Current Protocols in Protein Science

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1

Current Protocols in Protein Science

UNIT 14.2
(original pub 1996)

Analysis of Protein Acylation


This is the post
-
peer
-
reviewed (but not final) version of the following article:
Zeidman, R.
,
Jackson, C.S.
, and
Magee, A.I. 2009.
Analysis of Protein Acylat
ion.
Curr. Protoc. Protein Sci.

55:14.2.1
-
14.2.12. © 2009 by John Wiley & Sons, Inc., which has been published in final form
at
http://www.mrw.interscience.wiley.com/emrw/9
780471140863/cp/cpps/toc



Original authors:
Caroline S. Jackson and Anthony I. Magee

National Institute for Medical Research

London, United Kingdom


List of current authors and affiliations:

Ruth Zeidman
, Caroline S. Jackson

and Anthony I. Magee

Molecula
r Medicine

National Heart & Lung Institute

Imperial College London

London SW7 2AZ

UK


Author for correspondence, with full mailing address, tel, fax, email:

Anthony I. Magee

Molecular Medicine

National Heart & Lung Institute

Imperial College London

Sir

Alexander Fleming Building

South Kensington campus

London SW7 2AZ


2

UK

Tel. +44 (0)20 7594 3135


FAX +44 (0)20 7594 3015




2 figures

0 tables

0 multi
-
line equations


3
-
7 key terms for indexing:

Acylation

Palmitoylation

Myristoylation

Fatty acids

Protein mo
dification



Abstract of up to 150 words:


Proteins can be acylated with variety of fatty acids attached by different covalent bonds,
influencing among other things their function and intracellular localization. This unit describes
methods to analyse prote
in acylation, both the levels of acylation and also the identification of the
fatty acid and the type bond present in the protein of interest. Protocols are provided for metabolic
labelling of proteins with tritiated fatty acids, for the utilization of the

differential sensitivity to
cleavage of different types of bonds in order to distinguish between them, and for the separation
of the fatty acids associated with proteins by thin layer chromatography enabling their
identification.


3

INTRODUCTION

Protein acy
lation is the covalent attachment of fatty acids to a protein; the most commonly added
fatty acids are myristate (14:0) and palmitate (16:0).
Incorporation of radiolabeled fatty acids into
the protein of interest is still the “gold standard” for analysis o
f this modification.
First,
radiolabeled fatty acids are used to label eukaryotic cells in vitro (see
Basic Protocol 1
). The
radiolabeled material produced can then be analyzed by various methods: the type of fatty acid
linkage can be determined (see
Basic

Protocol 2
), the nature of the protein
-
bound label can be
determined to check for interconversion (see
Basic Protocol 3
), and the protein
-
bound fatty acid
can be identified (see
Basic Protocol 4
).


BASIC PROTOCOL 1:
BIOSYNTHETIC LABELING WITH FATTY ACIDS

To identify proteins that are modified with fatty acid groups, cultured cells are incubated first in
medium containing sodium pyruvate, which acts as a source of acetyl
-
CoA and minimizes
interconversion of the fatty acid to other metabolites, and then wit
h [
3
H]fatty acids. Fatty acids
tritiated at positions 9 and 10 provide the best combination of high specific activity and
detectability for
in vitro

labeling, and because the tritium label is
distant

from the carboxyl end
where

-
oxidation occurs, reincorp
oration of label is minimized.


Materials

Cells for culture

Complete tissue culture medium appropriate for cells

Labeling medium: complete tissue culture medium containing the relevant dialyzed serum and
1

mM sodium pyruvate, 37

C

5 to 10
µ
Ci/
µ
l [9,10(
n
)
-
3
H]fatty acid, e.g., [9,10(
n
)
-
3
H]palmitic acid or [9,10(
n
)
-
3
H]myristic acid (30
to 60 Ci/mmol; Amersham
GE Healthcare
, American Radiolabeled Chemicals, or NEN
PerkinElmer
) in ethanol

PBS, pH 7.2

(
APPENDIX 2E
), ice
-
cold


4

1% (w/v)
SDS

or
SDS sample buffer

(for

SDS
-
PAGE, when using adherent or nonadherent cells
respectively;
UNIT 10.1
)
or

RIPA lysis buffer

(for immunoprecipitation;
UNIT 13.2
)

5


SDS sample buffer

(see recipe)


Cell scrapers

Nitrogen gas


Additional reagents and equipment for immunoprecipitation (
UNIT 13.2
), SDS
-
PAGE (
UNIT
10.1
), treating a gel with sodium salicylate (
UNIT 14.3
) or DMSO/PPO solution (
UNIT 10.2
), and
fluorography
(
UNIT 10.2
)


NOTE:

All reagents and equipment coming into contact with live cells must be sterile, and proper
sterile technique should be used accordingly.


