Dynamic protein palmitoylation in cellular signaling

Progress in Lipid Research 48 (2009) 117–127

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Review

Dynamic protein palmitoylation in cellular signaling Tsuyoshi Iwanaga a, Ryouhei Tsutsumi a, Jun Noritake a, Yuko Fukata a,b, Masaki Fukata a,b,* a

Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan b PRESTO, Japan Science and Technology Agency, Chiyoda, Tokyo 102-0075, Japan

a r t i c l e

i n f o

Keywords: Protein palmitoylation DHHC protein Lipid modification Protein targeting Palmitoyl acyl transferase

a b s t r a c t Protein S-palmitoylation, the most common lipid modification with the 16-carbon fatty acid palmitate, provides an important mechanism for regulating protein trafficking and function. The unique reversibility of protein palmitoylation allows proteins to rapidly shuttle between intracellular membrane compartments. Importantly, this palmitate cycling can be regulated by some physiological stimuli, contributing to cellular homeostasis and plasticity. Although the enzyme responsible for protein palmitoylation had been long elusive, DHHC family proteins, conserved from plants to mammals, have recently emerged as palmitoyl acyl transferases. Integrated approaches including advanced proteomics, live-cell imaging, and molecular genetics are beginning to clarify the molecular machinery for palmitoylation reaction in diverse aspects of cellular functions. Ó 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of palmitoylation in protein trafficking and targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Shuttling between plasma membrane and endomembranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Targeting to lipid raft microdomains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DHHC palmitoyl acyl transferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Yeast DHHC proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Mammalian DHHC proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Method to identify physiological PAT–substrate pairs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Substrate specificity and consensus sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Regulatory mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological roles of DHHC PATs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: ABE, acyl-biotinyl exchange; AMPA, alpha-amino-3-hydroxy-5methyl-4-isoxazole propionic acid; AR, adrenergic receptor; DHHC, aspartatehistidine-histidine-cysteine; Erf, effect on Ras function; Ga, G protein a subunit; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; NCAM, neural cell adhesion molecule; PAT, palmitoyl acyl transferase; PM, plasma membrane; PPT, palmitoyl-protein thioesterase; PSD, postsynaptic density; RGS, regulator of G protein signaling. * Corresponding author. Address: Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan. Tel.: +81 564 59 5873; fax: +81 564 59 5870. E-mail address: [email protected] (M. Fukata). 0163-7827/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.plipres.2009.02.001

117 119 119 119 120 120 121 122 122 123 124 125 125 125

1. Introduction Posttranslational modification, such as phosphorylation, glycosylation, ubiquitination, and lipidation, provides proteins with additional functions. These modifications are essential for cells to maintain homeostasis and to rapidly respond to extracellular signals. Lipidation contributes to the precise targeting, subcellular trafficking, and function of proteins [1–4]. N-myristoylation, S-palmitoylation, and prenylation represent common lipid modifications occurring in the cytoplasmic face of membranes (Fig. 1).

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A

O

N-myristoylation N H

O

N-palmitoylation N H

O

S-palmitoylation S farnesylation S

B O

O

N

SH

CoA-S

palmitoyl-CoA

N

O

Cys

+

PPT

PAT CoA-SH

O

O

N

Cys

O

S N

Fig. 1. (A) Structures of common lipid modifications. N-myristoylation is the addition of the 14-carbon lipid myristate and occurs at the N-terminal glycine residue via stable amide linkage. N-palmitoylation is postulated to occur via a thioester intermediate utilizing the thiol of the cysteine residue, followed by a spontaneous rearrangement to generate the irreversible amide linkage. S-palmitoylation is the addition of the 16-carbon palmitate to a cysteine residue via a reversible thioester linkage. Prenylation is the addition of an isoprenoid, either a 15-carbon farnesyl (farnesylation) or a 20-carbon geranylneranyl (geranylgeranylation; not shown here) group, to a cysteine residue via a stable thioether linkage. (B) Reversible S-palmitoylation. Palmitate is transferred from palmitoyl-CoA, which is produced by acyl-CoA synthetase, to a protein by protein acyl transferase (PAT). In contrast, palmitate on proteins is cleaved by palmitoyl-protein thioesterase (PPT). Gray boxes indicate cysteine residues where palmitoylation occurs. Palmitoyl-CoA and palmitoyl-coenzymeA.

N-myristoylation is a co-translational attachment of 14-carbon myristic acid to an amino-terminal glycine through a stable amide linkage [5,6]. Prenylation is a posttranslational attachment of a prenyl group, 15-carbon farnesyl or 20-carbon geranylgeranyl group, to a carboxy-terminal cysteine-containing motif, CaaX (‘‘a” and ‘‘X” represent aliphatic and any residues, respectively) [7]. S-palmitoylation refers to the addition of palmitate, a 16-carbon saturated fatty acid, to cysteine residues through a labile thioester linkage [1–4]. Among these three lipid modifications, S-palmitoylation most efficiently increases protein hydrophobicity to facilitate membrane association of proteins. The labile thioester bond provides a unique feature, ‘‘reversibility”, with S-palmitoylation. Limited proteins, such as the secreted morphogen Sonic hedgehog, are subjected to irreversible N-palmitoylation at glycine and cysteine residues, which occurs in the luminal face of the secretory pathway [8]. The term protein palmitoylation will be used for ‘‘S-palmitoylation” in this review. Palmitoylation modifies numerous soluble and integral membrane proteins, including signaling proteins, enzymes, scaffolding proteins, ion channels, cell adhesion molecules, and neurotransmitter receptors [1–4,9]. Examples include trimeric G protein a subunits (Gas, Gai, Gaq) [10,11], small GTPases (H-Ras and N-Ras

