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Protein phosphorylation in response to PDGF stimulation in cultured neurons and astrocytes
Developmental Brain Research 99 Ž1997. 216–225
Research report
Protein phosphorylation in response to PDGF stimulation in cultured neurons and astrocytes Frank X. Zhang a b
a,1
, James B. Hutchins
a,b,)
Department of Anatomy, UniÕersity of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4500, USA Department of Neurology, UniÕersity of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4500, USA Accepted 26 November 1996
Abstract Platelet-derived growth factor ŽPDGF. is an important growth factor for a variety of cells, including neurons and glial cells. PDGF signal transduction pathways have been studied primarily in mesenchyme-derived cells Žsuch as fibroblasts and smooth muscle cells.. However, little is known about these pathways in the central nervous system ŽCNS.. It is believed that phosphorylation is a critical aspect of several steps in the signal transduction pathway. In this study, neurons and type 1 astrocytes in vitro were radiolabeled with 32 P-orthophosphate Ž 32 P-Pi .. The cells were lysed, and labeled proteins were separated by two-dimensional gel electrophoresis. Autoradiograms of PDGF-stimulated and control samples were compared. We found that in neurons and type 1 astrocytes in vitro, PDGF-BB greatly enhances protein phosphorylation while PDGF-AA has less of an effect on protein phosphorylation. Furthermore, because PDGF signal transduction pathways are likely to affect the cytoskeleton, we studied changes in actin-binding proteins induced by PDGF-BB. We found that PDGF-BB alters the expression, migration pattern andror avidity of some actin-binding proteins in neurons. In conclusion, protein phosphorylation is up-regulated by PDGF in mouse cortical neurons and type 1 astrocytes in vitro. PDGF’s effects on phosphorylation of cytoskeletal proteins might be a important mechanism by which PDGF affects the development and normal functions of central nervous system cells. q 1997 Elsevier Science B.V. All rights reserved. Keywords: Actin-binding protein; Cytoskeletal protein; Development; Growth factor; Nervous system; Signal transduction
1. Introduction Platelet-derived growth factor ŽPDGF. is a growth factor expressed in a variety of cells and seems to have multiple physiological and pathological effects. PDGF is involved in cell mitogenesis, differentiation, transformation and migration w57,58x. PDGF consists of homo- or heterodimers of two subunits termed A and B. It is well established that PDGF plays an important role in the development and biological functions of mesenchyme-derived cells, but its expression within, and effects on, the nervous system have only recently been described. Yeh et al. w80x demonstrated the expression of PDGF A-chain protein and message in embryonic and adult mouse neurons. Similarly, PDGF B-chain ) Corresponding author. Fax: q1 Ž601. 984-1655. E-mail: [email protected] 1 Current address: Dept. of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Room 264 JAH, Philadelphia, PA 19107.
protein and transcript are found within neurons of the monkey brain w65x. PDGF receptor comprises two subunits: a and b , which may also form homo- or heterodimers. PDGF receptor a subunit ŽPDGFR-a . has been found in human neuroblastoma cells w53x, in rat sciatic nerve and dorsal root ganglion cells w14x, in developing rat brain w61x, and in mouse brain tissue, cortical neurons and glia w81x. PDGF receptor b subunit ŽPDGFR-b . was found in the rat brain using immunohistochemical and Northern blotting techniques w61,72x. Hutchins and colleagues demonstrated with immunohistochemistry, Western blotting and receptor cross-linking that PDGF and its receptor are expressed in developing mouse brain w35,36x, and rat and mouse neuronal and astroglial cells in vitro w33,34x. While the location and expression of PDGF and its receptor have been characterized, the functions of PDGF in the nervous system have been explored less extensively. PDGF-AA inhibits premature differentiation of oligodendrocytertype 2 astrocyte progenitor ŽO2A. cells in rat optic nerve w50,54–56x. PDGF also stimulates chemotaxis
0165-3806r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 6 . 0 0 2 1 8 - 0
F.X. Zhang, J.B. Hutchinsr DeÕelopmental Brain Research 99 (1997) 216–225
in rat astrocytes w5x. PDGF-BB decreases potassium channel amplitude with no change in kinetics when PDGF receptor is expressed in Xenopus oocytes w75x. This suggests that PDGF has the capacity to influence neuronal activity. PDGF increases the survival of rat newborn cerebellar GABAergic interneurons and stimulates neurite outgrowth in rat cerebellar neurons in vitro w71,72x. The signal transduction pathways mediated by PDGF in fibroblasts have been extensively investigated, but little work has been done in the nervous system. PDGF receptor a and b subunits, which both belong to the receptor tyrosine kinase superfamily, form functional dimers Ž aa , ab and bb . when PDGF binds to them. Dimerization, in turn, initiates receptor auto- andror transphosphorylation. The conformational change which occurs activates other kinase sites on the receptor w3,46,47,64,77x. These events are obligatory initial steps in the PDGF signal transduction pathway w9,27,28x. The substrates of the receptor tyrosine kinases include phospholipase C g ŽPLCg ., phosphatidylinotitol-3-kinase ŽPI3 kinase., GTPase-activating factor ŽGAP., Grb2, Shc, Raf and protein tyrosine phosphatases. Other downstream elements of the PDGF signal transduction pathway include the Ras pathway, which is composed of Raf, Ras, 14-3-3, and MEK-1 w25,66x, or the JAK-STAT pathway w12,68x. The ultimate events in the PDGF signal pathway include activation of transcriptional factors affecting protein synthesis, posttranslational modification of proteins Žsuch as phosphorylation., and cytoskeletal reorganization. It has been shown that PDGF induces talin phosphorylation and reorganization in a skeletal muscle cell line w74x, and causes a change in the distribution of actin and vinculin in fibroblasts w26,30x and in vascular smooth muscle cells w31x. However, the effects of PDGF on protein phosphorylation, including cytoskeletal proteins, in nervous system cells have not yet been described. 2. Materials and methods 2.1. Neuronal cell culture All animal care and use procedures were carried out in accordance with federal and local guidelines. Pregnant C57B1 mice were sacrificed by cervical dislocation. Mouse embryos at embryonic day 13.5 to 15 w73x were killed by decapitation. Their cerebral cortices were dissected to remove the meninges and external blood vessels, and were dissociated with 0.25% trypsinr1 mM EDTA, ŽGibco. for 20 min at 378C. The dissociated cells were preplated onto a non-coated T-75 flask for 2 h to remove adherent cells Žmainly fibroblasts., then the floating cells were plated onto polylysine-coated or laminin-coated T-25 flasks at a density of 10 4 to 10 5 cellsrcm2 in DMEMrF-12 medium or Neurobasal medium. Either medium was supplemented with 20% serum and 100 Urml penicillin, 100 m grml streptomycin and 0.25 m grml amphotericin B Žall from