NOTE:

All culture incubations are performed in a humidified 37

C, 5% CO
2

incubator unless
otherwise

specified.


1. On the day before the labeling experiment, split the cells into fresh complete tissue culture
medium.


Set up the cells at two split ratios; then choose the culture closest to 70% to 80% confluency for
labeling.


2. The next day, replace th
e medium with a minimum volume of 37

C labeling medium. Incubate
1 hr.



5

Cells in suspension should be used at a cell density of 10
6

to 10
7

cells/ml. For adherent cells that
are 70% to 80% confluent, the minimum amount of medium necessary to cover the dish

e.g.,
1.5 ml for 60
-
mm dishes and 3
ml for 100
-
mm dishes

should be used.


3. Add
2

to 10
µ
Ci/
µ
l [9,10
(
n
)
-
3
H]fatty acid to a concentration of 50 to 500
µ
Ci/ml. Incubate up to
24 hr.


Cells vary in the rate and extent of incorporation (see
Critical Parameters
), so both the amount of
label and
the duration of incubation need to be optimized. Labeling cells overnight in the
presence of 200
µ
Ci/ml [
3
H]fatty acid will maximize the chances of detecting labeled proteins.
The amount of label and/or time of incubation can then be reduced if good incorp
oration of label
is achieved, or increased if poor incorporation is attained.


Short labeling times (e.g., pulses on the order of minutes up to 2 hr) require amounts of label at
the higher end of the indicated range. In this case, uptake is relatively low
and the medium plus
label can be reused one or more times. The level of label in the medium can be monitored by
scintillation counting. For longer incubations the interconversion of fatty acids becomes a greater
problem, and the protein
-
bound fatty acid la
bel should be analyzed (see Basic Protocols
3

and
4
).


If the [
3
H]fatty acid is not supplied in ethanol or if the concentration is too low, remove the solvent
by blowing nitrogen over the solution in its original container until dry
. Be careful to remove a
ll
traces of potentially toxic solvent e.g. toluene.

D
issolve the label in ethanol at a concentration of
2

to 10
µ
Ci/
µ
l. Do not transfer into another container or evaporate the solvent in a plastic
container, as this will cause a significant loss of label

that will adhere to the side of the container.


For adherent cells


6

4a. Place the dish on ice and aspirate the medium. Wash the cells twice with ice
-
cold PBS and
lyse the cells by adding 1% SDS for SDS
-
PAGE (
UNIT 10.1
)
or

RIPA lysis buffer for
immunoprecip
itation (
UNIT 13.2
), using 100
µ
l of 1% SDS for a 60
-
mm dish or 300
µ
l for a 100
-
mm dish, or 1 ml RIPA lysis buffer.


CAUTION: Radioactive medium and washes must be disposed of appropriately.


5a. Using a cell scraper, remove the lysed cells from the dish
and transfer them to a 1.5
-
ml
microcentrifuge tube. Add 20
µ
l lysate to 5
µ
l of 5


SDS
-
PAGE sample buffer. Use all of RIPA
lysate for immunoprecipitation. Resuspend immunoprecipitate in 20
µ
l SDS sample buffer.


For SDS
-
PAGE, use DTT at a concentration

20 mM, and do not boil the samples, but incubate
them only 3 min at 80

C.

T
his is n
ecessary because the thioester linkage of the fatty acid is
susceptible to cleavage

by nucleophiles
.

In this respect DTT is a safer option, but

-
mercapto
-
ethanol can be used with caution.


For nonadherent cells

4b. Microcentrifuge the cell suspension 1 mi
n at 6000 rpm, 4

C, to pellet the cells. Decant the
supernatant and wash the cell pellet once by resuspending it in 1 ml ice
-
cold PBS and
centrifuging again.


5b. Lyse the cells by resuspending the cell pellet in 100
µ
l SDS
-
PAGE sample buffer for
discontinuous SDS
-
PAGE (
U
NIT 10.1
)

or

1 ml RIPA lysis buffer for immunoprecipitation (
UNIT
13.2
) for 10
6

to 10
7

cells. Resuspend immunoprecipitate in 20
µ
l SDS sample buffer.


