Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells

Fukata, Yuko; Iwanaga, Tsuyoshi; Fukata, Masaki

doi:10.1016/j.ymeth.2006.05.015

Cited by 109 publications

(113 citation statements)

References 15 publications

Supporting

Mentioning

110

Contrasting

Order By: Relevance

“…1A). Because the expression levels of the 23 DHHCs have been variable when expressed in mammalian cells (38,39), we also assessed their expression in Xenopus oocytes by immunoblotting extracts of oocytes injected with cRNA encoding individual HA-DHHCs. Extracts of oocytes were subjected to immunoblotting with anti-HA antibodies and revealed bands of the expected size for each DHHC, except DHHC 21, although the ␤-actin signal by immunoblotting was relatively uniform for all the samples (Fig.…”

Section: Resultsmentioning

confidence: 99%

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

Mukherjee¹,

Wang²,

Kinlough³

et al. 2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

Edited by Henrik G. DohlmanThe epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (␣, ␤, and ␥). We previously reported that cytoplasmic residues on the ␤ (␤Cys-43 and ␤Cys-557) and ␥ (␥Cys-33 and ␥Cys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, ␥ subunit palmitoylation had a dominant role over ␤ subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when ␥ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking ␤ subunit palmitoylation sites. ␤ subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking ␤ and ␥ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCD cl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCD cl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na ؉ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.ENaCs 2 are amiloride-sensitive Na ϩ channels that are found in high resistance epithelia and other tissues. In the aldosterone-sensitive distal nephron (ASDN), ENaC-dependent Na ϩ transport has an important role in the maintenance of extracellular fluid volume and blood pressure, as well as extracellular K ϩ homeostasis. In the lung, ENaC has a role in regulating airway surface fluid volume, mucociliary clearance, and alveolar fluid volume (1-3). The channels are composed of three homologous subunits (termed ␣, ␤, and ␥), each with two transmembrane domains, a large extracellular loop and cytoplasmic N and C termini (3-5).ENaCs are regulated by signaling pathways that modulate its membrane trafficking, residency on the plasma membrane, and degradation (6 -8). ENaCs are also regulated by a variety of extracellular factors that affect its open probability (P o ). These include extracellular cations (H ϩ , Na ϩ , and other metals), anions (Cl Ϫ ), laminar shear stress, and proteases that cleave ENaC subunits at specific sites and release embedded inhibitory tracts (1, 2, 9 -14). Intracellular phosphorylation, inositol phospholipids, and cytoplasmic Cys palmitoylation also regulate ENaC P o (15-21).Cys palmitoylation of soluble and transmembrane proteins is a reversible post-translational modification that increases protein surface hydr...

show abstract

Section: Resultsmentioning

confidence: 99%

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

Mukherjee¹,

Wang²,

Kinlough³

et al. 2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…This method is commonly used to identify DHHCs capable of modifying individual protein substrates [35,[50][51][52][53]. CD86-HA S-palmitoylation was increased by DHHCs 2, 3, 6 and 7 ( Figure 2D, Additional file 2: Figure S7).…”

Section: Palmitoylation Of Cd86 and Tlr2 Can Be Increased By Specificmentioning

confidence: 99%

Chemoproteomics reveals Toll-like receptor fatty acylation

et al. 2014

View full text Add to dashboard Cite

Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells.

show abstract

“…The expression patterns obtained also highlighted transcripts for which a potential role in skeletal muscle development has not yet been defined. The zinc finger DHHC-type containing 23 (ZDHHC23), for example, codes for a membrane protein containing a palmitoyl transferase domain, which supposedly promotes protein palmitoylation, a crucial lipid modification in protein trafficking and function (Fukata et al, 2006). Substrates for palmitoylation include H-Ras, a GTP binding protein that regulates cell growth and differentiation (Fukata et al, 2006).…”

Section: Differentially Expressed Transcripts: a Statistical Approachmentioning

confidence: 99%

“…The zinc finger DHHC-type containing 23 (ZDHHC23), for example, codes for a membrane protein containing a palmitoyl transferase domain, which supposedly promotes protein palmitoylation, a crucial lipid modification in protein trafficking and function (Fukata et al, 2006). Substrates for palmitoylation include H-Ras, a GTP binding protein that regulates cell growth and differentiation (Fukata et al, 2006). A ZnFDHHC motif was found with an expression pattern similar to MyoD (muscle determination transcription factor) in the somitic mesorderm in Danio rerio (Nagaya et al, 2002).…”

Section: Differentially Expressed Transcripts: a Statistical Approachmentioning

confidence: 99%

Chicken skeletal muscle-associated macroarray for gene discovery

Jorge

Melo²,

Rosário³

et al. 2010

Genet. Mol. Res.

View full text Add to dashboard Cite

Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells

Cited by 109 publications

References 15 publications

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

Chemoproteomics reveals Toll-like receptor fatty acylation

Chicken skeletal muscle-associated macroarray for gene discovery

Contact Info

Product

Resources

About