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Gibco.. The purity of neuronal cultures was monitored with anti-neurofilament immunostaining. In previous studies, over 95% of cells were neurofilament-positive using identical techniques. 2.2. Type 1 astrocyte culture Type 1 astrocytes were obtained from postnatal day 1 to day 3 C57B1 mice, following the method described by Levison and McCarthy w45x. Briefly, after dissection and dissociation of the cerebral cortices, the cells were plated onto T-75 flasks in Basal Medium Eagle ŽBME. medium with 10% fetal bovine serum and 100 Urml penicillin, 100 m grml streptomycin and 0.25 m grml amphotericin B Žall purchased from Gibco; this mixture is termed BME-C.. After the cells reach confluence, the flask was shaken at 300 rpm overnight Ž378C.. The floating cells Žprocessbearing cells. were discarded. The adherent cells were trypsinized with 0.05% trypsin-EDTA ŽGibco. and replated onto T-25 flasks at 10 4 to 10 5 cellsrcm2 in BME-C medium. These type 1 astrocytes become confluent in about a week. Generally, over 95% of cells are anti-glial fibrillary acidic protein-immunoreactive. 2.3. Radiolabeling After two days in vitro, mouse cortical neurons were incubated in serum-free medium overnight. Therefore, radiolabeling took place at three days in vitro. The serum-free medium was G5rN3 medium including 5 nM hydrocortisone, 5 m grml insulin, 10 ngrml epidermal growth factor, 0.5 ngrml basic fibroblast growth factor, 100 m M putrescine, 20 nM progesterone, 30 nM sodium selenite, 50 m grml transferrin, 50 m grml ascorbic acid, 1 mgrml bovine serum albumin and 100 Urml penicillin-streptomycin w2x. After two rinses with phosphate-free DMEM medium ŽGibco., the neurons were incubated with 0.5 mCirml 32 P-orthophosphate Ž 32 P-Pi , Amersham. in phosphate-free DMEM at 378C for 2.5 h. Recombinant human PDGF-BB or -AA Ž30 ngrml, Promega. were added to experimental flasks for 2.5 h. PDGF has optimal effects at concentrations ranging from 10 to 50 ngrml in fibroblasts and smooth muscle cells. Confluent type 1 astrocytes were serum-starved with BME overnight, and then rinsed and labeled with 0.5 mCirml 32 P-Pi with Žexperimental group. or without Žcontrol group. 30 ngrml rPDGF-BB or -AA in GHCKS medium Ž11 mM glucose, 20 mM HEPES, 10.2 mM sodium citrate, 4 mM KCl, 109 mM NaCl and 0.0002% phenol red, pH 7.1. at 378C for 2.5 h. 2.4. Preparation of cell lysates After labeling, the neurons and type 1 astrocytes were rinsed twice with 50 mM Tris buffer, scraped and collected. After a brief microcentrifugation, the cell pellets were incubated with urea solubilization buffer Ž9 M urea,
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4% NP-40, 2% LKB ampholyte pH 3.5–10, 2% b-mercaptoethanol, pH 9.8. at room temperature for 2 h. Then the cell lysates were centrifuged at 12 000 = g, 48C for 3 h to remove insoluble material. The supernatant was collected and diluted for a dye-binding protein assay w4x and liquid scintillation counting to determine protein concentration and 32 P incorporation rate, respectively. 2.5. Two-dimensional gel electrophoresis 2-D gel electrophoresis was carried out based on the O’Farrell w51x method. Equal amounts of protein Ž20 m g. from experimental and control cell lysates were loaded onto tube gels Ž9.2 M urea, 1.33% LKB ampholyte pH 3.5–10, 0.67% Serva ampholyte pH 2–11, 2% NP-40 and 4% acrylamide.. Isoelectric focusing Žfirst dimension. was carried out for a total of 13,000 V-h. The proteins were then separated by molecular mass with standard SDS-
PAGE Ž4% acrylamide stacking gel and 12% acrylamide separating gel.. The pI calibration was conducted using two independent methods. In the first, a blank tube gel was run in parallel. This gel was cut into 1 cm long pieces and the slices were soaked in 1 ml distilled water overnight. The pH of the resulting buffer was measured. In the second method, a carbamylated G3PDH 2-D standard ŽLKB. was mixed in the protein sample and visualized along with cellular proteins. The slab gels were fixed, silver-stained w52x and dried. Conventional X-ray film autoradiography and PhosphorImager analysis ŽMolecular Dynamics. were carried out. Experiments were repeated several times to check for consistent PDGF effects. 2.6. Immunoprecipitation 32
P-labeled type 1 astrocytes were rinsed with 50 mM Tris buffer and lysed with immunoprecipitation buffer
Fig. 1. Silver-stained gel and autoradiograms of neuronal proteins. 32 P-Pi labeled mouse cortical neurons were lysed in urea buffer and separated by O’Farrell 2-D gel electrophoresis. Fig. 1A shows a silver-stained gel with the position of molecular weight standards indicated on the left and pI on the bottom. Actin Ža. and tubulin isoforms Žt. were identified by comparison to the literature. The horizontal spots ŽG, 36 kDa, pI 4.7–8.3. are carbamylated G3PDH pI calibration standards, and the vertical streaks are staining artifacts. Fig. 1B Žcontrol. and 1C ŽPDGF-BB stimulated. are autoradiograms from 32 P-Pi-labeled neuronal lysates. In general, PDGF-BB enhances protein phosphorylation in neurons in vitro. Several PDGF-stimulated phosphoproteins are labeled: a, b, c, and d . Their apparent molecular weight and pI are 110 kDa, 4.5; 130 kDa, 5.0; 20 kDa, 4.1; 16 kDa, 4.7; respectively. The protein labeled ‘d ’ is the mitochondrial protein F1 F0 ATPase d subunit w82x.
F.X. Zhang, J.B. Hutchinsr DeÕelopmental Brain Research 99 (1997) 216–225
Ž0.1% Triton X-100, 0.1% deoxycholate, 5 mM EDTA, 50 m M sodium vanadate, 1 m grml each leupeptin, aprotinin, pepstatin, 10 mM Tris pH 8, 140 mM NaCl and 0.025% sodium azide.. Anti-PDGF receptor Ž a q b . antibody Ž10 m l for 100 m g protein in total volume of 100 m l, UBI. was added to form an immunocomplex. A slurry of 10% Žwrv. Pansorbin ŽCalbiochem. was used to precipitate the immunocomplexes formed. The pellet was extensively rinsed with dilution buffer Ž0.1% Triton X-100, 0.1% BSA, 50 m M sodium vanadate, 0.025% sodium azide, 140 mM NaCl and 10 mM Tris, pH 8.0., TSA buffer Ž140 mM NaCl, 0.025% sodium azide and 50 m M sodium vanadate, 10 mM Tris, pH 8.0. and 50 mM Tris. Finally, Laemmli buffer was added to the pellets. After boiling and centrifugation, the supernatants from this step were resolved by SDS-PAGE Ž5% to 20% acrylamide exponential gradient gel.. The gel was fixed, dried, and set up for autoradiography.
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2.7. Actin-binding oÕerlay assay Neuronal lysates Ž100 m g. solubilized in urea solubilization buffer were separated by 2-D gel electrophoresis, and then transferred to a polyvinylidine difluoride ŽPVDF. m em brane in 10 m M 3- Ž cyclohexylam ino . -1propanesulfonic acid ŽCAPS. buffer, pH 11.0 with a field of 14 V at 48C overnight. The PVDF membranes were incubated in 2% non-fat dry milk in Tris-buffered saline ŽTBS. for 1 h. Biotinylated F-actin was formed by incubation of 0.1 mgrml biotinylated actin ŽCytoskeleton, Denver, CO. in 50 mM KCl, 2 mM MgCl 2 , 1 mM CaCl 2 and 1 mM ATP. The F-actin Ž5 m grml. was incubated with the membranes in ‘actin-binding buffer’ Ž50 mM Tris, 150 mM NaCl, 2 mM MgCl 2 , 1 mM CaCl 2 and 0.2 mM ATP. at room temperature for 1 h. The membranes were then rinsed and treated with avidin–HRP conjugate Ž2 m grml in actin-binding buffer. for 1 h. The actin binding protein
Fig. 2. Silver-stained gel and autoradiograms of type 1 astrocyte proteins. Urea lysates of radiolabeled type 1 astrocytes were resolved by 2-D electrophoresis. As in Fig. 1A, molecular weight and pI standards are labeled on the left and bottom of the silver-stained gel ŽFig. 2A.. Fig. 2B Žcontrol. and 2C ŽPDGF-BB-stimulated. are autoradiograms of 32 P-Pi-labeled cell lysates. As in Fig. 1, species marked a, b, c, d , e are several examples of proteins whose phosphorylation is increased by PDGF-BB, their estimated molecular weight and pI are 110 kDa, 4.5; 130 kDa, 5.0; 20 kDa, 4.1; 16 kDa, 4.7; 50 kDa, 6.0, respectively.
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complexes were visualized using 0.01% H 2 O 2 as substrate and diaminobenzidine Ž1 mgrml. as a chromogen. 2.8. Immunoblotting with ezrin antibody Neuronal lysate was prepared, electrophoresed and blotted onto PVDF as described in the ‘Actin-binding Overlay Assay’ method section. The PVDF membranes were incubated in ‘blocking solution’ Ž2% bovine serum albumin, 0.1% Tween-20 in TBS. for 1 h at room temperature. Rabbit antiserum against human ezrin, kindly provided by Dr. Tony Bretscher of Cornell University w7x, crossreacts with mouse ezrin. The membranes were incubated with anti-ezrin antibody at a 1:3,000 dilution in ‘blocking solution’ for 2 h at room temperature. After three washes with 0.1% Tween-20 in TBS, the membranes were incubated with HRP-conjugated-goat anti-rabbit Ž1:1,000, Jackson ImmunoResearch. for another hour. Then, the membranes were rinsed again, and reacted with 0.1% diaminobenzidine and 0.01% H 2 O 2 .