CAUTION: Radioactive medium and washes must be disposed of appropriately.


For analysis of total protein
-
bound fatty acid label, lyse the cells in 100
µ
l 1% SDS.


7


For SDS
-
PAGE, use DTT at a concentration

20 mM, and do not boil the samples, but incubate
them only 3 min at 80

C
. T
his is necessary because the thioester linkage of the fatty acid is
susceptible to cleavage

by nucleophiles
.

In this respect DTT is a safer option, but

-
mercapto
-
ethanol can be us
ed with caution.


6. Analyze whole
-
cell lysate or immunoprecipitate on an SDS
-
PAGE minigel, using 20
µ
l lysate
per lane. Store remaining lysate at

20

C.


7. Treat the gel with sodium salicylate (
UNIT 14.3
) or DMSO/PPO solution (
UNIT 10.2
). Using
preflashe
d film, fluorograph the gel (
UNIT 10.2
)

at

8
0

C.


Typical exposure times are overnight to 1 month.

Usually, a one week test exposure would be
done and subsequent exposure times are adjusted depending on the result.


BASIC PROTOCOL 2:
ANALYSIS OF FATTY AC
ID LINKAGE TO PROTEIN

To determine the type of linkage by which the [
3
H]fatty acid is attached to the protein (i.e.,
thioester, oxyester, or amide linkage), the fatty acid is selectively cleaved from the protein. The
most convenient method is to run replic
ate lanes on an SDS
-
PAGE gel, cut the lanes apart, and
analyze each lane separately.


Materials

Lysate or immunoprecipitate from [
3
H]fatty acid
-
labeled cells (see
Basic Protocol 1
, step 6)

0.2 M potassium hydroxide (KOH) in methanol

Methanol

1 M hydroxylam
ine

HCl, titrated to pH 7.5 with NaOH

1 M Tris

Cl, pH 7.5

(
APPENDIX 2E
)



8

Additional reagents and equipment for SDS
-
PAGE (
UNIT 10.1
), treating a gel with sodium
salicylate (
UNIT 14.3
) or DMSO/PPO solution (
UNIT 10.2
)
, and fluorography (
UNIT 10.2
)


1. Run an

SDS
-
PAGE gel (
UNIT 10.1
) using 20
µ
l lysate or immunoprecipitate from [
3
H]fatty
acid
-
labeled cells in each of four lanes.


2. Cut the four lanes apart and transfer each lane to a 15
-
ml tube containing one of the following
solutions:


0.2 M KOH in methanol

Methanol

1 M hydroxylamine

HCl

1 M Tris

Cl, pH 7.5.


Incubate 1 hr at room temperature with shaking.


The 0.2 M KOH in methanol will cleave thio
-

and oxyesters, but not amides; 1 M
hydroxylamine

HCl will rapidly cleave thioesters but will cleave oxyesters only poorly, and will not
cleave amides. Methanol and 1 M Tris

Cl serve as controls.


3. Wash each gel strip three times, 5 min each time, with water. Treat the strips with sodium
salicylate (
UNIT 14.3
) or DMSO/PPO solution (
UNIT 10.2
),
and fluorograph using preflashed film
at

8
0

C.


Typical exposure times are overnight to 1 month.

Usually, a one week test exposure would be
done and subsequent exposure times are adjusted depending on the result.

Cleavage is
measured as a reduction in the fluorographic signal compared to those for
controls, and can be
quantitated by densitometric scanning of the lane or scintillation counting of excised bands.


9

Bands with fatty acids linked to the protein by thioesters will be missing or greatly reduced in
lanes treated with 0.2 M KOH in methanol an
d 1 M hydroxylamine

HCl; oxyesters will be greatly
reduced or missing in the lane treated with 0.2 M KOH in methanol and may be slightly reduced
in the lane treated with 1 M hydoxylamine

HCl; and amide linkages will not be affected by any of
these treatmen
ts, so that proteins with amide
-
linked fatty acids will appear in all four lanes.


BASIC PROTOCOL 3:
ANALYSIS OF TOTAL PROTEIN
-
BOUND FATTY ACID LABEL IN
CELL EXTRACT

Due to problems of interconversion of fatty acids by

-
oxidation and chain elongation and

of
reincorporation of label into other metabolic precursors, the protein
-
bound label derived from
[
3
H]fatty acids should
ideally
be analyzed, especially for experiments with long labeling
incubations. This protocol is used to determine how much of the lab
el has been converted into
other fatty acids or metabolites during the incubation; a different procedure must be used to
determine whether the fatty acid on the protein of interest is different from that added during
labeling (see
Basic Protocol 4
).


