From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
The Acylation of Megakaryocyte Proteins: Glycoprotein IX Is Primarily
Myristoylated While Glycoprotein Ib Is Palmitoylated
By Paul K. Schick and Jean Walker
The acylation of megakaryocyte proteins was studied with
special emphasis on the myristoylationand palmitoylation
of the glycoprotein (GP) Ib complex. Guinea pig megakaryocytes were purified and separated into subpopulations at
different phases of maturation. Cells were incubated with
[3H]myristate, i3H1palmitate,or f3H1acetatet o study endogenous protein acylation. Cycloheximide was used t o distinguish between cotranslational and posttranslational acylation and hydroxylamine t o distinguish between thioester
and amide linkages. After incubations, delipidated proteins
or GPlb complex subunits, immunoprecipitated with PG-l,
AN-51, or FMC-25 monoclonal antibody, were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and assessed by fluorography. Radiolabeled
fatty acids bound t o GPlX and GPlb were also analyzed by
high pressure liquid chromatography (HPLC) and scintilla-
tion spectrometry. With [3H1myristic acid and r3HIacetate,
GPlX was found t o be a majormyristoylated protein in
megakaryocytes and CHRF-288 cells. Myristic acid was
linked t o GPlX by an amide bond, and this process occurred
cotranslationally. With I3H1acetate, GPlb was primarily palmitoylated, but with [3Hlmyristate, GPlb was acylated with
about equal amounts of myristic acid and palmitic acids.
Both fattyacids were linkedt o GPlb by thioester bonds, and
acylation was posttranslational.The myristoylation of GPlX
was found t o be most active in mature megakaryocytes,
while the palmitoylation
of GPlb occurred throughout megakaryocyte maturation. Myristoylation and palmitoylation
may have different functionsrelevant t o the assembly of the
GPlb complex in megakaryocytes.
0 1996 by The American Societyof Hematology.
T
with [3H]myristate and ['Hlpalmitate, [3H]acetatewas used
to study endogenous acylation of GPIb subunits. Cotranslational amide-linked myristoylation of GPIX was demonstrated. GPIX was primarily myristoylated and was found to
be one of the major myristoylated proteins in guinea pig
megakaryocytes. GPIb was primarily palmitoylated, and this
event occurred posttranslationally. The myristoylation of
GPIX was found to be most active at a later phase of megakaryocyte maturation than the palmitoylation of GPIb.
HE GLYCOPROTEIN (GP) Ib complex is a disulfidelinked heterodimer of GPIb-alpha and GPIb-beta that
forms a 1:l noncovalent complex with GPIX. GPV may also
be associated with the GPIb complex. The complex is a
receptor for von Willebrand factor (vWF), an adhesive molecule that is present in the subendothelium. A functional GPIb
complex expressed on the platelet surface is important for
mediating the initial phases of the adhesion of the platelets
to the subendothelium.' Abnormalities in the expression of
the GPIb complex or its subunits have been detected in
platelets from patients with the Bernard-Soulier syndrome.*
GPIb-beta and GPIX in human platelets have been shown
to be acylated by palmitic acid.' Palmitoylation is characterized by the linkage of palmitic acid to a cysteine through a
thioester bond and, therefore, can be cleaved by hydroxylamine or alkaline hydrolysis. Palmitoylation renders proteins
more hydrophobic and is believed to target proteins to specific cellular locations and to promote the anchorage of proteins to membrane^.^.^ Thus, it has been proposed that the
acylation of the GPIb complex may be animportant factor in
the expression of a functional GPIb complex on the platelet
s~rface.~
Proteins can also be acylated with myristic acid. In myristoylated proteins, the fatty acid is usually linkedto an
N-terminal glycine byan amide bond that is resistant to
hydroxylamine. The acylation of proteins with myristic acid
is usually a cotranslational event, while palmitoylation occurs po~ttranslationally.~-~
Platelets are not capable of significant protein synthesis and, therefore, would not be expected to be capable of cotranslational myristoylation.
However, thioester-linked posttranslational myristoylation
has been demonstrated in platelets.' Myristoylation and palmitoylation may have different biologic consequences, as
myristoylation has been reported to be important for the
assembly of large protein complexes by mediating proteinprotein interactions.6.8
We have studied protein acylation in megakaryocytes because megakaryocytes have the capacity for protein synthesis. This is the first study to delineate protein acylation in
megakaryocytes. In addition to assessing protein acylation
Blood, Vol 87, No 4 (February 15). 1996: pp 1377-1384
MATERIALS AND METHODS
Radiochemicals, Antibodies, and Reagents
The ['Hlmyristic acid (33.5 Ci/mmol), ['H]palmitic acid (39 Ci/
mmol), ['Hlacetate (4.13 Cilmmol), andEN'HANCE were purchased from Dupont/New England Nuclear (Boston, MA). Myristic
acid, palmitic acid, stearic acid, and their methyl esters were from
NuChek Prep (Elysian, MN). Mouse monoclonal antibody PG-l,
directed against the guinea pig von Willebrand receptor, was a gift
from Dr Johnathan Miller, and its characterization has beenreported? AK-l and SZ-l, both monoclonal antibodies against the
human GPIb-IX complex, were available commercially from Dako
(Carpinteria, CA) and Immunotech (Westbrook, ME), respectively.
