- Email: [email protected]
Phosphorylation and identification of phosphorylated forms of basic fibroblast growth factor
138
F I B R O B L A S T G R O W T H FACTOR
[13]
41,42 Evidently, one of the most exciting targets of the current biological research is analysis of the HBGF ligand-receptor systems, which may be involved in a number of important biological processes. laboratories
4I p. L. Lee, D. E. Johnson, L. S. Cousens, V. A. Fried, and L. T. Williams, Science 245, 57 (1989). 42 y . Hattori and M. Terada, unpublished results, 1990.
[13] P h o s p h o r y l a t i o n a n d I d e n t i f i c a t i o n o f P h o s p h o r y l a t e d F o r m s o f Basic F i b r o b l a s t G r o w t h F a c t o r B y JEAN-JACQUES F E I G E a n d A N D R E W BAIRD
Introduction Basic fibroblast growth factor (FGF) is a potent mitogen for endothelial cells and for a wide variety of mesoderm- and neuroectoderm-derived cells. 1-4 It has recently been suggested that the tight association (Kd --l0 nM) of basic FGF with the basement membrane regulates its bioavailability and controls its interaction with its high-affinity (Kd 10-20 pM) receptors. 5-7 It has thus been important to determine whether posttranslational changes in basic FGF participate in the regulation of its activity or modulate its extracellular and intracellular localization. Although many mechanisms might exist, we have studied the potential role of protein phosphorylation. Careful analysis of the primary structure of basic FGF revealed the presence of consensus sequences for the phosphorylation of basic FGF by both protein kinase C and cyclic AMP-dependent protein kinases (Table I). 8'9 Accordingly, we established that basic FGF is a substrate for these kinases and is synthesized as a phosphoprotein by bovine capillary endoJ W. H. Burgess and T. Maciag, Annu. Rev. Biochem. 58, 575 (1989). 2 D. Gospodarowicz, N. Ferrara, L. Schweigerer, and G. Neufeld, Endocr. Rev. 8, 95 (1987). 3 A. Baird and P. B6hlen, in "Handbook of Experimental Pharmacology" (M. B. Sporn and A. B. Roberts, eds.), p. 163. Academic Press, New York, 1990. 4 A. Baird, F. Esch, P. Morm~de, N. Ueno, N. Ling, P. B6hlen, S. Y. Ying, W. Wehrenberg, and R. Guillemin, Recent Prog. Horm. Res. 42, 143 (1986). 5 A. Baird and N. Ling, Biochem. Biophys. Res. Commun. 142, 428 (1987). 6 j. Folkman and M. Klagsbrun, Science 2.35, 442 (1987). 7 A. Baird and P. A. Waiicke, Br. Med. J. 45, 438 (1989). 8 j._j. Feige and A. Baird, Proc. Natl. Acad. Sci. U.S.A. 86, 3174 (1989). 9 j. j. Feige, J. D. Bradley, K. Fryburg, J. Farris, L. C. Cousens, P. Barr, and A. Baird, J. Cell Biol. 109, 3105 (1989).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[13]
DETECTION OF b F G F PHOSPHORYLATION
139
:> ,<
L~ .<
~
o
× Z
. ~
0
~:~
. ~ : ~,,~
~
~.~.~=
C
m 0 I<
I-
~"
~z
•
~:o,~-~._
~
~
~.
~
r.D
~z ~ ~ i
~
~'-~
.~ ~ -~ ~ ,~ ~. ~ ~ .r. .~
.1 e~
~1 z
0
~I
"~
. ¢~ ~ . -
~ L ~
Z
~
~'~
~ ~,~.-~
"~ ~
0
~,~OX
~
~
~
.
e
>,
<
~,
~.~
.~ 0
0
<,
<,
¢~-
..
c~
.< IZ
L~ L~
I....
0
~'~
~:~
0
~
~
..~< •
~
~
..
~
,.~ ~:~--
~o ~ ~ ~o' ~. - ~ • . ~. ~~ ~ ~ ~ ~ : z ~ z ~ = ~ , .
u~
~.~;
~
140
FIBROBLAST GROWTH FACTOR
[13]
thelial cells and human hepatoma cells in culture. 8 Although the posttranslational modification of basic FGF, which is modulated by components of the extracellular matrix, 9 has no known physiological function, it has been postulated to play a role in the control of basic FGF bioavailability. 7-9 The methods which were used to demonstrate that basic FGF is phosphorylated are the subject of this chapter. Source of Basic Fibroblast Growth Factor and Protein Kinases Basic FGF has been purified from a variety of sources, and recombinant forms have now been made available. We use routinely recombinant human basic FGF expressed in yeast or Escherichia coli. Stock solutions (10 mg/ml in 100 mM Tris-HC1, pH 7.4, buffer) are stored as aliquots at - 8 0 ° in Eppendorf microtubes and thawed only once to maintain stability and to avoid adsorption of the growth factor to the plastic tubes. Even then, irreversible polymerization of basic FGF to a 35-kDa species can be observed and must be monitored carefully by S D S - P A G E under reducing conditions. Protein kinase C (PK-C) is purified from bovine brain according to the procedure outlined by Walton et al. Jo The catalytic subunit of cyclic AMP-dependent protein kinase (PK-A) is purified from porcine skeletal muscle according to Zoller et al. ~j The specific activity of both enzyme preparations is 1000 units/mg protein, where 1 unit is defined as 1 nmol phosphate incorporated per minute into histones H~ (PK-C) or histones HII A (PK-A). Phosphorylation of Basic Fibroblast Growth Factor in Vitro by Purified Kinases Principle. The phosphorylation of recombinant human basic FGF by purified PK-C and PK-A is based on the ability of these enzymes to catalyze the transfer of the T-phosphate of ATP into specific amino acids (serine, threonine) of the substrate (see Table I). The growth factor can thus be radiolabeled with [7-32p]ATP and then separated from unreacted [T-32p]ATP by S D S - P A G E or by trichloroacetic acid (TCA) precipitation. Reagents
10 mM ATP in 10 mM Tris-HCl, pH 7.4 [T-32p]ATP (3000 Ci/mmol), 10 mCi/ml 5 mM CaClz i0 G. M. Walton, P. J. Bertics, L. G. Hudson, T. S. Vedvick, and G. N. Gill, Anal. Biochem. 161, 425 (1987). t1 M. J. Zoller, A. R. Kerlavage, and S. S. Taylor, J. Biol. Chem. 254, 2408 (1979).