Mater
ials

0.1 M HCl/acetone,

20

C

Lysate from [
3
H]fatty acid
-
labeled cells in 1% SDS (see
Basic Protocol 1
, step 4a or 5b)

1% (w/v)
SDS

2:1 (v/v) chloroform/methanol

Diethyl ether

6 M HCl (concentrated HCl diluted 1:1 with H
2
O)

Hexane

5 to 10
µ
Ci/
µ
l [9,10(
n
)
-
3
H]fatty acid standards (30 to 60 Ci/mmol; Amersham
GE Healthcare
,
American Radiolabeled Chemicals, or NEN
PerkinElmer
) in ethanol

90:10 (v/v) acetonitrile/acetic acid

EN
3
HANCE spray (
PerkinElmer
)


10


15
-
ml polypropylene centrifuge tubes

Mistral 3000i benchtop

centrifuge with swing
-
out four
-
bucket rotor or equivalent

Nitrogen gas

30
-
ml thick
-
walled Teflon container with an air
-
tight screw top

110

C oven

Thin
-
layer chromatography tank

RP18 thin
-
layer chromatography plate (e.g., Merck)

Kodak
BioMax MS

film, preflashed


Precipitate protein

1. Add 5 vol of 0.1 M HCl/acetone to 100
µ
l lysate from [
3
H]fatty acid
-
labeled cells in 1% SDS in
a 15
-
ml polypr
opylene tube. Incubate

1 hr at

20

C.


This will precipitate the protein.


2. Centrifuge 10 min at 1500


g

(1000 rpm in Mistral 3000i swing
-
out rotor), 4

C, to pellet the
precipitate. Remove the supernatant and allow the pellet to air dry gently.


Remove

free label

3. Dissolve the pellet in a minimum volume of 1% SDS and transfer to a 1.5
-
ml microcentrifuge
tube. Add 5 vol of 0.1 M HCl/acetone. Incubate

1 hr at

20

C.


4. Repeat steps 2 and 3.


These precipitation steps concentrate the protein and remove

much of the SDS and free label.



11

5. Add 500
µ
l of 2:1 chloroform/methanol and vortex. Centrifuge 10 min at 1000 rpm, 4

C, and
remove the supernatant. Repeat this step at least three times until no more free label is extracted
into the organic solvent, as
determined by scintillation counting of the supernatant.


6. Add 100
µ
l diethyl ether to the pellet and vortex. Centrifuge 10 min at 1000 rpm, 4

C, and
decant the supernatant. Dry the pellet by placing the microcentrifuge tube under a gentle stream
of nitr
ogen.


7. Place the tube into a 30
-
ml thick
-
walled Teflon container with a air
-
tight screw top containing 1
ml of 6 M HCl. Flush the tube and container with nitrogen. Close the lid tightly and incubate in an
oven 16 hr at 110

C.


This hydrolyzes the fatty
acids from the protein.


Extract hydrolyzed fatty acids

8. Extract the contents of the tube twice with 0.5 ml hexane and pool the extracts. Dissolve the
residue in 0.5 ml of 1% SDS. Determine the radioactivity in the hexane extracts and in the
residue.


Fa
tty acids will be extracted into hexane, while label incorporated into sugars and amino acids
will be mainly in the hexane
-
insoluble residue.


9. Evaporate the hexane extracts just to dryness with a gentle stream of nitrogen. Dissolve in 2 to
5
µ
l of 2:1 c
hloroform/methanol.


It is important not to overdry the sample because it may then be difficult to dissolve.


Identify fatty acids


12

10. Preequilibrate a thin
-
layer chromatography tank with 90:10 acetonitrile/acetic acid for 15 min.


11. Spot resuspended hex
ane extract onto an RP18 thin
-
layer chromatography plate. Dilute 1
µ
l
[9,10(
n
)
-
3
H]fatty acid standards in ethanol to give 1
µ
Ci/
µ
l and spot 0.5
µ
l in parallel lanes.
Develop the plate in 90:10 acetonitrile/acetic acid. Air dry the plate.


12. Detect the ra
dioactivity by spraying the plate with En
3
hance spray and exposing it to
preflashed Kodak
BioMax MS

film overnight

or longer

at

8
0

C. Identify the fatty acids.


See
Figure 14.2.1

for an example of a typical fluorogram.