AN-51, a monoclonal antibody against GPlb-alpha (Dako), and
FMC-25, a monoclonal antibody against GPIX (ICN, Costa Mesa,
CA), were also used.
From the Cardeza Foundation for Hematologic Research and the
Department of Medicine, Jefferson Medical College of the Thomas
Jefferson University, Philadelphia, PA.
Submitted April 18, 1995; accepted September 15, 1995.
Supported by National Institutes of Health Grants No. HL25455
and HL51481.
Address reprint requests to Paul K. Schick, MD, Cardeza Foundation for Hematologic Research, Jefferson Medical College, Thomas
Jefferson University, 1015 Walnut St, Philadelphia, PA 19107-5099.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advefiisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
ooO6-4971/96/8704-00$3.00/0
1377
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
1378
Isolation of Cells
Guinea pigs were used in most of the experiments in the study
because, in our experience, this species is the best available source
for the isolation of viable purified megakaryocytes and the preparation of megakaryocyte subpopulations at different phases of maturation. Human platelets were also studied and prepared as previously
described.' Guinea pig megakaryocytes and platelets were isolated
as previously described.'' Megakaryocytes were isolated to about
85% purity by cell number and greater than 98% by protein content,
as megakaryocytes are considerably larger than other bone marrow
cells. The viability of the isolated megakaryocytes was about 90%.
Megakaryocyte subpopulations at different phases of maturation
were prepared by the Celsep procedure." This procedure separates
megakaryocytes by size and can be used to prepare megakaryocyte
subpopulations that contain primarily mature megakaryocytes and
subpopulations that are highly enriched with immature megakaryocytes. The viability of the isolated subpopulations of megakaryocytes
is greater than 89%.and purity is greater than 98% by protein
content.
CHRF-288 cells" were obtained from Dr M. Lieberman (University of Cincinnati, Cincinnati, OH). The cells were grown in Fisher's
medium supplemented with 20%(vol/vol) horse serum and penicillin
(50 U/mL)/streptomycin (50 U/mL). Cultures were seeded at 3 X
IOh cells in 8 mLof culture medium in a 25-mL plastic flask and
were grown to 8 X IO' cells. All cell cultures were maintained at
37°C in a humidified incubator in the presence of 5% CO2 in air.
Incubation Conditions f o r the Acylation of Cells
Megakaryocytes. Megakaryocytes (2 X 105/mL)
were resuspended in Eagle's medium supplemented with 0.22% fatty acid-free
bovine serum albumin (BSA) and incubated with ['Hlmyristic acid
(300 pCi/mL, 8.9 pmol&), ['Hlpalmitic acid (300 pCi/mL, 7.7
pmol/L), or ['Hlacetate (66.7 pCi/mL, 16.3 pmol/L). To distinguish
between cotranslational and posttranslational acylation, cycloheximide ( I O pg/mL) was added to the incubation mediumof some
samples before the addition of radiolabeled fatty acids. Afterthe
labeling period, the mediumwas removed by Centrifugation,and
the cells were washed twice with calcium magnesium-free Hanks'
balanced salt solution with adenosine and theophylline.
CHRF-288 cells. At 54 hours after the initiation of the culture,
cells were transferred into a fresh flask and incubated with 1.3 mCi
['Hlmyristic acid (3.9 pmol/L) for 18 hours. Cells were seeded to
generate at least 8 X 10' cells after an 18-hour incubation with
the radioisotope. Cells were pelleted and washed twice with fresh
medium.
Processing and PuriJcation of Acylated Proteins
Delipidation in preparation for the assessment of total acylated
proteins. Washed and pelleted cells were incubated with 6 n L of
CHCI,:MeOH (2: I ) for 30 minutes at room temperature. The protein
was pelleted by centrifugation (4,50013 for 10 minutes at room temperature) and then extensively delipidated by sequential treatment
with 6 mL each ofCHC1':MeOH
(2:l). CHCI?:MeOH (l:2),
CHC13:MeOH:H20(l:l:O.3), and acetone.
Immunoprecipitationfor the assessment of the acylation of G P l b
subunits. The cells were first resuspended in 500 pL of calcium
magnesium-free Hank's, pH 7.4. Protease inhibitors were added to
a final concentration of 2.75 pmol/L aprotinin, 1 mmolL phenyl
methylsulfonyl fluoride (PMSF), and 1 mmoW iodoacetic acid.
Nonidet P-40 (NP-40) at 0.5% (vol/vol) was added to lyse the cells,
which were then incubated at4°C for 1 hour. Cell lysates were
centrifuged at l0,OOOg for 1 hour before immunoadsorption. Aliquots
SCHICK AND WALKER
of NP-40 lysates were incubated with 10 pg of various monoclonal
antibodies to the GPIb complex. PG-1, AN-51, and FMC-25 were
used for the lysates from guinea pig cells, and both SZ-l and AKI were used on the lysates from the human cell line. The antibodies
were precipitated with goat anti-mouse IgG conjugated to agarose
beads at4°C overnight. The beads werethenwashed extensively
with tris-buffered saline containing 0. I % NP-40. The bound material
was solubilized at 37°C with sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) sample buffer and then centrifuged
to remove the agarose beads. We compared the recovery of immunoprecipitated GPIb and GPIX by the method described above to that
in which Triton-l00 was used to lyse the cells, as the latter treatment
would recover glycoproteins associated with theskeletal membrane.'