PDGF-stimulated phosphoproteins are labeled in Figs. 1 and 2 Ža, b, c and d .. Their estimated molecular weight and pI are 110 kDa, 4.5; 130 kDa, 5.0; 20 kDa, 4.1; 16 kDa, 4.7, respectively. The protein d has been identified as F1 F0 ATPase d subunit by microsequencing, Western blotting and immunoprecipitation w82x. 3.3. Phosphorylation of type 1 astrocyte proteins by PDGF-BB It is obvious that the gel-staining pattern of neurons ŽFig. 1A. is different from type 1 astrocytes ŽFig. 2A.. PDGF-BB also increases protein phosphorylation in type 1 astrocytes ŽFig. 2B, control; Fig. 2C, PDGF-BB-stimulated.. Protein species whose phosphorylation responds to PDGF-BB stimulation include a,b,c, and d . These likely correspond to the same proteins in neurons and astrocytes. It seems that protein e Ž50 kDa, pI 6.0. is an astrogliaspecific protein. 3.4. PDGF receptor autophosphorylation
3. Results As expected, a relatively large number of neuronal and astroglial proteins are seen in silver-stained 2-D gels ŽFig. 1A and 2A.. A subset of these, but still a substantial number, are phosphoproteins which may be identified by 32 P-Pi labeling of cells in culture ŽFig. 1B and 2B.. The phosphoproteins of interest in the present paper are those whose labeling is either up- or down-regulated in response to PDGF Žcompare panels B and C in Figs. 1 and 2.. A number of such proteins can be seen Židentified by letters a–e. in autoradiograms when control and experimental cultures are compared.
Immunoprecipitation studies of radiolabeled type 1 astrocytes ŽFig. 3. demonstrated that PDGF-BB Žmiddle lane. is able to trigger PDGF receptor autophosphoryla-
3.1. Protein phosphorylation of CNS cells and PDGF-AA In contrast to PDGF-BB, treatment of either neurons or astroglia with PDGF-AA had only subtle and variable effects on protein phosphorylation Ždata not shown.. For this reason, the present study concentrates on PDGF-BBstimulated phosphorylation. 3.2. Phosphorylation of neuronal proteins by PDGF-BB It appears that PDGF-BB upregulates protein phosphorylation in neurons ŽFig. 1.. On a silver-stained gel ŽFig. 1A., actin Ža, 45 kDa, pI 5.4. and tubulin isoforms Žt, 54 kDa, pI 5.1. are identified by their apparent molecular weight, pI and migration pattern. The series of horizontal spots ŽG, 36 kDa, pI 4.7–8.3. are carbamylated G3PDH, and the vertical streaks are staining artifacts. By comparison of control ŽFig. 1B. and PDGF-BB-stimulated ŽFig. 1C. autoradiograms, it is clear that PDGF-BB enhances protein phosphorylation in neurons. Examples of these
Fig. 3. Immunoprecipitation with anti-PDGF receptor Ž a q b .. Cell lysates of 32 P-Pi labeled type 1 astrocytes were immunoprecipitated with anti-PDGF receptor Ž a q b . IgG and Pansorbin. PDGF-BB Žmiddle lane, BB. upregulates autophosphorylation of PDGF receptors Ždouble arrowheads., however, the level of phosphorylation of PDGF receptors after PDGF-AA treatment Žleft lane, AA. is indistinguishable from the control Žright lane, ctrl..
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a PDGF-stimulated neuronal culture.. Of the complex at 80 kD, the intermediate pI form Žjust above and to the right of the tip of the arrow. is increased in intensity between control and PDGF-treated lysates. More importantly, there is an apparent shift in pI of part of the 130 kDa complex to a more acidic pI Žthat is, to the left as shown.. This may result from posttranslational changes in the 130 kDa actin-binding protein, or increased expression of a related protein of lower pI, or both. The properties of actin binding, molecular weight, and pI are consistent with several known cytoskeletal proteins. Of the higher molecular weight set Žapparent M w about 130 kDa., the most basic ŽpI 6.2–6.5. is possibly vinculin. The molecular weight, the pI and the triplet structure Žthree isoforms of equal M w but different isoelectric points. are all characteristic of mammalian vinculin w20x which is found in neurons where it is tyrosine phosphorylated w37x. Note the upregulation of this complex of proteins in the presence of PDGF-BB ŽFig. 4B. relative to the levels in the control ŽFig. 4A..
Fig. 4. Actin-binding overlay assay. Actin-binding overlay blots were prepared from neuronal lysates as described in the Materials and Methods section. There are two major protein species of actin-binding proteins. In response to PDGF-BB treatment, vinculin-like protein species Ž130 kDa. have been shifted to the acidic side Žarrowhead.; and the avidity or expression of ezrin-like protein species Ž80 kDa. are increased Žarrow..
tion, and PDGF-AA Žleft lane. is not. Anti-PDGF receptor Ž a q b . precipitates two radiolabeled proteins. The higher molecular weight form is probably the PDGF receptor a-subunit Žtop band., while the other is likely to be the b-subunit Žbottom band.. 3.5. Actin-binding oÕerlay assay Fig. 4 shows the result of an actin overlay blotting experiment. As expected, only a few of the proteins found in a neuronal lysate are capable of binding actin in vitro after electroblotting. However, those proteins that do bind actin must do so with enough avidity to survive the multiple steps in the visualization process, and so are likely to represent proteins strongly associated with the actin cytoskeleton. The presence of PDGF significantly alters the expression, mobility andror avidity of neuronal actin-binding proteins relative to control cultures Žcompare Fig. 4A, actin-binding proteins in a control lysate, to Fig. 4B, from
Fig. 5. Immunoblotting with anti-ezrin antibody. Polyclonal antibody raised in rabbit against human ezrin from Dr. Tony Bretscher was used to probe PVDF blots of 2D gels. The blots indicate that the anti-ezrin antibody reacts with a 80 kDa protein species Žarrow. and that its pattern is quite similar to the 80 kDa protein in actin-binding overlay blots ŽFig. 4..
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The 80 kDa protein is possibly ezrin, or one of the ezrin-radixin-moesin family members. Ezrin has been found in developing neurons and has a molecular weight and predicted pI consistent with the species at this location. The spot at 80 kDa and pI 8.0 might be a ezrin-isoform which did not get into the gel. It is also possible that one or more of the actin-binding proteins visible using the overlay method in neurons are as yet unidentified proteins. In a total of 10 repetitions of this experiment, we see consistent effects of PDGF on actin-binding proteins in overlay blots. 3.6. Anti-ezrin immunoblotting Western blots ŽFig. 5. show that anti-ezrin antiserum reacts with an 80 kDa protein species. Comparing to Fig. 4, the 80 kDa protein in actin-binding overlay blots is very likely to be ezrin. An ezrin-like spot at the basic end is also visible in the immunoblot. 4. Discussion 4.1. The 2-D gel pattern of nerÕous system cells A limited amount of work has been done to establish a protein 2-D gel database w17x. While this should one day be helpful in identification of PDGF-stimulated phosphoproteins, at this time little is known about the 2-D map of mouse neurons and type 1 astrocytes. For this reason, most of the PDGF-regulated phosphoproteins on our silver staining gels remain unidentified. Fig. 1A, 2A and 4A show silver-stained 2-D gels of mouse neurons, type 1 astrocytes and neuronal cytoskeletal proteins, respectively. Identification of abundant proteins may be made by comparing our gels with the literature w11,16x. For example, actin and tubulin isoforms are relatively easy to identify. No obvious de novo protein synthesis was found in PDGF-stimulated neurons and type 1 astrocytes. 4.2. Effects of PDGF-AA on CNS cells PDGF-BB enhances protein phosphorylation in neurons and type 1 astrocytes ŽFigs. 1, 2 and 4., while PDGF-AA has little, if any, effect Ždata not shown.. Similarly, PDGFBB increases PDGF receptor autophosphorylation, and PDGF-AA does not ŽFig. 3.. It is widely accepted that in fibroblasts, PDGF-AA is a much less potent mitogen. In fact, most of the available information about the PDGF signal transduction pathway was deduced from PDGF-BB activities on fibroblasts. The best-known effect of PDGF-AA is that it regulates the timing of differentiation in oligodendrocytes by inhibiting premature differentiation of O2A cells w50,56x. Other researchers w41x have provided evidence that PDGF-AA modulates the PDGF-BB stimulation of smooth muscle cell migration: PDGF-AA has no stimulatory activity itself, but it can diminish the effect induced by PDGF-BB.