BASIC PROTOCOL 4:
ANALYSIS OF FATT
Y ACID LABEL IDENTITY

This protocol is used to identify the labeled fatty acid(s) associated with a specific protein band
on an SDS
-
PAGE gel. Following electrophoresis, the band of interest is located either by
comparison with molecular weight standards or

by fluorography of a sodium salicylate
-
treated gel
(see
UNIT 14.3
). DMSO/PPO
-
treated gels cannot be used. The labeled material is analyzed by
thin
-
layer chromatography.


Materials

SDS
-
PAGE gel of lysate from [
3
H]fatty acid
-
labeled cells

Additional reagent
s and equipment for analysis of protein
-
bound label (see
Basic Protocol 3
)


1. Excise the band(s) of interest from a wet or dried (fluorographed) SDS
-
PAGE gel. Wash three
times with shaking, 5 min each, with 0.5 ml water.


If the gel is fluorographed it sh
ould be treated with sodium salicylate (
UNIT 14.3
), not
DMSO/PPO solution. The dried gel piece will rehydrate and the salicylate will be washed out
during the washes.


13


2. Place the gel piece in a 1.5
-
ml microcentrifuge tube and lyophilize.


3. Hydrolyze th
e fatty acids in the band and identify them by thin
-
layer chromatography (see
Basic Protocol 3
, steps 7 to 12).


REAGENTS AND SOLUTIONS

Note

Use Milli
-
Q
-
purified water or equivalent in all recipes and protocol steps. For common stock
solutions, see
APPENDI
X 2E
; for suppliers, see
SUPPLIERS APPENDIX
.


SDS sample buffer (for discontinuous systems), 5



3.125 ml
1 M Tris

Cl, pH 6.8

(0.313 M final)

1 g SDS (10% final)

5 mg bromphenol blue (0.05% final)

5 ml glycerol (50% final)

H
2
O to 10 ml

Store at room temperature

Add
DTT

to appropriate concentration just before use


Warm the solution before use becau
se it tends to solidify.


COMMENTARY

Background Information

The two most common acyl groups that modify proteins are 14
-
and 16
-
carbon saturated fatty
acids,
myristic and palmitic acid
, respectively

(
Fig. 14.2.2
), and they occur both on different and
on ove
rlapping sets of proteins. By increasing the hydrophobicity of the protein, these fatty acid
moieties can play a role in localization of the protein to the membrane and sometimes to specific

14

types of membrane structures

e.g.
cholesterol
-

and sphingolipid
-
rich lipid rafts (
Zacharias

et al.,
2002)
. Identifying the type of acylation of a protein and determining whether the level of
modification can be affected by stimuli can provide more information on the mechanisms of
a
ction of proteins involved in signaling pathways.


Fatty acids are used in labeling cells
in vitro

because they will diffuse across the plasma
membrane and then be converted to acyl
-
CoA by the action of the enzyme acyl
-
CoA synthetase.
This activated form o
f the fatty acid is the substrate for protein
-
acyl transferases

(PATs)

that
transfer the acyl group to the protein. Tritiated fatty acids are most commonly used in biosynthetic
labeling of proteins, but fluorescent analogs of fatty acids have been used to
study palmitoylation
of rhodopsin (
Moench et al., 1994a,b
), and [

-
125
I]iodo
-
fatty acids have been used to study
myristoylation of v
-
src (
Peseckis et al., 1993
)

and palmitoylation of

Sonic hedgehog (Buglino and
Resh, 2008)
.
ω
-
azido
-
fatty acids can also be
used to metabolically label cells, followed by
labeling with phosphine
-
biotin and detection in a Western blot with streptavidin
-
HRP

(Hang et al.,
2007)
.

Acylation can also be detected by che
mically labe
ling the fatty acids
in lysed cells using
the acyl
-
bio
tin exchange (ABE) method
. After blocking free sulfhydryl groups palmitoyl
-
thioester
bonds are cleaved, generating a free
sulfhydryl group that is labe
led with a sulfhydryl
-
specific
biotin
-
conjugated compound (1
-
biotinamido
-
4
-
[4

-
(maleimidomethyl
)cyclohexa
necarboxamido]butane ;Btn

BMCC
), which subsequently can be
detected by streptavidin
-
HRP
(Drisdel and Green, 2004
, Drisdel at al., 2006, Wan et al., 2007
)
.