[12], Rap2b [13], RhoB [14], TC10 [15,16]), CLICK-III/CaMKIc [17], PSD-95 postsynaptic scaffolding protein [18], neural cell adhesion molecule (NCAM) [19], claudin [20], integrin [21], and CLIPR-59 cytoplasmic linker protein [22] (Table 1). Classical studies using metabolic labeling with [3H]palmitate showed that palmitoylation is a reversible modification in cells and the palmitate cycling of several substrates is dynamically regulated by extracellular signals [23–25]. For example, binding of isoproterenol to the b-adrenergic receptor (AR) markedly accelerates the depalmitoylation of the associated Gas, shifting Gas to the cytoplasm [26]. This receptor activation-induced depalmitoylation was also observed in a major postsynaptic PSD-95 scaffold, which anchors AMPA-type glutamate receptor (AMPA receptor) at the excitatory postsynapse through stargazin [27,28]. Upon glutamate receptor activation, accelerated depalmitoylation of PSD-95 dissociates PSD-95 from postsynaptic sites and causes AMPA receptor endocytosis [29]. Thus, palmitate turnover on Gas and PSD-95 is accelerated by the receptor activation, probably contributing to downregulation of the signaling pathway. In addition to these classic observations, advanced live-cell imaging and the recent discovery of DHHC-type palmitoyl acyl transferases (PATs) have provided new insights into the detailed molecular mechanism of dynamic palmitoylation.

T. Iwanaga et al. / Progress in Lipid Research 48 (2009) 117–127

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Table 1 Palmitoylated proteins and PAT candidates.a Palmitoylation motifs

Substrate

DHHC enzymes

Ref

DHHC9/18

[81]

Signaling proteins H-Ras N-Ras G

s

G

q

G

i2

Fyn

-KLN PPD ESG PG C M SC KC VLS -KLN SSD D G TQ G C M G LPC VVM M G C LG N SKTED Q RN EEKAQ R M TLESIM AC C LSEEAKEARR M G C TVSAED KAAAERSKM I D M G C VQ C KD KEATKLTEERD G 189

DHHC9/18

[81]

1

DHHC3/7

1

DHHC3/7

[35] [35]

1

DHHC2/3/7/21

[35]

1

DHHC21

189

Lck

1

M G C G C SSH PED D W M EN ID VC -

DHHC21

* [35]

eNOS

1

M G N LKSVAQ EPG PPC G LG LG LG LG LC -

DHHC2/3/7/8/21

[79]

R7BP

-PYPLVRRRKRRFFG LC C LVSS

GAP-43

1

M LC C M RRTKQ VEKN D D D Q KI -EN SISRLLFC C W FPW M LRAEM Q -LATG PC G C C SSC LN IG N KG KSS -

DHHC2/3/7/15

[59]

DHHC3

[17]

-TD LG KFC G LC VC PC N KLKSS -LTC C YC C C C LC C C FN C C C G KC KP -LVLTC C FC IC KKC LFKKKN K -

DHHC3/7/17

[59,77]

DHHC3/7/15/17

[80]

1

M D C LC IVTTKKYRYQ D ED TP -

DHHC2/3/7/15

[59]

1

M PG W KKN IPIC LQ AEEQ ERE-

DHHC3

[37]

CLIPR-59 CLICK-III/CaMKI Synaptic vesicle proteins SNAP-25 Cysteine-string protein Synaptotagmin Adaptor and scaffold proteins PSD-95 GRIP1b Transmembrane receptors Ion channels GluR1 GluR2 GABAA receptor subunit Kv1.1 Cell adhesion molecules Integrin 4 NCAM140

25 7

54 6

433

98

138

90

-RSESKRM KG FC LIPQ Q SIN E -LAM LVALIEFC YKSRAEAKR -YEC LD G KD C ASFFC C FED C RTG A -FAC PSKTD FFKN IM N FIPIVAII 85 2

845

43 6

DHHC3

[37]

DHHC3/7

[74]

DHHC3/7

[41]

DHHC2

[83]

263

-LLALLLLLC W KYC AC C KAC LA -ITC YFLN KC G LFM C IAVN LC M G EFN EKKTTC G TVC LKYLL- VVM VTG VLG C C ATFKERRN L - Q VFG M IFTC C LYRSLKLEH Y - AC AVIG M KC TRC AKG TPAKT - SLIG G TLLC LSC Q D EAPYRP 74 4

75 0

1

CD151

70

234 96

Claudin14

174

89

253

11 5

Fig. 2. Palmitoylation-dependent cycling of Ga between the plasma membrane (PM) and the Golgi apparatus. The PM targeting of Ga requires both interaction with the Gbc complex and subsequent palmitoylation of Ga at the Golgi. Although the Ga subunit seems to localize stably at the plasma membrane, recent reports [34,35] suggest that some Ga isoform shuttles rapidly between PM and the Golgi. Constitutive cycling of palmitate on Ga regulates this rapid Ga shuttling. This basal trafficking between the PM and the Golgi can also be observed in H-Ras and N-Ras [32]. PAT, palmitoyl acyl transferase; GPCR, G protein-coupled receptor; a, b, and c represent G protein a, b, and c subunits, respectively. Gc subunit is farnesylated or geranylgeranylated.

