Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Resh, Marilyn D.

doi:10.1016/j.ymeth.2006.04.013

Cited by 146 publications

(141 citation statements)

References 48 publications

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“…To confirm this finding, we treated COS7 cells transfected with CASP6 with 2-bromopalmitate (2-BP), an irreversible inhibitor of PAT activity. 40 Treatment of cells with increasing concentrations of 2-BP resulted in a dose-dependent reduction in palmitoylation (Figure 1b), confirming that CASP6 is indeed palmitoylated. Next, to determine whether HIP14 palmitoylates CASP6, we co-transfected COS7 cells with CASP6 and HIP14.…”

Section: Resultssupporting

confidence: 53%

Palmitoylation of caspase-6 by HIP14 regulates its activation

et al. 2016

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Caspase-6 (CASP6) has an important role in axonal degeneration during neuronal apoptosis and in the neurodegenerative diseases Alzheimer and Huntington disease. Decreasing CASP6 activity may help to restore neuronal function in these and other diseases such as stroke and ischemia, where increased CASP6 activity has been implicated. The key to finding approaches to decrease CASP6 activity is a deeper understanding of the mechanisms regulating CASP6 activation. We show that CASP6 is posttranslationally palmitoylated by the palmitoyl acyltransferase HIP14 and that the palmitoylation of CASP6 inhibits its activation. Palmitoylation of CASP6 is decreased both in Hip14 −/− mice, where HIP14 is absent, and in YAC128 mice, a model of Huntington disease, where HIP14 is dysfunctional and where CASP6 activity is increased. Molecular modeling suggests that palmitoylation of CASP6 may inhibit its activation via steric blockage of the substrate-binding groove and inhibition of CASP6 dimerization, both essential for CASP6 function. Our studies identify palmitoylation as a novel CASP6 modification and as a key regulator of CASP6 activity.

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Section: Resultssupporting

confidence: 53%

Palmitoylation of caspase-6 by HIP14 regulates its activation

et al. 2016

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“…2B). Functionally, pretreatment of HEK293 cells with the palmitoyl transferase inhibitor 2-bromopalmitate [2-BP (27)] also abolished membrane targeting of the STREX C terminus and CRD fusion proteins in the imaging assays (Fig. 2C) as for the palmitoylation-deficient C12:13A mutant.…”

Section: Strex Insert Is a Membrane-anchoring Domain Of The Cytosolic Cmentioning

confidence: 99%

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

Tian

Jeffries

McClafferty

et al. 2008

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

105

180

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Large conductance calcium-and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the poreforming ␣-subunits. However, the molecular mechanism(s) by which phosphorylation of the ␣-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single ␣-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.L arge conductance calcium-and voltage-gated potassium (BK) channels are potently regulated by protein phosphorylation (1) and are important determinants of neuronal, cardiovascular, endocrine, and epithelial function where channel dysfunction may lead to major disorders such as hypertension (2, 3), ataxia (4), epilepsy (5, 6), and incontinence (7). BK channels are potently regulated by phosphorylation, and several putative phosphorylation motifs on the pore-forming ␣-subunit have been identified (8-12). However, as for other potassium channels, the molecular basis through which phosphorylation of the ␣-subunit effects changes in BK channel activity is essentially unknown.BK channel pore-forming ␣-subunits are encoded by a single gene, KCNMA1 (13), and native BK channels show functional heterogeneity in their response to protein kinase A (PKA)-mediated phosphorylation. This diversity results, in large part, from the extensive alternative pre-mRNA splicing of the pore-forming ␣-subunits (10, 12). Previous studies have demonstrated that PKA phosphorylation of a conserved C-terminal phosphorylation motif. RQPS 899 results in BK channel activation (9,10,14). Inclusion of ...

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“…Traditionally, the main options for detection have been metabolic labelling with radiolabelled fatty acids or direct mass spectrometry of fatty-acylated proteins; both have significant disadvantages and limitations [134].…”

Section: Approaches For the Analysis Of Myristoylationmentioning

confidence: 99%

Protein myristoylation in health and disease

et al. 2009

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N-myristoylation is the attachment of a 14-carbon fatty acid, myristate, onto the N-terminal glycine residue of target proteins, catalysed by N-myristoyltransferase (NMT), a ubiquitous and essential enzyme in eukaryotes. Many of the target proteins of NMT are crucial components of signalling pathways, and myristoylation typically promotes membrane binding that is essential for proper protein localisation or biological function. NMT is a validated therapeutic target in opportunistic infections of humans by fungi or parasitic protozoa. Additionally, NMT is implicated in carcinogenesis, particularly colon cancer, where there is evidence for its upregulation in the early stages of tumour formation. However, the study of myristoylation in all organisms has until recently been hindered by a lack of techniques for detection and identification of myristoylated proteins. Here we introduce the chemistry and biology of N-myristoylation and NMT, and discuss new developments in chemical proteomic technologies that are meeting the challenge of studying this important co-translational modification in living systems.Keywords Post-translational modification . Drug design . Myristoylation . Lipidation . Chemical proteomics Post-translational modification and myristoylationThe proteome is a larger and more dynamic entity than the genome, a result of both increased sequence diversity at the mRNA level due to alternative splicing of genes, and increased chemical and functional complexity at the protein level due to post-translational modification (PTM). This explains to some extent why larger genomes do not necessarily translate into more complex organisms. Posttranslational modification allows the incorporation of chemistries and new molecular functions that cannot be directly encoded by the gene sequence. Myristoylation is the attachment of a 14-carbon saturated fatty acid, myristate, to the N-terminal glycine of a subset of eukaryotic proteins [8,16,17]. Although often referred to as a PTM, it usually occurs co-translationally [18], when fewer than 100 residues have been polymerised by the

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Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Cited by 146 publications

References 48 publications

Palmitoylation of caspase-6 by HIP14 regulates its activation

Palmitoylation of caspase-6 by HIP14 regulates its activation

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

Protein myristoylation in health and disease

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