In fibroblasts and monocytes, the chemotactic effect of PDGF has been well documented; however, PDGF-AA inhibits the chemotactic activity of PDGF-AB and -BB w70x. Giacobini and coworkers w21,22x also found that PDGF-BB had trophic effects on transplanted hippocampal tissue, whereas PDGF-AA seemed to inhibit growth of hippocampal grafts. Given the extensive interconnections between the PDGF-AA and -BB signal transduction pathways, it is not too surprising that we did not find strong and consistent effects of PDGF-AA alone on protein phosphorylation, including receptor autophosphorylation. PDGF receptor a subunit transduces a negative signal in some cell types, inhibiting smooth muscle cell migration w41x and fibroblast Ca2q influx w13x. One possibility currently under investigation is that PDGF-AA-mediated signal transduction in neurons does not involve phosphorylation in any significant way. The function of PDGF-AA may be as the ‘brake’ to a PDGF-BB-stimulated ‘accelerator’. This is consistent with our observation that PDGF-AA alone has little effect on protein phosphorylation. Furthermore, PDGF-AA binds only one receptor dimer combination, while PDGF-BB binds all three possible receptor dimers. It may simply be that PDGF-AA can not exert as large an effect as PDGF-BB. Our findings might also imply that previous descriptions of the expression of PDGF receptor a subunit in neurons are incorrect. This is considered unlikely because of the consistent observations of a number of laboratories indicating the existence of PDGF a receptor message and protein in CNS cells, both in developing tissue and in vitro w14,61,81x. 4.3. Effects of PDGF-BB on CNS cells Phosphorylation, a posttranslational covalent modification, is a key mechanism used by cells to convey intracellular signals. These signals may be initiated by growth factor binding to cell surface receptors, which in turn sets into motion a signal cascade impinging on the cytoplasm andror the nucleus in order to control cell growth and other cellular activities w40,76x. We show here that PDGFBB is able to stimulate protein phosphorylation in cortical neurons and type 1 astrocytes in vitro. PDGF receptors, signaling molecules, neurotransmitter-related enzymes, ion channel proteins, and cytoskeletal proteins are candidates for PDGF-stimulated phosphorylation. These proteins whose phosphorylation is enhanced by PDGF might be later identified using microsequencing, Western blotting and immunoprecipitation techniques. In fact, one of the PDGF-stimulated phosphoproteins ŽF1 F0 ATPase d subunit. has been provisionally identified using all three methods w82x. The significance of protein phosphorylation in the nervous system has been extensively explored. It has been suggested that phosphorylation of the neurotransmitters and their receptors is related to transmitter functions, and to synaptic plasticity and maturation w23,43,60x. Other
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studies suggest that phosphorylation of neuronal cytoskeletal proteins, such as neurofilaments, and the microtubule-associated protein tau, could affect axonal transport and neuronal connections w48x. Protein phosphorylation at the neuronal growth cone has also been described w8,79x. Vinculin was found to be tyrosine-phosphorylated in the growth cone-rich fraction of rat brain; its phosphorylation may be involved in the functions of growth cones w37,38x. We have reported that PDGF and its receptors are expressed in the central nervous system during development, especially when neurons begin process outgrowth w36x; we show in the present study the PDGF-BB enhances phosphorylation of neuronal cytoskeletal proteins. It is likely that PDGF plays an important role in neurite outgrowth by regulation of cytoskeletal phosphorylation and organization during the development of central nervous system. PDGF-BB also alters the expression, electrophoretic migration pattern andror actin-binding ability of some actin-binding proteins in mouse cortical neurons in vitro. Provisional identification was made based on molecular weight, pI and actin-binding properties. The 130 kDa, pI 6.2 protein is likely to be vinculin. Its expression or actin-binding capacity was increased by PDGF-BB. PDGF induces a rapid and transient increase in vinculin message production, and protein synthesis in 3T3 fibroblasts w1x. It might have a similar effect in neurons. We have prepared autoradiograms of 32 P-labeled neuronal lysates separated by 2D gel electrophoresis where the actin-binding proteins are identified by an actin overlay method. Combining autoradiography and the actin overlay method, we demonstrate that the 130 kDa protein is phosphorylated, and that its phosphorylation is not significantly regulated by PDGF stimulation. This is consistent with reports that vinculin is phosphorylated on either serine or tyrosine w67,78x. The complex at 80 kDa, pI 6.0 might be ezrin or one of the ezrin-radixin-moesin family members based on its actin binding properties, molecular weight and pI. The pattern of the anti-ezrin immunoreactive spots is almost identical to that of the 80 kDa protein on actin-binding blots. Either its expression or its avidity for actin have been enhanced by PDGF-BB treatment. Ezrin is phosphorylated in response to epidermal growth factor stimulation in mesenchyme-derived cells w7,15,24,32,42x. The autoradiogram from the blotting membrane, however, does not show that the 80 kDa protein is phosphorylated. PDGF-BB might not induce ezrin phosphorylation in neurons, or the degree of phosphorylation is below detectable limits. It is also possible that the 80 kDa protein complex is not ezrin but radixin or moesin since they share a high homology and their molecular weight and predicted pI are quite similar w18,29,44x. Ezrin has been demonstrated to be colocalized with actin in a wide variety of cells w6,7x. However, attempts to show binding of ezrin to native muscle a-actin in vitro have not been successful so far w6x. Recently, ezrin has been shown to bind b-actin columns, but not to columns
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of the full length molecules of a-actin w69x. Furthermore, Gary and Bretscher w19x have found that native ezrin has a normally masked C-terminal domain, and ezrin self-association involves unmasking this domain. Binding of ezrin to a-actin may also depend on this unmasking mechanism because the F-actin binding site of ezrin is also located in the normally masked C-terminal domain. They have also shown that SDS seems able to unmask the C-terminal domain by unfolding the ezrin molecule. In our system, ezrin is denatured by urea and SDS. This might be the reason that ezrin binds to a-actin relatively strongly in our actin binding assay, while others have failed to show native ezrin binding to a-actin in their cosedimentation and coprecipitation experiments w6x. Finally, PDGF may somehow involve unmasking the C-terminal domain, including the F-actin binding site, to regulate ezrin’s ability to bind actin. The actin-binding proteins form critical links in actin stress fibers and focal adhesions. Some actin-binding proteins are enriched in the growth cone particle fractions of fetal rat brains w39x. Rankin and Rozengurt w59x found that PDGF modulates tyrosine phosphorylation of focal adhesion kinase and paxillin in fibroblasts; PDGF also regulates assembly of focal adhesions, actin stress fibers and membrane ruffling in fibroblasts w62,63x. The effects of PDGF and other growth factors on focal adhesions and the cytoskeleton of non-neuronal cells have recently been described w10,49x. PDGF might play a role in neuronal outgrowth by regulating the organization of actin and actin-binding proteins in lamellopodia or focal adhesionlike structures. Our future work will be focused on identifying PDGFregulated phosphorylated proteins in order to dissect the elements of the PDGF signal transduction pathway in the nervous system, and to ultimately understand how PDGF influences the development of the nervous system. We could also study the time-course of PDGF-induced phosphorylation to examine initial PDGF signal transduction elements and later effects. It would also be of importance to know which of the PDGF-induced phosphoproteins are tyrosine-phosphorylated and which are modified on serine andror threonine. Acknowledgements We thank Drs. Jack Correia, Rosemary Hoffman and Sharon Lobert for providing advice during the course of this project. This work was partly supported by the Virginia Boyce Research Fund of the Fight for Sight. References w1x Ben-Ze’ev, A., Reiss, R., Bendori, R. and Gorodecki, B., Transient induction of vinculin gene expression in 3T3 fibroblasts stimulated by serum-growth factors, Cell Regul., 1 Ž1990. 621–636. w2x Bottenstein, J.E., Culture methods for growth of neuronal cell lines