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a Representative palmitoylated proteins, their palmitoylation sites, and reported DHHC PAT candidates are listed. Palmitoylated cysteine residues are shown in red. Myristoylated and prenylated residues are shown in blue and green, respectively. An asterisk ‘‘” indicates our unpublished data.

2. Role of palmitoylation in protein trafficking and targeting

Gaq, constitutively cycles between the PM and the Golgi in a de/ repalmitoylation-dependent manner (Fig. 2) [35]. Another representative example for the intracellular shuttling is R7BP, RGS7 binding protein. R7BP complexed with RGS7-Gb5 dynamically shuttles between the PM and the nucleus in an R7BP-palmitoylation-dependent manner [36].

2.1. Shuttling between plasma membrane and endomembranes

2.2. Targeting to lipid raft microdomains

H-Ras and N-Ras are representative palmitoylated proteins, which are also subjected to farnesylation. These lipid modifications are critical for H-Ras/N-Ras targeting to the plasma membrane (PM) and the Golgi apparatus [12]. Although Golgi resident Ras is thought to represent a trafficking intermediate of newly synthesized protein en route to the PM [30,31], Rocks et al. [32,33] elegantly revealed that H-Ras/N-Ras constitutively cycle between the PM and the Golgi membrane (Golgi) by a constitutive palmitoylation–depalmitoylation cycle. Fluorescence recovery after photobleaching analysis showed that YFP-H-Ras fluorescence in the Golgi rapidly recovered with a half-life of 6 min [32]. Photoactivated H-Ras at the PM clearly shows retrograde PM-Golgi trafficking. Importantly, an inhibitor of protein palmitoylation, 2-bromopalmitate, blocks this constitutive cycling and redistributes H-Ras into all the intracellular membranes. These observations indicate that the Golgi pool of H-Ras is continuously exchanged with the PM pool, and that this exchange requires the transient trapping at the Golgi mediated by repalmitoylation (probably occurring at the Golgi). This study [32] has raised the novel concept that the continuous cycle prevents Ras from non-specific residence on the other compartments, thereby maintaining the specific PM-Golgi compartmentalization. In addition, another palmitoylated substrate, Gao, which has been thought to localize stably at the cytosolic face of the PM, was also shown to shuttle rapidly between the PM and intracellular membranes [34]. Taking advantage of Dendra2, a green-to-red photoconvertible fluorescent protein, we also found that another trimeric G protein a subunit,

As shown in Table 1, many integral membrane proteins undergo palmitoylation at the cytoplasmic juxtamembrane cysteine residue. These include NCAM [19], integrins (b4, a3, and a6) [21], claudins [20], G protein-coupled receptors (GPCR) [1], and AMPA-type glutamate receptors [37]. One may wonder why many integral membrane proteins get palmitoylated even though integral membrane proteins by themselves target to the PM without palmitoylation. The function of palmitoylation of integral membrane proteins goes beyond a simple membrane anchor. Accumulating evidence indicates that palmitoylation targets proteins to lipid rafts, subdomains of the PM that are enriched in sphingolipids and cholesterol [38,39]. Blocking palmitoylation by site-directed mutagenesis of the modified cysteine residue or treating with an inhibitor of palmitoylation delocalizes proteins from raft microdomains. For example, disruption of NCAM140 [40,41] palmitoylation prevents its association with lipid rafts and abolishes neurite outgrowth. Instead of lipid rafts, palmitoylation of integrin (b4, a3, and a6) promotes assembly of a novel type of signaling platform enriched for palmitoylated tetraspanins (CD9, CD81, and CD63), known as tetraspanin-enriched microdomains, and regulates cell morphology and cell spreading [21]. Thus, palmitoylation is critical for not only simple protein trafficking to the PM but also (1) constitutive shuttling between intracellular compartments and (2) precise microdomain partitioning. The speed and kinetics of the palmitoylation–depalmitoylation cycle could be altered in response to specific extracellular signals, providing proteins with the attractive mechanism for dynamic

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relocalization. However, the molecular machinery conferring palmitate cycling had been unknown due to the difficulty in identification of PATs and palmitoyl-protein thioesterases (PPTs), which mediate palmitoylation and depalmitoylation, respectively. 3. DHHC palmitoyl acyl transferases 3.1. Yeast DHHC proteins Until recently, identifying PATs and PPTs was an elusive goal in biochemistry and cell biology: numerous biochemists have encountered difficulties developing a specific in vitro palmitoylation assay and purifying PATs and PPTs. Some results indicated that palmitoylation in cells might occur non-enzymatically because the formation of palmitoyl thioester linkage on proteins (such as Ga subunit) can occur spontaneously in vitro in the presence of palmitoyl-coenzymeA [42]. Consequently, identification of PATs has been a challenging issue. Elegant forward genetic studies in yeast have recently provided a breakthrough in the field. Deschenes’s group [43] made a non-farnesylated Ras2 mutant protein in which the CaaX box was replaced by a stretch of basic residues. This mutant protein was palmitoylated but not farnesylated, and yeast cells expressing this mutant Ras depend on palmitoylation for its viability. A synthetic lethal genetic screen using this mutant yeast identified two candidate genes involved in the Ras2 palmitoylation [44]; Erf2 (effect on Ras function) and Erf4, both of which are ER-resident proteins. Mutations of one or both genes resulted in diminished palmitoylation of Ras2 and mislocalization of green fluorescent protein (GFP)-Ras2 from the PM to the vacuole [44]. A subsequent study demonstrated that a recombinant Erf2 and Erf4 complex mediates Ras2 palmitoylation in vitro [45]. Davis’s group [46] also identified Golgi resident Akr1, which mediates palmitoylation of yeast casein kinase 2 (Yck2). Deletion