The results of these experiments revealed that there was comparable
recovery by both methods
SDS-PAGE, Hydroxvlamine Treatment, and F1uorograph.y
of Gels
Delipidated samples or immunopurified GPIb subunits were
placed in treatment buffer for SDS-PAGE. Most samples were analyzed under nonreducing conditions, but some samples were reduced
by treatment with I % beta-mercaptoethanol. Samples were also reduced by 20 mmol/L dithiothreitol ( D V ) and heated at 80°C for 3
minutes. SDS-PAGE was performed on 5% to 15% gradient gels
and 10% gels. Gels were soaked inEN'HANCEand
dried. and
acylated proteins were detected by fluorography.
To identify a thioester-linkage, gels were washed three times for
I O minutes in dHzO. Then, gels were treated with 1 molL hydroxylamine, pH 7, or 1 mol& Tris-HCI, pH 7 (as a control), for 6 hours,
stained with Coomassie Blue, and analyzed by fluorography as described above. To identify an 0-ester linkage, gels were treated with
1 mol/L hydroxylamine, pH10,and
in other experiments, were
subjected to alkaline hydrolysis with 0.2 m o l n KOH in methanol.
Identification of Fatty Acid Linked to Protein by High
Pressure Liquid Chromatography
Methanolysis of acylated proteins. Radiolabeled gel slices containing the protein of interest were excised from slab gels after SDSPAGE, washed four times with10% MeOH, homogenized in 50
mmol/L ammonium bicarbonate containing 0.1% SDS, and incubated with 0. I mg TPCK-treated trypsin (Sigma, St Louis, MO) for
24 hours. After 6 and I8 hours, fresh trypsin at 0.1 mg/mLwas
added to the homogenate to completely digest the protein. The gel
mass was filtered from the solution using a 0.2-pm membrane, 200
pg of BSA was added as a camer, and the solution was evaporated
to dryness in a Savant Speed Vac Concentrator (Forma Sci, Marietta.
OH). Two milliliters of 83% MeOH, 17%HCI was added to each
sample, andthey were maintained under N2 for 24 hours. Each
sample was extracted three times with petroleum ether and dried.
Acetonitrile containing nonradioactive fatty acid standards palmitate,
myristate, stearate, methyl myristate, methyl palmitate, and methyl
stearate were added to each sample in preparation for analysis by
high pressure liquid chromatography (HPLC).
HPLC. Separation of the fatty acids and fatty acid methyl esters
was achieved by reverse-phase HPLC (Waters 600E System, Milford, MA) on a C18 column (Econosil 5 pm, 25mm X 4.6 mm;
Alltech Scientific Inc, Deerfield, IL) using 84% or 94% acetonitrile:
HzO at a flow rate of 1 mL/min. Detection was by ultraviolet monitoring at 205 nm. One-milliliter fractions were collected when the
mobile phase was 95% acetonitrile, while 2-mL fractions were collected whenthe mobile phase was 84%acetonitrile. The radioactivity
in the collected fractions was estimated by scintillation spectrometry.
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
1379
ACYLATION OF MEGAKARYOCYTEGLYCOPROTEIN le-IX
The retentiontimes of the radiolabeled productswere determined
by coelution with the unlabeled standards.
200 -
RESULTS
After the incubation of megakaryocytes with [3H]myristic
acid and [.'H]palmitic acid, delipidated proteins were separated by 10% unreduced SDS-PAGE, and acylated proteins
were identified by fluorography. Incubations with palmitic
acid demonstrated that the most prominent acylated proteins
were 24,27 to 29.36 to 38,61,87 to 97, and I40 kD proteins.
Similar species of proteins were found to be palmitoylated in
guinea pig platelets. There is a species difference in the
acylation of GPIX in human and guinea pig platelets. Our
experiments confirmed that in human platelets, GPIX and
GPIb are palmitoylated. GPlX is palmitoylated to a greater
extent than GPIb. However, in guinea pig platelets, GPIb
but not GPIX is palmitoylated.
With myristic acid, a 19-kD band was the most intensely
acylated protein in megakaryocytes, and other prominently
acylated proteins were 16, 40 to 48, 55 to 59, 97, and 170
kD. Both 2-hour and overnight incubations were performed,
and the results showed that there were no significant differences in the protein species that were acylated at these two
time periods. Thus, several megakaryocyte proteins were
shown to be acylated. These data are consistent with the
demonstration that a number of proteins are acylated in most
cells, although fewer proteins are acylated than phosphorylated or glyco~ylated.~
The palmitoylation and myristoylation of megakaryocyte
proteins were compared under unreduced and reduced conditions. After the incubation of megakaryocytes with radiolabeled fatty acids, the delipidated proteins were separated on
a 5% to 15% SDS-PAGE gradient gel, and acylated proteins
were detected by fluorography. Samples were reduced by
the addition either of 1 % beta-mercaptoethanol or 20 mmoll
L DTT rather than the standard 5% beta-mercaptoethanol to
avoid disruption of thioester bondsi4Figure 1 demonstrates
that several megakaryocyte proteins had been acylated. One
of the most intensely myristoylated proteins is a 19-kD protein that was evident in both unreduced and reduced gels.
This behavior is consistent with that of GPIX. In experiments
with [.'H]palmitic and ["Hlmyristic acids, a protein of about
23 kD is only evident in the reduced gel, and this band
represents GPIb-beta. In the experiment shown in Fig 1, gel
samples hadbeen reduced with 1% beta-mercaptoethanol,
but those data were similar to those of other experiments in
which gels had been reduced with DTT.