With
methods that rely on a chemical reaction for detection of acylation

there is a concern that
the
level
of
acylation
might be underestimated
due to inefficiency of the chemical
reaction
.
Furthermore,

in the ABE method,
palmitoylation may be below the detection threshold if the level
of palmitoylation is low or the turnover rate high, as

only steady sta
te levels
are measured
. The
ABE method can also give false positives, e.g. with other types of this ester. The reader is refered
to the original publications for these methods.Therefore, metabolic labe
ling with tritiated fatty
acids is still the most relia
ble and commonly used acylation detection method.



15

A wide variety of proteins are myristoylated, including viral structural proteins and many proteins
involved in cell signaling, such as the


subunits of trimeric G proteins
,
cytoskeletal
-
bound
anchoring proteins,

and the Src family of tyrosine kinases (
Resh, 1999
). Myristoylation
,
usually
an
irreversible modification,

most commonly occurs co
-
translationally via an amide linkage to an
NH
2
-
term
inal glycine residue

but it has also been reported to occur post
-
translationally
for

several
proteins,
including

PAK2 (Vilas et al., 2006)
,

gelsolin (
Sakurai

and
Utsumi
, 2006), actin (Utsumi et
al., 2003) and BID (Zha et al., 2000) following caspase cleavage.

Myristoylation is dependent on
the removal of the initiator methionine and has been s
hown to occur by the time the nascent
polypeptide is 100 amino acids long. Inhibitors of protein synthesis will therefore block
myristoylation. The enzyme responsible for NH
2
-
terminal myristoylation,
N
-
myristoyl transferase
(NMT), was first isolated from
S
accharomyces cerevisiae
; both the yeast and human homologs
have been well characterized and show different protein substrate specificities . Substrate
specificity of yeast NMT is determined by recognition of a sterically unhindered NH
2
-
terminal
glycine fol
lowed by an amino acid sequence that conforms to the following criteria:
no
charged
residue or proline at position 2; any amino acid at positions 3 and 4; serine, alanine,
glycine,
cysteine, asparagine
or threonine at position 5;
and
no proline at position

6
(
Farazi et al. 2001)
.
Residues C
-
terminal to this region appear to be important in recognition of the


subunits of
trimeric G proteins and the Src family of tyrosine kinases (
Glover et al., 1988
;

Gordon et al.,
1991
).


The NMT enzymatic reaction procee
ds by formation of a myristoyl
-
CoA
-
enzyme complex,
subsequent binding of the peptide, transfer of the myristate moiety to the peptide, release of CoA,
and release of the myristoylated peptide (
Rudnick et al., 1991
). Several assays have been
developed for t
his enzyme (
e.g., King and Sharma, 1991
;

Rudnick et al., 1992
;

French et al.,
1994
, Pennise et al. 2002; Takamune et al., 2002
).
A large number of NMT inhibitors have been
identified

(reviewed in
Selvakumar

et al., 2007)
, includ
ing proteins, histidine anal
og
s,
myristic
acid
analog
s and

myristoyl
-
CoA

variants

which
inhibit acyl CoA synthetase and therefore block the

16

conversion of fatty acids to acyl CoA;

including 2
-
hydroxymyristic acid that is converted
to
2
-
hydroxymyristoyl
-
CoA, a potent inhibitor of NMT
,
and other synthetic organic compounds
.


Myristoylation of a protein can be necessary for its activity, e.g., the transforming activity of Src
(
Kamps et al., 1986
). Myristoylation alone may not be sufficient for a protein to be localized to the
membrane; fo
r this further lipid modification, such as palmitoylation
, prenylation

or cooperative
interaction with protein sequences, is required (
Resh,
2006
a
).


Palmitoylation (with and without myristoylation) occurs on many signaling molecules, including
rhodopsin,

-
subunits of G proteins,
Ras, G
-
protein coupled receptors
and Src
-
family tyrosine
kinases. Palmitoylation is a post
-
translational event occurring via a thioester linkage to a cysteine
residue
. Sometimes, the thioester bond can be chemically rearranged to
form a
stable attachment
of the palmitate through an amide bond
to an immediately adjacent N
-
terminal glycine
instead
.
This is seen in, for
instance, Hh/Shh (Pepinsky et al., 1998) and
G

s

(Kleus
s and Krau
se, 2003).