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Research report
Protein phosphorylation in response to PDGF stimulation in cultured neurons and astrocytes Frank X. Zhang a b
a,1
, James B. Hutchins
a,b,)
Department of Anatomy, UniÕersity of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4500, USA Department of Neurology, UniÕersity of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4500, USA Accepted 26 November 1996
Abstract Platelet-derived growth factor ŽPDGF. is an important growth factor for a variety of cells, including neurons and glial cells. PDGF signal transduction pathways have been studied primarily in mesenchyme-derived cells Žsuch as fibroblasts and smooth muscle cells.. However, little is known about these pathways in the central nervous system ŽCNS.. It is believed that phosphorylation is a critical aspect of several steps in the signal transduction pathway. In this study, neurons and type 1 astrocytes in vitro were radiolabeled with 32 P-orthophosphate Ž 32 P-Pi .. The cells were lysed, and labeled proteins were separated by two-dimensional gel electrophoresis. Autoradiograms of PDGF-stimulated and control samples were compared. We found that in neurons and type 1 astrocytes in vitro, PDGF-BB greatly enhances protein phosphorylation while PDGF-AA has less of an effect on protein phosphorylation. Furthermore, because PDGF signal transduction pathways are likely to affect the cytoskeleton, we studied changes in actin-binding proteins induced by PDGF-BB. We found that PDGF-BB alters the expression, migration pattern andror avidity of some actin-binding proteins in neurons. In conclusion, protein phosphorylation is up-regulated by PDGF in mouse cortical neurons and type 1 astrocytes in vitro. PDGF’s effects on phosphorylation of cytoskeletal proteins might be a important mechanism by which PDGF affects the development and normal functions of central nervous system cells. q 1997 Elsevier Science B.V. All rights reserved. Keywords: Actin-binding protein; Cytoskeletal protein; Development; Growth factor; Nervous system; Signal transduction
1. Introduction Platelet-derived growth factor ŽPDGF. is a growth factor expressed in a variety of cells and seems to have multiple physiological and pathological effects. PDGF is involved in cell mitogenesis, differentiation, transformation and migration w57,58x. PDGF consists of homo- or heterodimers of two subunits termed A and B. It is well established that PDGF plays an important role in the development and biological functions of mesenchyme-derived cells, but its expression within, and effects on, the nervous system have only recently been described. Yeh et al. w80x demonstrated the expression of PDGF A-chain protein and message in embryonic and adult mouse neurons. Similarly, PDGF B-chain ) Corresponding author. Fax: q1 Ž601. 984-1655. E-mail: [email protected] 1 Current address: Dept. of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Room 264 JAH, Philadelphia, PA 19107.
protein and transcript are found within neurons of the monkey brain w65x. PDGF receptor comprises two subunits: a and b , which may also form homo- or heterodimers. PDGF receptor a subunit ŽPDGFR-a . has been found in human neuroblastoma cells w53x, in rat sciatic nerve and dorsal root ganglion cells w14x, in developing rat brain w61x, and in mouse brain tissue, cortical neurons and glia w81x. PDGF receptor b subunit ŽPDGFR-b . was found in the rat brain using immunohistochemical and Northern blotting techniques w61,72x. Hutchins and colleagues demonstrated with immunohistochemistry, Western blotting and receptor cross-linking that PDGF and its receptor are expressed in developing mouse brain w35,36x, and rat and mouse neuronal and astroglial cells in vitro w33,34x. While the location and expression of PDGF and its receptor have been characterized, the functions of PDGF in the nervous system have been explored less extensively. PDGF-AA inhibits premature differentiation of oligodendrocytertype 2 astrocyte progenitor ŽO2A. cells in rat optic nerve w50,54–56x. PDGF also stimulates chemotaxis
0165-3806r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 3 8 0 6 Ž 9 6 . 0 0 2 1 8 - 0
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in rat astrocytes w5x. PDGF-BB decreases potassium channel amplitude with no change in kinetics when PDGF receptor is expressed in Xenopus oocytes w75x. This suggests that PDGF has the capacity to influence neuronal activity. PDGF increases the survival of rat newborn cerebellar GABAergic interneurons and stimulates neurite outgrowth in rat cerebellar neurons in vitro w71,72x. The signal transduction pathways mediated by PDGF in fibroblasts have been extensively investigated, but little work has been done in the nervous system. PDGF receptor a and b subunits, which both belong to the receptor tyrosine kinase superfamily, form functional dimers Ž aa , ab and bb . when PDGF binds to them. Dimerization, in turn, initiates receptor auto- andror transphosphorylation. The conformational change which occurs activates other kinase sites on the receptor w3,46,47,64,77x. These events are obligatory initial steps in the PDGF signal transduction pathway w9,27,28x. The substrates of the receptor tyrosine kinases include phospholipase C g ŽPLCg ., phosphatidylinotitol-3-kinase ŽPI3 kinase., GTPase-activating factor ŽGAP., Grb2, Shc, Raf and protein tyrosine phosphatases. Other downstream elements of the PDGF signal transduction pathway include the Ras pathway, which is composed of Raf, Ras, 14-3-3, and MEK-1 w25,66x, or the JAK-STAT pathway w12,68x. The ultimate events in the PDGF signal pathway include activation of transcriptional factors affecting protein synthesis, posttranslational modification of proteins Žsuch as phosphorylation., and cytoskeletal reorganization. It has been shown that PDGF induces talin phosphorylation and reorganization in a skeletal muscle cell line w74x, and causes a change in the distribution of actin and vinculin in fibroblasts w26,30x and in vascular smooth muscle cells w31x. However, the effects of PDGF on protein phosphorylation, including cytoskeletal proteins, in nervous system cells have not yet been described. 2. Materials and methods 2.1. Neuronal cell culture All animal care and use procedures were carried out in accordance with federal and local guidelines. Pregnant C57B1 mice were sacrificed by cervical dislocation. Mouse embryos at embryonic day 13.5 to 15 w73x were killed by decapitation. Their cerebral cortices were dissected to remove the meninges and external blood vessels, and were dissociated with 0.25% trypsinr1 mM EDTA, ŽGibco. for 20 min at 378C. The dissociated cells were preplated onto a non-coated T-75 flask for 2 h to remove adherent cells Žmainly fibroblasts., then the floating cells were plated onto polylysine-coated or laminin-coated T-25 flasks at a density of 10 4 to 10 5 cellsrcm2 in DMEMrF-12 medium or Neurobasal medium. Either medium was supplemented with 20% serum and 100 Urml penicillin, 100 m grml streptomycin and 0.25 m grml amphotericin B Žall from
