspikes, lamellopodia, and filopodia (Wood and Martin, 2002). However, factors that regulate the formation and maintenance of specific classes of membranous processes in neuronal cells remain poorly understood.Dendritic filopodia, cell surface extensions filled with tight parallel bundles of actin filaments, are thought to be precursors for developing synapses (Dailey and Smith, 1996;Small, 1988). Filopodia are typically 5-35 m in length and rapidly alter their position and shape. In developing axons, filopodia occur at the tips of growth cones and may aid the axon in finding its appropriate target. In dendrites, filopodia act as precursors to spines, short bulbous protrusions of the dendrite that form excitatory postsynaptic contacts on many neurons (Jontes and Smith, 2000). Thus, changes in membrane structure that result in the formation of filopodia are likely important in axonal guidance and spine formation. In contrast, dendritic branching is thought to be mediated via an actin and/or microtubule-dependent mechanisms (Jan and Jan, 2003). Recent studies showed that in early neuronal development dendrites originate from lamellopodia and short neurites (Jan and Jan, ...

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“…In fact, a palmitoyl transferase has recently been purified (Lui et al, 1996). Nonenzymatic palmitoylation can also occur in vitro (O'Brien et al, 1987;Ross and Braun, 1988;Sudo et al, 1992;Duncan and Gilman, 1996). In studies of Ga, protein (Duncan and Gilman, 1996) and proteolipid protein (Bizzozero et al, 1987), the cysteine residues that are nonenzymatically palmitoylated in vitro are the same residues that are palmitoylated in vivo.…”

Section: Introductionmentioning

confidence: 99%

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

Caron

1997

MBoC

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It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(a,3)-methylenediphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.

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Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43.

Cited by 111 publications

References 51 publications

Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae

Regulation of Dendritic Branching and Filopodia Formation in Hippocampal Neurons by Specific Acylated Protein Motifs

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

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