To determine whether the GPIb complex and its subunits
were acylated. megakaryocytes were incubated with radiolabeled fatty acids, the intact GPIb complex was immunoprecipitated, GPIb subunits were separated by 10% unreduced
SDS-PAGE, and acylation was estimated by fluorography.
PG- 1, a monoclonal antibody specific to intact GPlb complex
in guinea pig platelets and megakaryocytes, was used in the
experiments shown in Fig2. Figure 2A demonstrates the
palmitoylated megakaryocyte proteins immunoprecipitated
with PG-!. GPIb ( I 70 kD) was found to be palmitoylated.
The identity of the two fainter bands has notbeen deter-
116
-
97 -
66 45
-
31 21
14:O16:O
U
14:O16:O
U
REDUCED UNREDUCED
Fig 1. Palmitoylation and myristoylation in guinea pig megakaryocytes: reducingand nonreducing conditions. Guinea
pig megakaryocytes in Eagle's medium supplemented with 0.22% BSA (200.0001
mL) were incubated with ['Hlmyristate or ['Hlpalmitate 1300 pCilmL1
for 17 hours. Proteins were delipidated and separated on a 5% to
15% SDS-PAGE gradient gel, andacylated proteins were detected by
fluorography.Proteins were separated under unreduced
and reduced
conditions. Samples were reduced by the addition of 1% beta-mercaptoethanol.To each lane,200 F g protein was applied. 160, incubation with ['Hlpalmitate; 140, incubation with ['Hlmyristate.
mined. Cycloheximide, an inhibitor of protein synthesis, did
not block the palmitoylation of GPIb, as shown in Fig 2A.
Figure 2B demonstrates the myristoylated megakaryocyte
proteins immunoprecipitated with PG-l. GPIX (19 kD) and
GPIb were found to be myristoylated, but GPIX was considerably more myristoylated than GPIb. After treatment with
cycloheximide, myristoylation of GPIX was blocked, while
myristoylation of GPIb was unchanged. This suggests that
the myristoylation of GPIX is cotranslational, while the myristoylation of GPIb is a posttranslational event.
The acylation of subunits of the GPIb complex in guinea
pig megakaryocytes was also studied with AN-51, which
recognizes GPIb-alpha, and with MC-25, which can immunopurify the GPIX subunit. Both of these antibodies reacted
with their respective epitopes in guinea pig megakaryocytes.
The results of these experiments are not shown but confirmed
that GPIX was intensely myristoylated in guinea pig megakaryocytes.
The myristoylation of GPlb subunits was also studied in
CHRF-288 cells, a tumor cell line with megakaryocytic char-
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
1380
SCHICK AND WALKER
-
45
-
- 21.5 -
e (GplX)
CHCONTCHCONT
acteristics." CHRF-288 cells were studied to determine
whether the myristoylation of GPIX occurs in this cell line
and, thus, to provide indirect evidence for this event in
human megakaryocytes. Figure 3 is a fluorogram of an ex-
200 116-
97 -
66 45 Fig 3. Myristoylated GPlX in CHRF288 cells. CHRF cells were labeled with
1.3 mCi [3Hlmyristate for 18 hours and
lysed, and theGPlb complex was immunoprecipitatedby SZ-l antibody. The
immunoprecipitated sample was separated byunreduced SDS-PAGE on a 5%
t o 15% gradient gel, and acylated proteins were detected by fluorography.
31 21
-
Figthe
2. myristoylation
Palmitoylation
ofand
GPlX and GPlb complex in guinea pig megakaryocytes: effect of cycloheximide. Guinea pig megakaryocytes in Eagle's medium supplemented with 0.22%
BSA (200,00O/mL) were incubated with (AI f3H1palmitate or(B) I3H1myristate(300pCi/mL) for17 hours
in the presence or absence of cycloheximide (l0p g l
mL). Cells were lysed and immunoprecipitatedwith
PG-l antibody. The immunoprecipitated samples
were separated by unreduced 1096 SDS-PAGE, and
acylated proteins were detected by fluorography.
CONT. megakaryocytes not treated with cycloheximide; CH, megakaryocytes treated with cycloheximide.
periment in which CHRF-288 cells wereincubatedwith
['Hlmyristic acid, andthe GPIb complex wasimmunoprecipitated with SZ-l monoclonal antibody. GPlX was
heavily myristoylated, while GPlb was minimally myristoylated.
Endogenous protein acylation was studied by the incubation of megakaryocytes with ['Hlacetate, which revealed that
GPIX and GPlb were acylated. To study the acylation of
GPIb subunits, immunoprecipitated GPIb subunits were separated by SDS-PAGE, and acylated GPIb and GPlX were
eluted from the gels and subjected to acid methanolysis to
produce fatty acid methyl esters thatwere analyzed by
HPLC. Radioactivity wasmeasured by scintillation spectrometry. These experiments revealed that the principal fatty
acid linked to GPIX was myristic acid (Fig. 4C). and the
main fatty acid linked to GPIb was palmitic acid (Fig 4B).