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Gibco.. The purity of neuronal cultures was monitored with anti-neurofilament immunostaining. In previous studies, over 95% of cells were neurofilament-positive using identical techniques. 2.2. Type 1 astrocyte culture Type 1 astrocytes were obtained from postnatal day 1 to day 3 C57B1 mice, following the method described by Levison and McCarthy w45x. Briefly, after dissection and dissociation of the cerebral cortices, the cells were plated onto T-75 flasks in Basal Medium Eagle ŽBME. medium with 10% fetal bovine serum and 100 Urml penicillin, 100 m grml streptomycin and 0.25 m grml amphotericin B Žall purchased from Gibco; this mixture is termed BME-C.. After the cells reach confluence, the flask was shaken at 300 rpm overnight Ž378C.. The floating cells Žprocessbearing cells. were discarded. The adherent cells were trypsinized with 0.05% trypsin-EDTA ŽGibco. and replated onto T-25 flasks at 10 4 to 10 5 cellsrcm2 in BME-C medium. These type 1 astrocytes become confluent in about a week. Generally, over 95% of cells are anti-glial fibrillary acidic protein-immunoreactive. 2.3. Radiolabeling After two days in vitro, mouse cortical neurons were incubated in serum-free medium overnight. Therefore, radiolabeling took place at three days in vitro. The serum-free medium was G5rN3 medium including 5 nM hydrocortisone, 5 m grml insulin, 10 ngrml epidermal growth factor, 0.5 ngrml basic fibroblast growth factor, 100 m M putrescine, 20 nM progesterone, 30 nM sodium selenite, 50 m grml transferrin, 50 m grml ascorbic acid, 1 mgrml bovine serum albumin and 100 Urml penicillin-streptomycin w2x. After two rinses with phosphate-free DMEM medium ŽGibco., the neurons were incubated with 0.5 mCirml 32 P-orthophosphate Ž 32 P-Pi , Amersham. in phosphate-free DMEM at 378C for 2.5 h. Recombinant human PDGF-BB or -AA Ž30 ngrml, Promega. were added to experimental flasks for 2.5 h. PDGF has optimal effects at concentrations ranging from 10 to 50 ngrml in fibroblasts and smooth muscle cells. Confluent type 1 astrocytes were serum-starved with BME overnight, and then rinsed and labeled with 0.5 mCirml 32 P-Pi with Žexperimental group. or without Žcontrol group. 30 ngrml rPDGF-BB or -AA in GHCKS medium Ž11 mM glucose, 20 mM HEPES, 10.2 mM sodium citrate, 4 mM KCl, 109 mM NaCl and 0.0002% phenol red, pH 7.1. at 378C for 2.5 h. 2.4. Preparation of cell lysates After labeling, the neurons and type 1 astrocytes were rinsed twice with 50 mM Tris buffer, scraped and collected. After a brief microcentrifugation, the cell pellets were incubated with urea solubilization buffer Ž9 M urea,
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4% NP-40, 2% LKB ampholyte pH 3.5–10, 2% b-mercaptoethanol, pH 9.8. at room temperature for 2 h. Then the cell lysates were centrifuged at 12 000 = g, 48C for 3 h to remove insoluble material. The supernatant was collected and diluted for a dye-binding protein assay w4x and liquid scintillation counting to determine protein concentration and 32 P incorporation rate, respectively. 2.5. Two-dimensional gel electrophoresis 2-D gel electrophoresis was carried out based on the O’Farrell w51x method. Equal amounts of protein Ž20 m g. from experimental and control cell lysates were loaded onto tube gels Ž9.2 M urea, 1.33% LKB ampholyte pH 3.5–10, 0.67% Serva ampholyte pH 2–11, 2% NP-40 and 4% acrylamide.. Isoelectric focusing Žfirst dimension. was carried out for a total of 13,000 V-h. The proteins were then separated by molecular mass with standard SDS-
PAGE Ž4% acrylamide stacking gel and 12% acrylamide separating gel.. The pI calibration was conducted using two independent methods. In the first, a blank tube gel was run in parallel. This gel was cut into 1 cm long pieces and the slices were soaked in 1 ml distilled water overnight. The pH of the resulting buffer was measured. In the second method, a carbamylated G3PDH 2-D standard ŽLKB. was mixed in the protein sample and visualized along with cellular proteins. The slab gels were fixed, silver-stained w52x and dried. Conventional X-ray film autoradiography and PhosphorImager analysis ŽMolecular Dynamics. were carried out. Experiments were repeated several times to check for consistent PDGF effects. 2.6. Immunoprecipitation 32
P-labeled type 1 astrocytes were rinsed with 50 mM Tris buffer and lysed with immunoprecipitation buffer
Fig. 1. Silver-stained gel and autoradiograms of neuronal proteins. 32 P-Pi labeled mouse cortical neurons were lysed in urea buffer and separated by O’Farrell 2-D gel electrophoresis. Fig. 1A shows a silver-stained gel with the position of molecular weight standards indicated on the left and pI on the bottom. Actin Ža. and tubulin isoforms Žt. were identified by comparison to the literature. The horizontal spots ŽG, 36 kDa, pI 4.7–8.3. are carbamylated G3PDH pI calibration standards, and the vertical streaks are staining artifacts. Fig. 1B Žcontrol. and 1C ŽPDGF-BB stimulated. are autoradiograms from 32 P-Pi-labeled neuronal lysates. In general, PDGF-BB enhances protein phosphorylation in neurons in vitro. Several PDGF-stimulated phosphoproteins are labeled: a, b, c, and d . Their apparent molecular weight and pI are 110 kDa, 4.5; 130 kDa, 5.0; 20 kDa, 4.1; 16 kDa, 4.7; respectively. The protein labeled ‘d ’ is the mitochondrial protein F1 F0 ATPase d subunit w82x.
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Ž0.1% Triton X-100, 0.1% deoxycholate, 5 mM EDTA, 50 m M sodium vanadate, 1 m grml each leupeptin, aprotinin, pepstatin, 10 mM Tris pH 8, 140 mM NaCl and 0.025% sodium azide.. Anti-PDGF receptor Ž a q b . antibody Ž10 m l for 100 m g protein in total volume of 100 m l, UBI. was added to form an immunocomplex. A slurry of 10% Žwrv. Pansorbin ŽCalbiochem. was used to precipitate the immunocomplexes formed. The pellet was extensively rinsed with dilution buffer Ž0.1% Triton X-100, 0.1% BSA, 50 m M sodium vanadate, 0.025% sodium azide, 140 mM NaCl and 10 mM Tris, pH 8.0., TSA buffer Ž140 mM NaCl, 0.025% sodium azide and 50 m M sodium vanadate, 10 mM Tris, pH 8.0. and 50 mM Tris. Finally, Laemmli buffer was added to the pellets. After boiling and centrifugation, the supernatants from this step were resolved by SDS-PAGE Ž5% to 20% acrylamide exponential gradient gel.. The gel was fixed, dried, and set up for autoradiography.
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2.7. Actin-binding oÕerlay assay Neuronal lysates Ž100 m g. solubilized in urea solubilization buffer were separated by 2-D gel electrophoresis, and then transferred to a polyvinylidine difluoride ŽPVDF. m em brane in 10 m M 3- Ž cyclohexylam ino . -1propanesulfonic acid ŽCAPS. buffer, pH 11.0 with a field of 14 V at 48C overnight. The PVDF membranes were incubated in 2% non-fat dry milk in Tris-buffered saline ŽTBS. for 1 h. Biotinylated F-actin was formed by incubation of 0.1 mgrml biotinylated actin ŽCytoskeleton, Denver, CO. in 50 mM KCl, 2 mM MgCl 2 , 1 mM CaCl 2 and 1 mM ATP. The F-actin Ž5 m grml. was incubated with the membranes in ‘actin-binding buffer’ Ž50 mM Tris, 150 mM NaCl, 2 mM MgCl 2 , 1 mM CaCl 2 and 0.2 mM ATP. at room temperature for 1 h. The membranes were then rinsed and treated with avidin–HRP conjugate Ž2 m grml in actin-binding buffer. for 1 h. The actin binding protein
Fig. 2. Silver-stained gel and autoradiograms of type 1 astrocyte proteins. Urea lysates of radiolabeled type 1 astrocytes were resolved by 2-D electrophoresis. As in Fig. 1A, molecular weight and pI standards are labeled on the left and bottom of the silver-stained gel ŽFig. 2A.. Fig. 2B Žcontrol. and 2C ŽPDGF-BB-stimulated. are autoradiograms of 32 P-Pi-labeled cell lysates. As in Fig. 1, species marked a, b, c, d , e are several examples of proteins whose phosphorylation is increased by PDGF-BB, their estimated molecular weight and pI are 110 kDa, 4.5; 130 kDa, 5.0; 20 kDa, 4.1; 16 kDa, 4.7; 50 kDa, 6.0, respectively.
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complexes were visualized using 0.01% H 2 O 2 as substrate and diaminobenzidine Ž1 mgrml. as a chromogen. 2.8. Immunoblotting with ezrin antibody Neuronal lysate was prepared, electrophoresed and blotted onto PVDF as described in the ‘Actin-binding Overlay Assay’ method section. The PVDF membranes were incubated in ‘blocking solution’ Ž2% bovine serum albumin, 0.1% Tween-20 in TBS. for 1 h at room temperature. Rabbit antiserum against human ezrin, kindly provided by Dr. Tony Bretscher of Cornell University w7x, crossreacts with mouse ezrin. The membranes were incubated with anti-ezrin antibody at a 1:3,000 dilution in ‘blocking solution’ for 2 h at room temperature. After three washes with 0.1% Tween-20 in TBS, the membranes were incubated with HRP-conjugated-goat anti-rabbit Ž1:1,000, Jackson ImmunoResearch. for another hour. Then, the membranes were rinsed again, and reacted with 0.1% diaminobenzidine and 0.01% H 2 O 2 .