of Akr1 in yeast mislocalizes Yck2 from the PM to the cytoplasm. Recombinant Akr1 palmitoylates Yck2 in vitro. Importantly, Erf2 and Akr1 have four- and six-pass transmembrane domains, respectively, and share a conserved domain referred to as a DHHC-CRD, a cysteine-rich domain with an aspartate-histidine-histidine-cysteine signature motif (Fig. 3) [2,47,48]. This DHHC-CRD, which oriented to the cytoplasmic side [49], is necessary for the palmitoylating activity of Erf2 and Akr1, suggesting that DHHC-CRD-containing proteins (DHHC proteins) may function as PATs. In fact, other DHHC proteins (Pfa3, Pfa4, and Swf1) in yeast have been reported to have the PAT activity. Pfa3 [50,51] and Pfa4 [52,53] mediate Vac8 and Chs3 palmitoylation, respectively, and regulate their subcellular distributions. Swf1 palmitoylates yeast SNARE proteins such as Snc1, Syn8, and Tlg1, and prevents their ubiquitination and degradation [54]. Erf4 does not have a DHHC-CRD or any known motifs. As Ras2-PAT activity of Erf2 depends on the presence of Erf4, and Erf2 tightly associates with Erf4 in yeast [45], it has been thought that Erf4 is an auxiliary protein for Erf2 to fully function as a Ras2-PAT. Importantly, this Erf2/Erf4 complex is conserved in mammalian cells (Section 3.4). On the other hand, Akr1 does not require the other protein to palmitoylate Yck2 [46]. Does another class of PATs besides DHHC proteins exist in cells, or does a non-enzymatic mechanism contribute to certain substrates? Roth et al., using systematic proteomic analyses, clearly answered this question [53]. They developed a powerful method (acyl-biotinyl exchange (ABE)) to systematically purify palmitoylated proteins from yeast and to subsequently identify palmitoylated proteins by mass spectrometry [53,55]. This method allowed them to identify 35 new palmitoyl yeast proteins including many SNARE proteins, amino acid permeases, and signaling proteins. They applied this proteomic ABE method to mutant yeast strains either singly or multiply deficient for the seven yeast DHHC proteins. When six of seven DHHC genes in yeast were deleted, 29 of the 30 surveyed palmitoyl proteins were no longer detected

Fig. 3. DHHC protein family in mammals. Domain structures of representative DHHC proteins, DHHC3, DHHC9, and DHHC17. These proteins have four or six transmembrane domains and a conserved cysteine-rich domain (CRD) containing the DHHC motif in the cytoplasmic loop. The DHHC sequence is essential for palmitoylating activity. DHHC3 and DHHC17 have a PDZ-binding motif and ankyrin repeats, respectively. DHHC9 forms a complex with GCP16, which is a homologue yeast Erf4 and is palmitoylated [90]. The consensus sequence of DHHC-CRD is indicated (green and red). DHHC, aspartate-histidine-histidine-cysteine and X, a variety of amino acids.

T. Iwanaga et al. / Progress in Lipid Research 48 (2009) 117–127

by the ABE method [53]. Thus, DHHC family proteins are responsible for most of the palmitoylation within the yeast cell. Also, this loss-of-function study indicates that the DHHC protein family has partially overlapping and distinct PAT specificities. Besides yeast (seven genes), the DHHC family is conserved from nematode Caenorhabditis elegans (15 genes, predicted) and fruit fly Drosophila melanogaster (22 genes) [56–58] to mammals (23 genes) [59,60]. DHHC proteins are also found in plants such as Arabidopsis thaliana, Zea mays, and Solanum tuberosum [61]. In A. thaliana, TIP1, a homologue of yeast Akr1, was identified as a causative gene of tip growth defective1 mutant, displaying one tenth the length of wild-type root hairs [62]. As inhibition of palmitoylation in wild-type Arabidopsis roots by 2-bromopalmitate reproduces the TIP1 mutant phenotype and TIP1 complements mutant yeast lacking Akr1, it is conceivable that TIP1 regulates plant cell growth as a PAT. Furthermore, DHHC PAT in the intestinal protozoan parasite Giardia lamblia has been recently reported [63]. Thus, the DHHC protein is genetically conserved between various species. 3.2. Mammalian DHHC proteins There are 23 DHHC genes in the mouse and human genome database (Fig. 4). Several human DHHC proteins have been isolated as associated genes with human diseases or binding partners with neuronal receptors. DHHC2/REAM (reduced expression associated with metastasis) was isolated as a novel gene whose expression is reduced significantly in human colorectal cancers with high potential for metastasis. Also, mutations of DHHC2/REAM have been found in several human tumors including a colorectal cancer, hepatocellular carcinoma, and non-small cell lung cancer [64]. DHHC3/GODZ (Golgi apparatus-specific protein with the DHHC