W
here it occurs with NH
2
-
terminal myrist
oylation, the myristoylation is usually a prerequisite for
palmitoylation. This may be because the enzyme responsible for palmitoylation recognizes the
myristoylated protein or, more likely, because myristoylation brings the protein to the correct cell
loc
ation for palmitoylation to occur. Palmitoylation is also found in conjunction with
prenyl

modification at the C
-
terminus of proteins belonging to the Ras superfamily; it is responsible for
the localization of these proteins to the membrane (
Newman and Mag
ee, 1993
). This
palmitoylation is dependent on prior modification of the protein by
prenylation
. G protein subunits
such as the

1

subfamily and members of the Src family of tyrosine kinases have an NH
2
-
terminal amino acid sequence of Met
-
Gly
-
Cys, where th
e initiator methionine is removed and
replaced by myristate and the cysteine is palmitoylated (
Resh, 1994
). Palmitoylation is a
reversible modification and has been shown to be dynamic in vivo, with the level of palmitoylation
changing in response to vario
us stimuli such as receptor activation, insulin, and growth factors
(
James and Olsen, 1989
;

Jochen et al., 1991
;

Wedegaertner et al., 1995
). This phenomenon is
thought to play a role in switching on or off signaling molecules by altering either the localiz
ation

17

of the molecules or their presentation to other signaling molecules with which they interact.

Proteins can be auto
-
palmitoylated; alternatively the process can be catalyzed by
protein
-
acyl
transferases (PATs) and there has been p
rogress in the ident
ification and characterization of
PATs in recent years
. The DHHC family of PATs are transmembrane proteins, all having
cystein
e
-
rich domains containing a conserved aspar
t
ate
-
histidine
-
histid
i
ne
-
cystein
e

(DHHC)
motif, which is required for the PAT activity.

Originally discovered in yeast (Lobo et al., 2002, Roth
et al., 2002), there are now 23
mammalian

DHHC proteins

known

(Fukata et al. 2004)
and work
is emerging describing their function and substra
tes, which are intracellular proteins, for example
Ras, G
protein subunits, eN
OS, vacuole proteins, G protein
-
coupled receptors
and the neuronal
PSD
-
95 protein

(Smotrys and Linder, 2004)
.


Some secreted proteins

and peptides
, like Hed
gehog (Hh)/Sonic hedgehog (Shh),
Wnt
s and
ghrelin
, require acylation for their f
unction and are palmitoylated by a different group of PATs
called MBOAT (membrane
-
bound
-
O
-
acyltransferase) family, which do not have any apparent
sequence homology and where only a small subset of members are known to transfer fatty acids
and other lipids
to proteins

(Miura and Treisman, 2006)
.

Rasp/Skinny hedgehog (Ski)/Hedgehog
acyl transferase (Hhat) is the PAT for Hh/Shh
(
Buglino and Resh, 2008
)
,
Porcupine (Porc) is the
PAT for Wnt (Zhai et al., 2004)

and GOAT is the PAT for ghrelin (Yang et al, 2008)
.


Proteins are depalmitoylated by acyl protein thioesterases (TEs). So far, two have been
described, APT1, which has been shown to depalmit
oyl
ate Gi

, H
-
Ras and eNOS (Duncan and
Gilman, 1998, Duncan and Gilman, 2002 and Yeh et al.
, 1999), and PPT1, which r
emoves

palmitate as a step in the protein degradation process (
Verkruyse

and Hofmann
,

1996).

I
nhibitors of
palmit
oylation include

fatty acid analog
ue
s
, including 2
-
bromopalmitate
, a non
-
metabolizable palmitate analogue
, and natural antibiotics, like ceruleni
n, which inhibits fatty acid
synthesis, and tunicamycin, which is structurally similar to palmitoyl
-
CoA


(Resh
, 2006b).

More
specific small molecule inhibitors of individual PAT subgroups are also emerging

(Ducker et al.,
2006).


18


Analysis of the type of fatty acid linkage present should always be performed. Post
-
translational
myristoylation via a thioester linkag
e has been found in platelets (
Muszbek and Laposata, 1993
).
The term "palmitoylation" is not strictly accurate because other long
-
chain fatty acids, such as
stearate (18:0) and oleate (18:1), can also be thioesterified to proteins; "
S
-
acylation"

or
“thioac
ylation”

are

becoming more commonly used to describe this modification. The acylating
activit
ies

seem to be relatively unspecific for chain length and degree of unsaturation, and utilize
acyl
-
CoAs partly in proportion to their abundance in the cell



henc
e the predominance of
palmitate. This and the potential for interconversion of labeled fatty acids necessitate analysis of
the chain length of the attached label. Other more complicated methods can be used to identify
the type of fatty acid attached to the

protein; these include gas chromatography, reversed
-
phase
HPLC, and mass spectroscopy (
Aitken, 1992
).
Mass spectrometry

can also be used for

identifying acyl chains attached to proteins,
by comparison of acylated and deacylated peptides

from the digested
protein of interest
,
giving the
stoichiometry

of the acylation and the exact mass
of the modifying group
.