The synthesis of fatty acid species from ['Hlacetate is shown
in Fig 4A, and the data were derived from the extraction of
megakaryocyte total lipids, the production of fatty acid
methyl esters, and analysis by HPLC and scintillation spectrometry. The data indicate that with ['Hlacetate as a precursor, palmitic acid is the primary radiolabeled fatty acid along
with smaller amounts of stearic acid, and only trace amounts
of ['Hlmyristic acid could be detected in megakaryocyte
lipids. Although the pool of radiolabeled myristic acid with
acetate as a precursor is extremely small, GPIX appears to
be selectively acylated by myristic acid.
Itwas important to identify the fatty acid that acylated
GPlb and GPIX after incubation with exogenously supplied
['Hlmyristic and ['H]palmitic acids because megakaryocytes
have the capacity to elongate these precursors. Therefore,
after incubation with ['Hlmyristate or [.'H]palmitate, radiolabeled fatty acids linked to GPlX and GPlb were assessed by
HPLCand scintillation spectrometry, as described above.
After the incubation of megakaryocytes with [ZH]palmitic
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
ACYLATION
GLYCOPROTEIN
OF MEGAKARYOCYTE
IE-IX
1381
I3H1Acetate-MK Lipids
[3H1 Acetate-Gplb
I3HIMyristate-Gplb
5n 200
n
1000
MS
0
20
40
0
60
f3Hl Acetate-GDIX
40
60
v H I Myristate-GplX
400F
MM
h
300
5
20
0
Y
X
200
100
I
0
20
40
FRACTION
60
FRACTION
Fig 4. Identification of radiolabeled fatty
acids bound t o GPlb,
GPlX by HPLC and scintillationspectrometry. Guinea pig megakaryocytes in Eagle's medium supplemented with 0.22% BSA (200.0001
mL) were incubatedwith ['Hlacetate (66.7 pCilmL) or ['Hlmyristate
1300 pCilmL) for 17 hours. Megakaryocyte lipids were extracted or
GPlb subunits were immunoprecipitated
with PG1 antibody and separated by 10% unreduced SDS-PAGE, and acylated proteins were
eluted. Extracted megakaryocyte lipids and immunoprecipitated
GPlb subunits were subjected t o acid methanolysis. the resultant
fatty acid methyl esters were separated by HPLC, and radioactivity
was measured by scintillation spectrometry. In panels A through C,
the mobile phase was 9546 acetonitrile, and in panels D and E, 84Y0
acetonitrile. (A) The fatty acids synthesized from acetate found in
total megakaryocyte lipids. (B1 The fatty acids synthesized from acetate found covalently linkedt o GPlb. (Cl the fattyacids synthesized
from acetate covalently linkedt o GPIX. After the incubation
of megakaryocytes with ['Hlmyristate, w e used the approach described
above t o demonstrate the radiolabeled fatty
acids bound t o GPlb (Dl
and t o GPlX (E). MM, methyl myristate; MP, methyl palmitate; MS,
methyl stearate; MA, myristic acid; PA, palmitic acid.
acid, essentially only palmitic acid was found to be covalently linked to GPIb (data ndt shown). After the incubation
with['Hlmyristicacid.
about 48% ofthefattyacid
covalently bound to megakaryocyte GPTb was myristic acid, and
52%was palmitic acid(Fig4D).However,
myristic acid
was found to be the primary fatty acid covalently linked to
GPlXafterthe
incubationof megakaryocytes with ['H]myristic acid (Fig 4E).
After the incubationwith[3H]myristic
acid, fatty acids
covalently linked to GPlX were resistant to hydrolysis with
I m o l L hydroxylamine, pH 7 and pH 10, or under alkaline
hydrolysis, indicating that the fatty acid waslinked to protein
by an amide bond and not to thioester or 0-ester linkages.
However. fatty acids linked to GPlb were sensitive to hydroxylamineat pH 7, suggesting that thislinkagewas
a
thioester bond (data not shown).
Megakaryocytes atdifferent phases of maturationwere
prepared by the Celsep procedure. and the acylation of GPlb
subunitswas studied by incubation with ['Hlmyristic and
['Hlpalmitic acids. Three different subpopulations of megakaryocytes were prepared: a mature subpopulation that contained 92% stage 111 and 1V (mature based on cytoplasmic
maturation) and 99% 16N and 32N ploidy megakaryocytes;
an intermediate maturity subpopulation that contained 7 1 %
stage 111 and IV and 74% 16N megakaryocytes: and an immature subpopulation that contained 67%- stage I and I1 (immature based oncytoplasmic maturation)and 66% 8Nmegakaryocytes.Equivalent
amounts of protein frommature,
intermediate maturity, and immaturemegakaryocyte subpopulations were applied to each lane. There is a considerable
difference in the size of mature and immature megakaryocytes, and thus. biochemical activities cannot be compared
on the basis of cell number but can be compared on the basis
of protein content or cell volume." Figure SA demonstrates
that themyristoylationof GPlX occurredprimarily in the
mature and intermediate maturity subpopulations. Figure SB
demonstrates thatthepalmitoylation
of GPIb occurred in
each of the three subpopulations. After incubation with ['HImyristic acid, acylation of GPlb also occurred similarly in
each of the cell subpopulations (datanot shown). Densitometry indicated that the ratio of myristoylation of GPIX in
mature to immature subpopulations shown in Fig SA was
greater than 1 0 0 : 1, while the ratio of palmitoylation of GPIb
in mature to immature subpopulations shown in Fig SB was
about 2.5: 1.