PDGF-stimulated phosphoproteins are labeled in Figs. 1 and 2 Ža, b, c and d .. Their estimated molecular weight and pI are 110 kDa, 4.5; 130 kDa, 5.0; 20 kDa, 4.1; 16 kDa, 4.7, respectively. The protein d has been identified as F1 F0 ATPase d subunit by microsequencing, Western blotting and immunoprecipitation w82x. 3.3. Phosphorylation of type 1 astrocyte proteins by PDGF-BB It is obvious that the gel-staining pattern of neurons ŽFig. 1A. is different from type 1 astrocytes ŽFig. 2A.. PDGF-BB also increases protein phosphorylation in type 1 astrocytes ŽFig. 2B, control; Fig. 2C, PDGF-BB-stimulated.. Protein species whose phosphorylation responds to PDGF-BB stimulation include a,b,c, and d . These likely correspond to the same proteins in neurons and astrocytes. It seems that protein e Ž50 kDa, pI 6.0. is an astrogliaspecific protein. 3.4. PDGF receptor autophosphorylation
3. Results As expected, a relatively large number of neuronal and astroglial proteins are seen in silver-stained 2-D gels ŽFig. 1A and 2A.. A subset of these, but still a substantial number, are phosphoproteins which may be identified by 32 P-Pi labeling of cells in culture ŽFig. 1B and 2B.. The phosphoproteins of interest in the present paper are those whose labeling is either up- or down-regulated in response to PDGF Žcompare panels B and C in Figs. 1 and 2.. A number of such proteins can be seen Židentified by letters a–e. in autoradiograms when control and experimental cultures are compared.
Immunoprecipitation studies of radiolabeled type 1 astrocytes ŽFig. 3. demonstrated that PDGF-BB Žmiddle lane. is able to trigger PDGF receptor autophosphoryla-
3.1. Protein phosphorylation of CNS cells and PDGF-AA In contrast to PDGF-BB, treatment of either neurons or astroglia with PDGF-AA had only subtle and variable effects on protein phosphorylation Ždata not shown.. For this reason, the present study concentrates on PDGF-BBstimulated phosphorylation. 3.2. Phosphorylation of neuronal proteins by PDGF-BB It appears that PDGF-BB upregulates protein phosphorylation in neurons ŽFig. 1.. On a silver-stained gel ŽFig. 1A., actin Ža, 45 kDa, pI 5.4. and tubulin isoforms Žt, 54 kDa, pI 5.1. are identified by their apparent molecular weight, pI and migration pattern. The series of horizontal spots ŽG, 36 kDa, pI 4.7–8.3. are carbamylated G3PDH, and the vertical streaks are staining artifacts. By comparison of control ŽFig. 1B. and PDGF-BB-stimulated ŽFig. 1C. autoradiograms, it is clear that PDGF-BB enhances protein phosphorylation in neurons. Examples of these
Fig. 3. Immunoprecipitation with anti-PDGF receptor Ž a q b .. Cell lysates of 32 P-Pi labeled type 1 astrocytes were immunoprecipitated with anti-PDGF receptor Ž a q b . IgG and Pansorbin. PDGF-BB Žmiddle lane, BB. upregulates autophosphorylation of PDGF receptors Ždouble arrowheads., however, the level of phosphorylation of PDGF receptors after PDGF-AA treatment Žleft lane, AA. is indistinguishable from the control Žright lane, ctrl..
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a PDGF-stimulated neuronal culture.. Of the complex at 80 kD, the intermediate pI form Žjust above and to the right of the tip of the arrow. is increased in intensity between control and PDGF-treated lysates. More importantly, there is an apparent shift in pI of part of the 130 kDa complex to a more acidic pI Žthat is, to the left as shown.. This may result from posttranslational changes in the 130 kDa actin-binding protein, or increased expression of a related protein of lower pI, or both. The properties of actin binding, molecular weight, and pI are consistent with several known cytoskeletal proteins. Of the higher molecular weight set Žapparent M w about 130 kDa., the most basic ŽpI 6.2–6.5. is possibly vinculin. The molecular weight, the pI and the triplet structure Žthree isoforms of equal M w but different isoelectric points. are all characteristic of mammalian vinculin w20x which is found in neurons where it is tyrosine phosphorylated w37x. Note the upregulation of this complex of proteins in the presence of PDGF-BB ŽFig. 4B. relative to the levels in the control ŽFig. 4A..
Fig. 4. Actin-binding overlay assay. Actin-binding overlay blots were prepared from neuronal lysates as described in the Materials and Methods section. There are two major protein species of actin-binding proteins. In response to PDGF-BB treatment, vinculin-like protein species Ž130 kDa. have been shifted to the acidic side Žarrowhead.; and the avidity or expression of ezrin-like protein species Ž80 kDa. are increased Žarrow..
tion, and PDGF-AA Žleft lane. is not. Anti-PDGF receptor Ž a q b . precipitates two radiolabeled proteins. The higher molecular weight form is probably the PDGF receptor a-subunit Žtop band., while the other is likely to be the b-subunit Žbottom band.. 3.5. Actin-binding oÕerlay assay Fig. 4 shows the result of an actin overlay blotting experiment. As expected, only a few of the proteins found in a neuronal lysate are capable of binding actin in vitro after electroblotting. However, those proteins that do bind actin must do so with enough avidity to survive the multiple steps in the visualization process, and so are likely to represent proteins strongly associated with the actin cytoskeleton. The presence of PDGF significantly alters the expression, mobility andror avidity of neuronal actin-binding proteins relative to control cultures Žcompare Fig. 4A, actin-binding proteins in a control lysate, to Fig. 4B, from
Fig. 5. Immunoblotting with anti-ezrin antibody. Polyclonal antibody raised in rabbit against human ezrin from Dr. Tony Bretscher was used to probe PVDF blots of 2D gels. The blots indicate that the anti-ezrin antibody reacts with a 80 kDa protein species Žarrow. and that its pattern is quite similar to the 80 kDa protein in actin-binding overlay blots ŽFig. 4..
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The 80 kDa protein is possibly ezrin, or one of the ezrin-radixin-moesin family members. Ezrin has been found in developing neurons and has a molecular weight and predicted pI consistent with the species at this location. The spot at 80 kDa and pI 8.0 might be a ezrin-isoform which did not get into the gel. It is also possible that one or more of the actin-binding proteins visible using the overlay method in neurons are as yet unidentified proteins. In a total of 10 repetitions of this experiment, we see consistent effects of PDGF on actin-binding proteins in overlay blots. 3.6. Anti-ezrin immunoblotting Western blots ŽFig. 5. show that anti-ezrin antiserum reacts with an 80 kDa protein species. Comparing to Fig. 4, the 80 kDa protein in actin-binding overlay blots is very likely to be ezrin. An ezrin-like spot at the basic end is also visible in the immunoblot. 4. Discussion 4.1. The 2-D gel pattern of nerÕous system cells A limited amount of work has been done to establish a protein 2-D gel database w17x. While this should one day be helpful in identification of PDGF-stimulated phosphoproteins, at this time little is known about the 2-D map of mouse neurons and type 1 astrocytes. For this reason, most of the PDGF-regulated phosphoproteins on our silver staining gels remain unidentified. Fig. 1A, 2A and 4A show silver-stained 2-D gels of mouse neurons, type 1 astrocytes and neuronal cytoskeletal proteins, respectively. Identification of abundant proteins may be made by comparing our gels with the literature w11,16x. For example, actin and tubulin isoforms are relatively easy to identify. No obvious de novo protein synthesis was found in PDGF-stimulated neurons and type 1 astrocytes. 4.2. Effects of PDGF-AA on CNS cells PDGF-BB enhances protein phosphorylation in neurons and type 1 astrocytes ŽFigs. 1, 2 and 4., while PDGF-AA has little, if any, effect Ždata not shown.. Similarly, PDGFBB increases PDGF receptor autophosphorylation, and PDGF-AA does not ŽFig. 3.. It is widely accepted that in fibroblasts, PDGF-AA is a much less potent mitogen. In fact, most of the available information about the PDGF signal transduction pathway was deduced from PDGF-BB activities on fibroblasts. The best-known effect of PDGF-AA is that it regulates the timing of differentiation in oligodendrocytes by inhibiting premature differentiation of O2A cells w50,56x. Other researchers w41x have provided evidence that PDGF-AA modulates the PDGF-BB stimulation of smooth muscle cell migration: PDGF-AA has no stimulatory activity itself, but it can diminish the effect induced by PDGF-BB.