121

zinc finger domain) was identified as a GluR1 (an AMPA-type glutamate receptor subunit)-interacting protein by yeast two-hybrid analysis [65]. DHHC8 was proposed as a candidate schizophrenia susceptibility gene [66–68]. DHHC9 [69] and DHHC15 [70] were reported to be associated with X-linked mental retardation. DHHC17, a mammalian counterpart of yeast Akr1 and plant TIP1, was isolated as a huntingtin-interacting protein, HIP14, by the yeast two-hybrid screening [71]. However, it was unclear whether mammalian DHHC proteins function as PATs. Although knowledge obtained from yeast genetic studies [44,46] urged investigators to identify PATs toward mammalian palmitoyl proteins, the large family of mammalian DHHC proteins has prevented investigators from easily identifying specific enzyme–substrate pairs. In 2004, three different groups, including ours, reported that mammalian DHHC proteins mediate protein palmitoylation [59,72,73]. Keller et al. [73] first identified DHHC3/GODZ as a binding protein with the c-aminobutyric acid (GABA)A receptor c2 subunit by yeast two-hybrid screening. DHHC3/GODZ palmitoylates the GABAA receptor c2 subunit in heterologous cells, whose palmitoylation plays an essential role in the clustering and cell surface stability of GABAA receptors [73–75]. This study raises the possibility that we may identify the specific PAT–substrate pair by exploring the DHHC protein associated with the substrate. In fact, GluR1, an AMPA receptor subunit, interacts with DHHC3/GODZ [65] and DHHC3 palmitoylates GluR1 [37]. DHHC17/HIP14, identified as a huntingtin-interacting protein [71], palmitoylates huntingtin [76]. However, most enzyme reactions are mediated by transient interaction of the enzyme with its substrate and does not necessarily require such a tight interaction. In other words, this approach may be applied to only limited substrates. Huang et al. [72] noticed the predominant neuronal expression of DHHC17/HIP14 and

Fig. 4. Phylogenetic tree of the mouse DHHC protein family and a summary of PAT–substrate pairs. The tree is based on alignment of the DHHC-CRD core domains. DHHC proteins are classified into several subfamilies and apparently have substrate specificity. DHHC3 and DHHC7 palmitoylate PSD-95, Ga, SNAP-25, GABAA receptor c2 subunit (GABAARc), NCAM, and so on, suggesting that DHHC3 and DHHC7 show a broad substrate specificity. DHHC3 is also known as GODZ; DHHC17 as HIP14; DHHC11 as NIDD; and DHHC2 as REAM. GenBank accession numbers for each DHHC clone are listed in Fukata et al. [59]. Sc, Saccharomyces cerevisiae.

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Fig. 5. Palmitoylation of PSD-95 is essential for its postsynaptic targeting. Wild-type PSD-95-GFP, which is palmitoylated at cysteines 3 and 5, clearly targets to the postsynaptic membrane in hippocampal neurons and clusters at synaptic sites (green points; left panel). By contrast, palmitoylation deficient mutant of PSD-95 (C3,5S) diffusely localizes in somato-dendrites (right panel). GFP, green fluorescent protein.

examined whether DHHC17/HIP14 palmitoylates representative neuronal palmitoyl proteins, such as SNAP-25, Synaptotagmin I, PSD-95, GAD65, huntingtin, H-Ras, Lck, and paralemmin by in vitro palmitoylation assay [72]. DHHC17/HIP14 showed PAT activity toward SNAP-25, Synaptotagmin I, PSD-95, GAD65, and huntingtin. Because the databases of mRNA expression imply that most DHHC proteins express ubiquitously, it is difficult to determine physiological PATs only based on the expression profile of DHHC proteins. A more generic screening method is necessary for determining the specific PATs in mammalian cells.

palmitoylation of the endogenous substrate is affected when the candidate DHHC clone is inhibited by the dominant negative mutant or the knockdown approach. This screening method has been used to identify various enzyme–substrate pairs. Examples include NCAM [41], eNOS [79], the GABAA receptor c2 subunit [74], SNAP-25 [77], Ga [35], Lck tyrosine kinase, GAP-43, and H-Ras (Fig. 4). So far, the palmitoylation levels of investigated substrates (more than 30 kinds of substrates) were all enhanced by some DHHC proteins (14 of 23 DHHC proteins exhibited PAT activity toward at least one substrate), demonstrating that the DHHC protein family represents the main PATs in mammals as well as in yeast [53].

3.3. Method to identify physiological PAT–substrate pairs 3.4. Substrate specificity and consensus sequences To identify the candidate PAT for specific substrates without any bias, we isolated all mouse DHHC clones and established a simple, systematic, and straightforward screening method [59,77]. As a model substrate, we selected PSD-95, which is a representative neuronal scaffolding protein targeted to the postsynaptic membrane in a palmitoylation-dependent manner (Fig. 5) [18,29,78]. The method includes three steps: (1) transient transfection of PSD-95 together with an individual DHHC clone into HEK293 cells, (2) metabolic labeling with [3H]palmitic acid, and (3) SDS–PAGE and fluorography. Unlike in a conventional metabolic labeling procedure, we did not purify the substrate by immunoprecipitation for detection of DHHC clone-induced palmitoylation. Immunoprecipitated protein does not necessarily reflect the total pool of palmitoylated proteins. Typical extraction buffer using Triton X-100 or even Radioimmunoprecipitation assay (RIPA) buffer may not extract all of the palmitoylated proteins, which strongly associate with membranes and are found in the insoluble fraction. If immunoprecipitation is necessary, 1% SDS (followed by dilution with Triton X-100) is suitable for the extraction of palmitoylated proteins. This method allowed us to evaluate the individual PAT activity relatively and quantitatively. Using our systematic method, we found that a subset of DHHC proteins (DHHC2, 3, 7, and 15: PSD-95-PAT (P-PAT)) quantitatively enhance palmitoylation of PSD-95 in cells. After this screening, the candidate DHHC clones were verified by (1) expression pattern and (2) loss-of-function analysis. In the case of PSD-95, the responsible PATs should be expressed in the brain where PSD-95 is expressed. Subsequently, we examined whether