Several issues should be considered in interpretingthe
datafromCelsep
subpopulations. Immature andmature
megakaryocyte subpopulationsdiffered significantly in cytoplasmic maturation and in ploidy. Both of these parameters
have been considered to reflect megakaryocyte maturation.
While the study didnot distinguish between the roleof cytoplasmic maturation and ploidy in the differences in acylation,
previous studies indicated that arachidonic acid uptake,15 the
expression of surface-exposed sialoglycoproteins.'" and the
expression ofP-selectin mRNA"are related primarily to
cytoplasmic maturation rather than to ploidy. This is consis-
A
M
B
IT
IM
M
IT
IM
Fig 5. Myristoylated GPlX and GPlb in guinea pig megakaryocytes
of differing maturities.Guinea pig megakaryocyte subpopulations at
different phases of maturation prepared by the Celsep procedure
were incubated for 17 hours with ['Hlmyristate or ['Hlpalmitate in
Eagle's medium (300 pCilmL). The GPlB complex in megakaryocyte
subpopulations was immunoprecipitated with PG-l, proteins were
separated by 10% unreduced SDS-PAGE, and acylated GPlX and GPlb
were assessed by fluorography. Equivalent amounts of protein from
immature, intermediate, and mature subpopulations were applied.
(A) Myristoylationof GPIX. (B) Palmitoylation of GPlb. M,mature; IT,
intermediate maturity; IM, immature subpopulations.
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
1382
SCHICK AND WALKER
likely is involved in these exceptions, as myristoyl CoA:protent with the concept that ploidy is established before cytoplasmic maturation." It is not surprising that GPIX myristoytein N-myristoyltransferase has no activity against 1y~ine.l~
lation is active in the intermediate maturity subpopulation,
GPIX has one lysine side chain near the N-terminus that
as it contains 74% cytoplasmically mature megakaryocytes."
may be the site of amide-linked myristoylation."
The myristoylation of GPIX in megakaryocytes is cotransHowever, the virtual absence of evidence of myristoylation
inthe immature subpopulation is surprising, because this
lational and tightly coupled with protein synthesis, as it was
fraction contains 33% mature megakaryocytes based on morinhibited by cycloheximide. Myristoylation is usually cophologic criteria.12 Nonviability of the immature fraction
translational in cases of linkage of myristic acid to N-termidoes not account for this finding, because this fraction is
nal glycine4-' and in the linkage of myristic acid to a lysine
active in proteoglycan synthesis,12 in lipid ~ynthesis,'~~'"~"residue in the insulin receptor."
and in the expression of mRNA for vWF and GPIb-alpha.'7
The investigation of palmitoylation revealed that similiar
Although the immature subpopulation has been classified on
proteins were palmitoylated in guinea pig platelets and
the basis of morphologic evidence of cytoplasmic maturamegakaryocytes as had beenreported in human platelets with
tion, other, as yet undefined characteristics may better distinthe exception of GPIX.28.29
Palmitoylation of GPIX occurs in
guish the immature from the mature subpopulations.
human platelets but not in guinea pig platelets. GPIb-beta
subunit is the site of palmitoylation of GPIb in both human
DISCUSSION
and guinea pig platelets and megakaryocytes.
Our study has demonstrated that megakaryocyte GPIb can
The study showed that a 19-kD protein is one of the most
be palmitoylated after incubation with ['Hlpalmitic acid or
prominently myristoylated proteins in guinea pig megakary['Hlmyristic acid. When megakaryocytes were incubated
ocytes. This protein was identified as GPIX using monowith exogenous ['Hlmyristic acid, 48% of the fatty acids
clonal antibodies PG- 1, AN-5 1, and FMC-25. The myristoybound to GPIb was myristic acid, and 52% was palmitic
lated 19-kD protein was evident in both reduced and
acid. Both of these fatty acids were boundto GPIb by a
unreduced gels as would be expected for GPIX. GPIb was
thioester linkage and wereposttranslational events. The demalso found to be acylated after the incubation of megakaryoonstration of thioester-linked myristic acid to GPIb in megacytes with myristic acid, and reduced SDS-PAGE revealed
karyocytes is consistent with similar observations in human
that GPIb-beta was the site of acylation. The study indicated
platelets.' This most likely accounts for the finding that
that megakaryocyte GPIX was considerably more acylated
GPIb-beta was the site of acylation in megakaryocytes that
than GPIb when megakaryocytes had been incubated with
had been incubated with ['Hlmyristic acid (Fig 1). In human
myristic acid.
platelets, myristic and palmitic acids bind to the same set of
GPIX was also found to be myristoylated in CHRF-288
platelet proteins, but palmitic acid is preferred over myristic
cells, a tumor cell line with megakaryocytic characteristics.13
acid. Also, the rate and intensity of the binding of fatty acids
Because the SZ-l monoclonal antibody that recognizes the
to proteins was considerably greater with palmitic acid than
intact GPIb complex was used for immunoprecipitation, the
with
myristic acid in human platelets7
demonstration of acylated GPIX suggests that the intact
It was important to study endogenous protein acylation,
GPIb complex exists in CHRF cells. This information indibecause differences in the acylation of proteins have been
cates that the myristoylation of GPIX is not limited to guinea
noted
in other cells when the fatty acid hadbeenderived
pig megakaryocytes but may also occur in human megakaryfrom acetate versus exogenously supplied myristic or palocytes.
mitic acids." The study of endogenous acylation of GPIX
The study showed that myristic acid is covalently linked
using acetate confirmed the data from exogenously supplied
to GPIX by an amide bond, because hydroxylamine at pH
myristic acid to study protein acylation. With [3H]acetateas
7 and pH 10 did not result in the loss of myristic acid from
a precursor, GPIX was acylated with primarily myristic acid.