In fibroblasts and monocytes, the chemotactic effect of PDGF has been well documented; however, PDGF-AA inhibits the chemotactic activity of PDGF-AB and -BB w70x. Giacobini and coworkers w21,22x also found that PDGF-BB had trophic effects on transplanted hippocampal tissue, whereas PDGF-AA seemed to inhibit growth of hippocampal grafts. Given the extensive interconnections between the PDGF-AA and -BB signal transduction pathways, it is not too surprising that we did not find strong and consistent effects of PDGF-AA alone on protein phosphorylation, including receptor autophosphorylation. PDGF receptor a subunit transduces a negative signal in some cell types, inhibiting smooth muscle cell migration w41x and fibroblast Ca2q influx w13x. One possibility currently under investigation is that PDGF-AA-mediated signal transduction in neurons does not involve phosphorylation in any significant way. The function of PDGF-AA may be as the ‘brake’ to a PDGF-BB-stimulated ‘accelerator’. This is consistent with our observation that PDGF-AA alone has little effect on protein phosphorylation. Furthermore, PDGF-AA binds only one receptor dimer combination, while PDGF-BB binds all three possible receptor dimers. It may simply be that PDGF-AA can not exert as large an effect as PDGF-BB. Our findings might also imply that previous descriptions of the expression of PDGF receptor a subunit in neurons are incorrect. This is considered unlikely because of the consistent observations of a number of laboratories indicating the existence of PDGF a receptor message and protein in CNS cells, both in developing tissue and in vitro w14,61,81x. 4.3. Effects of PDGF-BB on CNS cells Phosphorylation, a posttranslational covalent modification, is a key mechanism used by cells to convey intracellular signals. These signals may be initiated by growth factor binding to cell surface receptors, which in turn sets into motion a signal cascade impinging on the cytoplasm andror the nucleus in order to control cell growth and other cellular activities w40,76x. We show here that PDGFBB is able to stimulate protein phosphorylation in cortical neurons and type 1 astrocytes in vitro. PDGF receptors, signaling molecules, neurotransmitter-related enzymes, ion channel proteins, and cytoskeletal proteins are candidates for PDGF-stimulated phosphorylation. These proteins whose phosphorylation is enhanced by PDGF might be later identified using microsequencing, Western blotting and immunoprecipitation techniques. In fact, one of the PDGF-stimulated phosphoproteins ŽF1 F0 ATPase d subunit. has been provisionally identified using all three methods w82x. The significance of protein phosphorylation in the nervous system has been extensively explored. It has been suggested that phosphorylation of the neurotransmitters and their receptors is related to transmitter functions, and to synaptic plasticity and maturation w23,43,60x. Other
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studies suggest that phosphorylation of neuronal cytoskeletal proteins, such as neurofilaments, and the microtubule-associated protein tau, could affect axonal transport and neuronal connections w48x. Protein phosphorylation at the neuronal growth cone has also been described w8,79x. Vinculin was found to be tyrosine-phosphorylated in the growth cone-rich fraction of rat brain; its phosphorylation may be involved in the functions of growth cones w37,38x. We have reported that PDGF and its receptors are expressed in the central nervous system during development, especially when neurons begin process outgrowth w36x; we show in the present study the PDGF-BB enhances phosphorylation of neuronal cytoskeletal proteins. It is likely that PDGF plays an important role in neurite outgrowth by regulation of cytoskeletal phosphorylation and organization during the development of central nervous system. PDGF-BB also alters the expression, electrophoretic migration pattern andror actin-binding ability of some actin-binding proteins in mouse cortical neurons in vitro. Provisional identification was made based on molecular weight, pI and actin-binding properties. The 130 kDa, pI 6.2 protein is likely to be vinculin. Its expression or actin-binding capacity was increased by PDGF-BB. PDGF induces a rapid and transient increase in vinculin message production, and protein synthesis in 3T3 fibroblasts w1x. It might have a similar effect in neurons. We have prepared autoradiograms of 32 P-labeled neuronal lysates separated by 2D gel electrophoresis where the actin-binding proteins are identified by an actin overlay method. Combining autoradiography and the actin overlay method, we demonstrate that the 130 kDa protein is phosphorylated, and that its phosphorylation is not significantly regulated by PDGF stimulation. This is consistent with reports that vinculin is phosphorylated on either serine or tyrosine w67,78x. The complex at 80 kDa, pI 6.0 might be ezrin or one of the ezrin-radixin-moesin family members based on its actin binding properties, molecular weight and pI. The pattern of the anti-ezrin immunoreactive spots is almost identical to that of the 80 kDa protein on actin-binding blots. Either its expression or its avidity for actin have been enhanced by PDGF-BB treatment. Ezrin is phosphorylated in response to epidermal growth factor stimulation in mesenchyme-derived cells w7,15,24,32,42x. The autoradiogram from the blotting membrane, however, does not show that the 80 kDa protein is phosphorylated. PDGF-BB might not induce ezrin phosphorylation in neurons, or the degree of phosphorylation is below detectable limits. It is also possible that the 80 kDa protein complex is not ezrin but radixin or moesin since they share a high homology and their molecular weight and predicted pI are quite similar w18,29,44x. Ezrin has been demonstrated to be colocalized with actin in a wide variety of cells w6,7x. However, attempts to show binding of ezrin to native muscle a-actin in vitro have not been successful so far w6x. Recently, ezrin has been shown to bind b-actin columns, but not to columns
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of the full length molecules of a-actin w69x. Furthermore, Gary and Bretscher w19x have found that native ezrin has a normally masked C-terminal domain, and ezrin self-association involves unmasking this domain. Binding of ezrin to a-actin may also depend on this unmasking mechanism because the F-actin binding site of ezrin is also located in the normally masked C-terminal domain. They have also shown that SDS seems able to unmask the C-terminal domain by unfolding the ezrin molecule. In our system, ezrin is denatured by urea and SDS. This might be the reason that ezrin binds to a-actin relatively strongly in our actin binding assay, while others have failed to show native ezrin binding to a-actin in their cosedimentation and coprecipitation experiments w6x. Finally, PDGF may somehow involve unmasking the C-terminal domain, including the F-actin binding site, to regulate ezrin’s ability to bind actin. The actin-binding proteins form critical links in actin stress fibers and focal adhesions. Some actin-binding proteins are enriched in the growth cone particle fractions of fetal rat brains w39x. Rankin and Rozengurt w59x found that PDGF modulates tyrosine phosphorylation of focal adhesion kinase and paxillin in fibroblasts; PDGF also regulates assembly of focal adhesions, actin stress fibers and membrane ruffling in fibroblasts w62,63x. The effects of PDGF and other growth factors on focal adhesions and the cytoskeleton of non-neuronal cells have recently been described w10,49x. PDGF might play a role in neuronal outgrowth by regulating the organization of actin and actin-binding proteins in lamellopodia or focal adhesionlike structures. Our future work will be focused on identifying PDGFregulated phosphorylated proteins in order to dissect the elements of the PDGF signal transduction pathway in the nervous system, and to ultimately understand how PDGF influences the development of the nervous system. We could also study the time-course of PDGF-induced phosphorylation to examine initial PDGF signal transduction elements and later effects. It would also be of importance to know which of the PDGF-induced phosphoproteins are tyrosine-phosphorylated and which are modified on serine andror threonine. Acknowledgements We thank Drs. Jack Correia, Rosemary Hoffman and Sharon Lobert for providing advice during the course of this project. This work was partly supported by the Virginia Boyce Research Fund of the Fight for Sight. References w1x Ben-Ze’ev, A., Reiss, R., Bendori, R. and Gorodecki, B., Transient induction of vinculin gene expression in 3T3 fibroblasts stimulated by serum-growth factors, Cell Regul., 1 Ž1990. 621–636. w2x Bottenstein, J.E., Culture methods for growth of neuronal cell lines
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