Do DHHC PATs have a substrate specificity as shown in protein kinases? Our screening results using the DHHC clone library (Fig. 4) and loss-of-function analysis in yeast [53] clearly indicate that DHHC proteins show exquisite substrate specificity. Among DHHC proteins, DHHC3 and the closely related DHHC7 have broad substrate specificity. DHHC3 and DHHC7 enhance the palmitoylation of various substrates, including PSD-95 [59], eNOS [79], GABAA receptor c2 subunit [74], SNAP-25 [59,77], CSP [80], Ga [35,59], NCAM [41], and GAP-43 [59]. In contrast, DHHC2 and DHHC15 are more specific to PSD-95 and GAP-43 [59]. DHHC9 and DHHC18 are specific to H-Ras and N-Ras [81]. DHHC21 preferably palmitoylates Lck, Fyn (Fukata et al., unpublished data) and eNOS [79]. These systematic screenings should allow us to classify 23 DHHC proteins into several subfamilies. Phylogenetic tree alignment of the DHHC protein family (Fig. 4) suggests that there may be some structural correlations between each DHHC motif and its substrate specificity. Individual DHHC proteins also have potential regulatory domains such as the SH3 domain in DHHC6, the ankyrin repeats in DHHC17 and 20, and the PDZ-binding motif at the C-terminal in DHHC3, 5, 8, and 14 (Fig. 3). This is akin to protein kinases, which share a core catalytic region and differ in regulatory domains that afford differential control systems. It is conceivable that these regulatory regions may recruit specific substrates or regulators to DHHC proteins. Whether DHHC PAT-specificity might be regulated by these potential regulatory domains requires future studies.

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As described in Section 3.1, Erf2, the first identified DHHC protein in yeast, functions as a PAT toward yeast Ras2 in a complex with an auxiliary protein, Erf4. Linder’s group [81] identified GCP16 as a mammalian functional ortholog of Erf4. They found that DHHC9, a mammalian counterpart of Erf2, requires GCP16 for its PAT activity toward H/N-Ras and for its protein stability. This study [81] raises the possibility that some DHHC proteins require auxiliary subunit proteins like Erf4 and GCP16. Because several GCP16 family proteins are predicted in the mammalian genome, some other DHHC proteins may complex with GCP16 family proteins. The GCP16 family may play a significant role in the substrate specificity, subcellular localization, and stability of the DHHC protein. The consensus sequence of protein palmitoylation remains unknown, whereas that for myristoylation (N-terminal glycine in the sequence MGXXXS/T (M: Met; G: Gly; S: Ser; T: Thr; X: a variety of amino acids)) or isoprenylation (cysteine in the C-terminal CaaX motif) is well characterized. Why can we not expect the rule of palmitoylation sites from the many previous examples? Palmitoylation occurs at the various cysteine residues located at (1) the amino-terminal region in PSD-95, GAP-43, and Ga, (2) the internal region in SNAP-25, CSP, and GAD65, (3) the carboxyl terminal region in H-Ras and RhoB, and (4) the juxtamembrane region of various transmembrane proteins in NCAM and GluRs (Table 1). Our recent systematic screening may solve this complicated question. DHHC9/DHHC18 and DHHC21 favor dual-lipidated substrates with palmitoylated cysteines near the C-terminal isoprenylated cysteine (H-Ras and N-Ras) and the N-terminal myristoylated glycine (Lck, Fyn, and eNOS), respectively. Exclusively palmitoylated substrates PSD-95 and GAP-43, which DHHC2 and 15 specifically palmitoylate, have hydrophobic residues and basic residues surrounding the modified cysteine residues. We propose that the individual DHHC subfamily (Fig. 4) recognizes a specific pattern for palmitoylation. The large family of DHHC proteins may explain for the diverse palmitoylation motifs. Further identification of enzyme– substrate pairs will contribute to clarifying the DHHC subfamilyspecific consensus sequence. 3.5. Regulatory mechanisms Palmitoylation is a unique lipid modification in that it is a reversible modification and is dynamically regulated by the extracellular stimulus, like phosphorylation. Palmitoylation level is determined by a finely tuned balance between PAT and PPT activities. The most intensively analysed substrates whose palmitoylation levels are regulated by the extracellular signal are heterotrimeric G protein a subunit Gas [26] and neuronal scaffolding protein PSD-95 [29]. In 1994, Wedegaertner and Bourne [26] reported that activation of b-AR by isoproterenol markedly accelerates the palmitate cycling on Gas (depalmitoylation and subsequent repalmitoylation of Gas) and induces PM-to-cytosol translocation of Gas in S49 cyc lymphoma cells. This receptor activation-induced removal of palmitate on Gas contributes to downregulation of the signaling pathway. Another report using Gaq-GFP showed that Gaq-GFP is recruited at the PM in a palmitoylation-dependent manner and Gaq-GFP at the PM does not change upon a2A-AR agonist stimulation in HEK293T cells [82]. Our recent photoconversion analysis using Gaq-Dendra2 and fluorescence recovery after photobleaching analysis revealed that Gaq rapidly shuttles between the PM and the Golgi in HeLa cells, and this shuttling requires palmitoylation by DHHC3/7 at the Golgi [35]. Although we attempted to decipher whether a1A-AR agonist stimulation affects Gaq dynamic relocalization in HeLa cells, our imaging resolution could not detect a significant change. Given that Gas and Gaq palmitoylation is specifically mediated by common Golgi resident PAT, DHHC3/7 ([35];