GPIX. This agent at pH 7 hydrolyzes thioester bonds and at
Megakaryocytes can synthesize palmitic and stearic acid
pH 10 hydrolyzes 0-ester bonds but not amide bonds. Amwith acetate as a precursor. However, with ['Hlacetate as a
ide-linked myristoylation has been shown to usually occur
precursor, radiolabeled palmitic acid was the primary fatty
at an N-terminal glycine in viruses, yeasts, and mammalian
acid, while only trace amounts of radiolabeled myristic acid
cell^.^.^ However, GPIX appears to be linked to myristic acid
were found in megakaryocytes. Thus, there appears to be a
at a different site, because it does not have an N-terminal
selective demand for myristic acid for the acylation of GPIX.
glycine.*' There are several exceptions to the absolute need
With acetate as a precursor, GPIb wasmainly acylated
for an N-terminal glycine as the site for amide linkage of
with palmitic acid. However, as noted above with exogenous
myristoylated protein^.^ The insulin receptor, immunoglobumyristic acid, GPIb is acylated with both myristic and pallin heavy chain, the interleukin (1L)-l-alpha and IL-l-beta
mitic acids. In the experiments with exogenous myristic acid,
precursors, and tumor necrosis factor lack glycine residues
myristic and palmitic acids are ester-linked at similar sites,
correctly positioned for classical myristoylation.22-26
In these
and the linkage of myristic acid to GPIb may be related to
exceptions, myristic acid was considered to be amide-linked
high concentrations of the precursor. The results of the acetoan internal lysine. Myristoylation at a specific internal
tate experiments most likely reflect in vivo protein acylation
lysine residue in IL-3-alpha and -beta precursors has been
of GPIb. The information from the acetate and exogenous
demonstrated using anin vitro myristoylation assay.24 A
fatty acid experiments are, nevertheless, consistent in the
specific peptide-lysine amino myristoyltransferase most
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
ACYLATION OF MEGAKARYOCYTE GLYCOPROTEIN le-IX
conclusion that GPIX is primarily myristoylated and GPIb
is palmitoylated in megakaryocytes.
The studyindicatesthat the palmitoylationofGPIb is
established in immature megakaryocytes, but GPIX is primarily myristoylated in mature megakaryocytes. The possibility that GPIX is expressed duringlater phases of megakaryocyte maturation has beenpostulated3’and may explain
why the myristoylation of GPIX was not observed inimmature megakaryocytes.
Differences in other biochemical activities have been observed in mature and immature megakaryocytes. For example, mRNAs for fibr~nectin,~’
vWF, and G P I b - a l ~ h a ’are
~
expressed in immature megakaryocytes, and most aspects of
lipid synthesis are establishedinimmature
megakaryocytes.15.19.20 However, de novo fattyacid synthesis occurs
primarily in mature megakaryocytes.20 Surface-exposed sialoglycoproteins’6 andthe expression of mRNA for P-selectin’7 are evident mainly in mature megakaryocytes. Platelets
are mostlikely assembled during the terminal phases of
megakaryocyte maturation. Therefore, the myristoylation of
GPIX as well as the biochemical activities mentioned above
appear to be characteristicsand markers oftheterminal
phases of megakaryocyte maturation and may b e important
for platelet production.
Myristoylated proteins may be responsible for the mediation of protein-protein interactions and the assembly of large
protein
For example, many myristoylated proteins are subunits of large protein complexes, such as protein
kinase A or alpha subunitsof G proteins6 Inaddition, myristoylation can stabilize the interaction between capsid proteins in picorna viruses.* The myristoylation of GPIX may
be important for the interaction of GPIb subunits and the
formation of a functional complex in mature megakaryocytes.
ACKNOWLEDGMENT
We appreciate receiving the PG-l monoclonal antibody from Dr
Jonathan Miller and the CHRF-288 cells from Dr Michael Lieberman. We also appreciate Drew Lickens’ expertise and efforts in
preparing the illustrations and would like to thank Angela Balduzzi
for her assistance in preparing the manuscript.
