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. 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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. 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