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Table 1), palmitate cycling of each Ga member may depend on the cellular/agonist contexts or may be regulated mainly by alteration of PPT activity. Taking advantage of 2-bromopalmitate, an inhibitor of palmitoylation reaction, El-Husseini et al. [29] demonstrated that palmitate on PSD-95 cycles dynamically in neurons. PSD-95 is accumulated at the postsynapse in a palmitoylation-dependent manner and thereby gives a platform for the postsynaptic clustering of AMPA receptors (Fig. 5) [18]. They showed that treatment of primary hippocampal neurons with 2-bromopalmitate for several hours markedly blocks the palmitoylation of PSD-95 and disperses PSD-95/AMPA receptor clusters, indicating that palmitate continuously turns over on PSD-95 and on-going palmitoylation of PSD-95 is required to maintain PSD-95/AMPA receptor clustering at the postsynapse. They also showed that glutamate-induced synaptic activity accelerates the depalmitoylation of PSD-95 and causes AMPA receptor endocytosis [29], suggesting that unidentified PPT seems to be the primary target of downstream regulation of glutamate stimulation. Thus, palmitate turnover on Gas and PSD-95 is accelerated by receptor activation, contributing to downregulation of the signaling pathway. In contrast, we recently found that a certain situation, synaptic activity blockade, enhances PSD-95 palmitoylation level by newly occurring palmitoylation (Noritake et al., unpublished data). Also, a previous report showed that the half-life of palmitate on PSD-95 increases (from 2 to 4 h) upon activity blockade [29]. These observations indicate that when synaptic activity is reduced, PAT activity increases and PPT activity decreases, leading to incremental increases in palmitoylated PSD95. Because increased palmitoylated PSD-95 recruits more AMPA receptors, regulated palmitate cycling on PSD-95 by the activity blockade may be involved in homeostasis of glutamate receptors. How is PAT activity regulated by extracellular signals? Several regulatory mechanisms of DHHC PATs considered include (1) posttranslational modifications, such as phosphorylation and S-nitrosylation, (2) some second messengers, such as phospholipids, cAMP, and metal ions, and (3) some regulatory proteins. In fact, in yeast, Akr1 palmitoylating activity toward Yck2 casein kinase is increased by ATP in vitro [46]. Further intensive studies are required to address this important issue. The subcellular localization of DHHC PATs should provide another regulatory mechanism for PATs. Igarashi’s group [60] systematically characterized the subcellular localization of tagged DHHC proteins in HEK293T cells and found that most of the DHHC proteins are localized in the ER and/or Golgi compartment. Although such relative examination is very important, the localization of the overexpressed protein is not necessarily the same as that of the endogenous one. Also uncertain is the cellular locus of DHHC proteins in highly polarized neurons, which extend two different kinds of processes: a long, thin axon and relatively short dendrites (Fig. 6). Keller et al. [73] first reported that endogenous DHHC3 is localized at the Golgi in primary cultured neurons. Recently, our group confirmed this localization by siRNA knockdown of DHHC3 [35]. Given that DHHC3 localizes exclusively to the Golgi and mediates much of protein palmitoylation (Fig. 4), it is conceivable that DHHC3 mainly mediates the protein palmitoylation during or just after protein synthesis rather than the synaptic activity-regulated palmitoylation that may occur near the postsynapse. The other PAT, which is localized near the synapse, may be responsible for the activity-regulated PSD-95 palmitoylation. DHHC17/HIP14 is reported to be associated with several vesicular structures, including the Golgi as well as sorting/recycling and late endosomal structures in neurons [72], and be primarily localized at presynaptic terminals [57,58,72,80]. Since DHHC17/HIP14 preferentially palmitoylates presynaptic proteins, such as SNAP-25 and CSP, DHHC17/HIP14 may serve as an axonal PAT in neurons [57,58,72,80]. The detailed analysis of PAT localization by the

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Fig. 6. Differential localization of DHHC proteins in neurons. Neurons contain two distinct types of processes, a single axon (orange) and multiple dendrites (gray) (lower right). Axons and dendrites differ from each other in their structural components and the composition of their proteins and organelles. Axons are typically long and thin, whereas dendrites are relatively short and thick. Large DHHC family proteins differentially localize in polarized neurons. DHHC3 is specifically localized at the Golgi and mediates palmitoylation of various substrates, suggesting that DHHC3 mainly mediates protein palmitoylation during or just after protein synthesis (upper left). By contrast, DHHC17/HIP14 preferentially localizes at the axon terminal and palmitoylates presynaptic proteins, such as SNAP-25 and CSP (lower left) [57,58,72,80]. Some DHHC proteins (indicated as DHHC-X, orange) may be localized in dendrites and palmitoylate postsynaptic scaffolds such as PSD-95, which determines the number of synaptic AMPA receptor/stargazin complex (upper right).