REFERENCES
1. Charo IF, Kieffer N, Phillips DR: Platelet membrane glycoproteins, in Colman RW, Hirsh J, Marder VJ, Salzman EW (eds): Hemostasis and Thrombosis: Basic Principles and Clinical Practice,
3rd ed. Philadelphia, PA, Lippincott, 1994, p 489
2. George JN, Nurden AT: Inherited disorders of the platelet
membrane: Glanzmann thrombasthenia, Bernard-Soulier syndrome,
and other disorders, in Colman RW, Hirsh J, Marder VJ, Salzman
EW (eds): Hemostasis and Thrombosis: Basic Principles and Clinical
Practice, 3rd ed. Philadelphia, PA, Lippincott, 1994, p 652
3. Muszbek L, Laposata M: Glycoprotein Ib and glycoprotein IX
in human platelets are acylated with palmitic acid through thioester
linkages. J Biol Chem 264:9716, 1989
4. Grand RJA: Acylation of viral and eukaryotic proteins. Biochem J 258:625, 1989
5 . Schmidt MFG: Fatty acylation of proteins. Biochim Biophys
Acta 988:411, 1989
1383
6. McIlhinney RAJ: The fats of life: The importance and function
of protein acylation. Trends Biochem Sci 15:387, 1990
7. Muszbek L, Laposata M: Myristoylation of proteins in platelets
occurs predominantly through thioester linkages. J Biol Chem
268:8251, 1993
8. Chow M, Newman JFE, Filman D, Hogie JM, Rowlands DJ,
Brown F: Myristylation of picornavirus capsid protein VP4 and its
structural significance. Nature 327:482, 1987
9. Kupinski JM, Miller JL: Synthesis by guinea pig megakaryocytes of platelet glycoprotein receptors for fibrinogen and vonWillebrand factor. Thromb Res 43:345, 1986
10. Schick PK, Yu B P Methylene blue-induced serotonin release
in human platelets. J Lab Clin Med 82:546, 1973
11. Schick PK, Schick B: Methods for studying the biochemistty
of recognizable megakaryocytes, in Colman RW, Smith JB (eds):
Modem Methods in Pharmacology: Methods for Studying Platelets
and Megakaryocytes, v01 4. New York, W ,Liss, 1987, p 19
12. Schick PK, Schick BP, Williams-Gartner K Characterization
of guinea pig megakaryocyte subpopulations at different phases of
maturation prepared with Celsep separation system. Blood 73: 1801,
1989
13. Fugman DA, Witte DP, Jones CLA, Aronow BJ, Lieberman
MA: In vitro establishment and characterization of a human megakaryoblastic cell line. Blood 75:1252, 1990
14. Magee AI, Wootton J, DeBony J: Detecting radiolabelled
lipid-modified proteins in polyacrylamide gels. Methods Enzymol
250:330, 1995
15. Schick PK: Arachidonic acid is preferentially incorporated by
immature megakaryocytes. J Lab Clin Med 113:79, l989
16. Schick PK, Filmyer W Jr: Sialic acid in mature megakaryocytes: Detection by wheat germ agglutinin. Blood 65:1120, 1985
17. Schick PK, Konkle B, He X, Thornton R P-selectin mRNA
is expressed at later phases of megakaryocyte maturation than
mRNAs for GPIb-alpha and vWF. J Lab Clin Med 121:714, 1993
18. Paulus JM: DNA metabolism and development of organelles
in guinea pig megakaryocytes: A combined ultrastructural, autoradiographic and cytophotometric study. Blood 35:298, 1970
19. Schick PK, He X: Composition and synthesis of glycolipids
in megakaryocytes and platelet: Differences in synthesis in megakaryocytes at different stages of maturation. J Lipid Res 31:1645, 1990
20. Schick PK, Williams-Gamer K, He X: Lipid composition
and metabolism in megakaryocytes at different stages of maturation.
J Lipid Res 31:27, 1990
21. Hickey MJ, Williams SA, Roth GJ: Human platelet glycoprotein D(: An adhesive protoype of leucine-rich glycoproteins with
flank-center-flank structures. Proc Natl Acad Sci USA 86:6773,1989
22. Hedo JA, Collier E, Watkinson A: Myristyl and palmityl
acylation of the insulin receptor. J Biol Chem 262:954, 1987
23. Pillai S, Baltimore D: Myristoylation andthe post-translational acquisition of hydrophobicity by the membrane immunoglobulin heavy-chain polypeptide in B lymphocytes. Proc Natl Acad Sci
USA 84:7654, 1987
24. Stevenson FT, Bursten SL, Fanton C, Locksley RM, Lovett
DH: The 31-kDa precursor of interleukin-l-alpha is myristoylated
on specific lysines within the 16-kDa N-terminal propiece. Proc Natl
Acad Sci USA 90:7245, 1993
25. Bursten SL, Locksley RM, Ryan JL, Lovett DH: Acylation
of monocyte and glomerular mesangial cell proteins. J Clin Invest
82:1479, 1988
26. Stevenson FT, Bursten SL, Fanton C , Locksley RM, Lovett
DH: Myristyl acylation of the tumor necrosis factor alpha precursor
on specific lysine residues. J Exp Med 176:1053, 1992
27. Towler DA, Eubanks SR, Towery DS, Adams SP, Glaser L:
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
1384
Amino-terminal processing of proteins by N-myristoylation. J Biol
Chem 262:1030, 1987
28.Muszbek L, Laposata M: Covalent modification of platelet
proteins by palmitate. Blood 74:1339, 1989
29. Huang EM: Palmitoylation of platelet proteins. Platelets 5:61.
I994
30. Towler D, Glaser L: Acylation of' cellular proteins with
SCHICK AND WALKER
endogenouslysynthesizedfattyacids.Biochemistry
25:878,
1986
31. Hickey MJ, Roth GJ: Characterization ofthe gene encoding
human platelet glycoprotein IX. J Biol Chem 268:3438, 1993
32. Courtney M-A, Stoler MH, Marder VJ, Haidaris PJ: Developmental expression of mRNAs encoding platelet proteins in rat megakaryocytes. Blood 77560. 1991
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
1996 87: 1377-1384
The acylation of megakaryocyte proteins: glycoprotein IX is primarily
myristoylated while glycoprotein Ib is palmitoylated
PK Schick and J Walker
Updated information and services can be found at:
http://www.bloodjournal.org/content/87/4/1377.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.