(
Liang et al., 2004)


Further information about the cellular localization of acylated proteins can be found by
detergent
extraction of the cell lysate
. Extraction with Triton X
-
114
(
which has the property of phase
separation at 30

C
)

can distinguish between hydrophobic and hydrophilic proteins (
Aitken, 1992
).
Extraction of membranes with
non
-
ionic detergents

at 4

C and 37

C can identify proteins that are
associated with
detergent resistant membranes (DRMs)

and provide preliminary e
vidence for
association with lipid rafts (LR) or membrane
lipid

microdomains

(Janes et al, 1999)
. Full
characterization of lipid raft/membrane lipid microdomain association requires several
complementary techniques and is beyond the scope of this review
.


Critical Parameters and Troubleshooting

Cells should be subconfluent; it is recommended that the cells be plated at two split ratios so the
culture closest to 70% to 80% confluency can be selected to be used for labeling.


19


The pH of all solutions should be


7.5 in order to avoid hydrolysis of labile thioesters; the high pH
of SDS
-
PAGE buffers does not seem to be a problem when using minigels, where the running
times are relatively short. Dithiothreitol (DTT) or 2
-
mercaptoethanol should be used with care, as

these will also cleave thioester bonds; for SDS
-
PAGE a maximum concentration of 20 mM DTT
should be used and samples should not be boiled but incubated only 3 min at 80

C.


The use of short labeling times (especially for palmitoylation) will reduce reinco
rporation of the
label.


The ability to detect myristoylation and/or palmitoylation of a protein using these methods will
depend on the ability of the cells to take up radiolabeled compounds and incorporate them into
metabolic precursors; the pool sizes of

endogenous fatty acids and fatty acyl
-
CoA esters; the
expression level
and activities

of

the
NMT
, PATs and thioesterases
; the abundance, rate of
synthesis, and turnover of the protein(s) and modification(s) of interest; and the efficiency of
antibodies fo
r immunoprecipitation.


Anticipated Results

Typically, it is possible to detect myristoylated proteins using fluorographic exposure times
ranging from

1 week for high
-
level expression of protein in transformed cells (e.g., Lck in
LSTRA cells) to 1 to 4 we
eks for a well
-
expressed endogenous protein.

For a poorly expressed
protein, exposure times of 1 to 3 months have been used.


Time Considerations

In vitro labeling experiments require 2 to 3 days for growing and labeling the cells. Harvesting the
cells, pr
eparing the lysate for SDS
-
PAGE (with or without prior immunoprecipitation) and SDS
-
PAGE require 1 to 2 days plus time for fluorography.



20

Linkage analysis of the labeled proteins takes 1 to 2 hr after the gel has been run. Analysis of the
label in cell lys
ates requires 1 day to precipitate the protein, remove free label, and hydrolyze the
sample. A second day is required to extract the label and perform thin
-
layer chromatography, and
fluorography requires an overnight exposure. Analysis of the protein
-
bound

label in gel bands
takes ~3 days after fluorography (dried gel) and a similar amount of time from a wet gel, except
the analysis begins after the gel has been run.


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27



Figure 14.2.1

Fluorogram of thin
-
layer chromatography plate showing analysis of acylated nerve
growth factor (NGF) receptor. Outside lanes, migration of 0.5
µ
Ci [
3
H]palmitate and [
3
H]myristate
standards. Lane 1, NGF

receptor immunoprecipitated from cells labeled with [
3
H]palmitic acid.
Lane 2, NGF receptor immunoprecipitated from cells labeled with [
3
H]myristic acid. Although the
cells were labeled with different fatty acids, the protein was labeled with palmitic aci
d due to
chain elongation of [
3
H]myristic acid to [
3
H]palmitic acid by the cells. Exposure for standards, 1
week; exposure for lanes 1 and 2, 1 month.




28





Figure 14.2.2

Structures of myristic and palmitic acids.