specific PAT antibodies combined with live-cell imaging will clarify the intracellular site of palmitoylation and spatio-temporal regulation of PAT-mediated palmitoylation. 4. Physiological roles of DHHC PATs The genetic studies in model organisms have elucidated the physiological significance of some DHHC proteins. Very recently, Drosophila DHHC17/HIP14 (dHIP14) was identified as an essential gene for synaptic transmission [57,58]. In flies with dHIP14 mutant photoreceptors, the synaptic components of electroretinograms are completely absent [58]. Also, loss of dHIP14 causes mislocalization of palmitoylated presynaptic proteins SNAP-25 and CSP, suggesting that dHIP14 palmitoylates these presynaptic proteins in vivo and regulates their presynaptic targeting and functions. Importantly, dHIP14 primarily localizes in presynaptic terminals. Ohyama et al.

[57] isolated dHIP14 during forward genetic screening for novel proteins that affect synaptic transmission in electroretinograms. In the dHIP14 mutant fly, evoked neurotransmitter release at the neuromuscular junction is impaired and that CSP is severely mislocalized because of loss of palmitoylation. Expression of wild-type dHIP14 in neurons (i.e., presynaptic expression) could rescue the mutant phenotypes. These works clearly established dHIP14 protein as a presynaptic PAT and dHIP14’s role in controlling neurotransmitter release by trafficking CSP and SNAP-25 to the presynapse. Systematic knockdown of DHHC genes should provide us with unexpected physiological functions of DHHC proteins. When DHHC2 (but not DHHC3, 4, 8, 17, 18, and 21) was knocked down, A431 epithelial cells showed dispersed and scattered phenotype, indicating that DHHC2 is essential for cell–cell association through its substrates, tetraspanins CD9 and CD151 [83]. As mutations or

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reduced expression of DHHC2 were found in various human cancers with metastasis [64], misregulation of DHHC2 may be involved in cancer metastasis. Human genetic evidence suggests that ZDHHC8/DHHC8 is associated with schizophrenia [66,68], although several reports do not support this linkage [84–86]. ZDHHC8/DHHC8 knockout mice had a deficiency in prepulse inhibition and a decrease in exploratory activity in a new environment [68]. This abnormal behavior of knockout mice supports the hypothesis that DHHC8 may be a risk factor for schizophrenia. Further human genetic linkage studies and identification of physiological substrates of DHHC8 will contribute to clarifying the function of DHHC8. Further genetic loss-of-function mutants in the DHHC family in various model organisms will be indispensable for elucidating the physiological roles of PATs. 5. Future perspectives Two important questions remain unanswered in the field of protein palmitoylation. The most frequently asked question is what are the physiological PPTs? Ten years ago, Duncan and Gilman [87] purified a potential PPT from rat liver cytosol fraction based on the thioesterase activity toward [3H]palmitoyl-Gai. APT1 depalmitoylates Gas and H-Ras in vitro, and expression of APT1 accelerates the depalmitoylation of Gas in HEK293 cells [87]. Overexpressed APT1 was also shown to accelerate the depalmitoylation of eNOS in COS7 cells [88]. To clarify the physiological significance of APT1, loss-of-function analyses in mammalian cells are required. Although APT1 is a promising candidate for cytosolic PPT, it remains unknown whether APT1 and its family molecules are general PPTs as DHHC proteins for PATs. Because palmitoyl proteins are tightly anchored to the PM, we wonder whether some PPTs are membrane-bound or transmembrane proteins. Now, we can see the dynamic relocalization of several palmitoyl proteins from membrane to cytosol upon 2-bromopalmitate treatment. If the candidate gene for PPTs is knocked down, the relocalization induced by 2-bromopalmitate should be inhibited. This scenario may allow us to identify the physiological PPTs by forward genetic screening or genome-wide knockdown. As another difficult and important issue, how can we visualize the dynamic cycling of palmitoylated–depalmitoylated states of proteins? As the development of the phospho-specific antibody greatly contributed to the field of protein phosphorylation, specific visualization of the palmitoylated protein should be helpful. However, nobody has succeeded in generating such antibodies, possibly due to the difficulty of preparing the antigen, which is modified by lipid palmitate. New inventions on bioprobes to visualize spatiotemporal dynamics of endogenous palmitoylated proteins are awaited. Very recently, global palmitoyl proteomes have revealed numerous palmitoyl substrates in yeast and mammals [53,89]. Because key molecules of essential signal transduction pathways are often regulated by palmitoylation, defining molecular mechanisms that control palmitoylation cycles will lay the foundation for better understanding and treatment of various diseases. In fact, mutations of DHHC proteins in humans are associated with various diseases, including cancers and neurological disorders (Section 3.2). The DHHC enzyme family and unidentified PPTs may become ideal therapeutic targets. Acknowledgements We thank Dr. David S. Bredt (Eli Lilly and Company) for the kind gift of PSD-95 cDNA, and Dr. Franck Perez (Curie Institut) for valuable discussion. R.T. and J.N. are supported by the Japan Society for

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