[13]
DETECTIONOF bFGF PHOSPHORYLATION
141
3 M MgC12 160/xg/ml phosphatidylserine and 3.2/xg/ml dioctanoylglycerol in 100 mM Tris-HCl, pH 7.4 [phospholipids are first dissolved in chloroform/methanol under nitrogen and solubilized into 100 mM Tris buffer (pH 7.4) using sonication at 4° for 10 min] 5 × sample buffer [10% sodium dodecyl sulfate (SDS), 50% glycerol (v/v), 5% mercaptoethanol (v/v), 0.25% bromphenol blue (w/v), 250 mM Tris, pH 6.8] 50% TCA 1 N NaOH Using these stock solutions, a radioactive ATP mix is prepared containing the following: (1) for PK-C, 100/xCi/ml [7-32p]ATP, 2 × 10 -5 M ATP, 1.2 mM CaClz, 20 mM MgCl2 ; (2) for PK-A, 100/xCi/ml [y-32p]ATP, 2 × l0 -5 M ATP, 20 mM MgC12. Procedure. When performing the phosphorylation assays, it is important to include the appropriate controls. They include omitting the kinase, its activator (phospholipids for PK-C), or the substrate. The final assay volume is 20/zl and contains 2/zl basic FGF (0.25 mg/ml), 5/zl of the phospholipid mixture, and 3 /zl of PK-C (0.02 units) or PK-A (0.06 units). All the reagents are kept on ice while the assay is being set up, and the reaction is started by adding the ATP mix (10/xl), vortexing, and placing the Eppendorf microtube in a 30° water bath. The reaction is stopped by adding of 5 /zl of a 5 × Laemmli's sample buffer, and the phospho-FGF is analyzed by polyacrylamide gel electrophoresis. After heating at 95 ° for 3 min, the sample is loaded onto a 0.8-mm thick 0.1% SDS- 15% polyacrylamide gel (8 × 10 cm, Idea Scientific, Corvallis, OR), and phosphoproteins are electrophoresed according to Laemmli 12 and visualized by autoradiography of the dried gel. An example of this result is shown in Fig. 1A, where basic FGF phosphorylated by purified PK-C is shown. Identification of Phosphoamino Acids Phosphoamino acids are identified in phosphorylated basic FGF using the procedure of Cooper et al. 13Briefly, 3zP-labeled basic FGF is extracted from the polyacrylamide gel in 50 mM ammonium bicarbonate (pH 7.3-7.6) supplemented with 0.1% SDS and I% mercaptoethanol. After precipitation with 50% TCA, the pellet is dissolved in 6 N HCI, and the protein is hydrolyzed for 60 min at 110°. 3Zp-Labeled amino acids are then I2 U. K. Laemmli, Nature (London) 227, 680 (1970). 13 j. A. Cooper, B. M. Sefton, and T. Hunter, this series, Vol. 99, p. 387.
142
FIBROBLAST GROWTH FACTOR
1 2
[13]
34 m
43-
261814-
b
6-
A
IB
FIG. 1. Detection of phosphorylated human basic FGF. (A) In this example, recombinant human basic FGF was phosphorylated for l0 min by purified PK-C and [y-3zp]ATPas described in the text. The 32p-labeled proteins were separated by SDS-PAGEand visualized by autoradiography. Lane 1, PK-C, no phospholipid, no basic FGF; lane 2, PK-C, phospholipids, no basic FGF; lane 3, PK-C, no phospholipid, basic FGF; lane 4, PK-C, phospholipids, basic FGF. (B) Phosphoamino acid analysis was performed on the 18-kDaband from lane 4 in (A) (corresponding to 32P-labeled basic FGF), which was excised from the gel and hydrolyzed in 6 N HC1 as described in the text. The radiolabeled amino acids were mixed with standard phosphoamino acids, analyzed by two-dimensional electrophoresis on cellulose plates, and visualized by autoradiography. The asterisk indicates the position of sample loading. The position of phosphoamino acids is indicated as follows: P-ser, phosphoserine; P-thr, phosphothreonine; P-tyr, phosphotyrosine.
mixed with standard p h o s p h o a m i n o acids and are subsequently separated by two-dimensional electrophoresis on cellulose thin-layer plates. The first dimension is run for 20 min at 1.5 kV in p H 1.9 buffer, and the second dimension is run for 16 min at 1.3 kV in pH 3.5 buffer. 13Standard phosphoamino acids are revealed by ninhydrin staining and used to identify the radiolabeled amino acids, which are visualized by autoradiography (Fig. 1B).
Kinetic Analyses of Phosphorylation When the stoichiometry of phosphorylation is to be determined, the reaction volume is scaled up to 200/zl with the final reagent concentrations kept unchanged, except for the concentration of ATP which is raised to 5 × 10 - 4 M. At appropriate time intervals, 20-/zl aliquots are removed from the reaction tube and the radiolabeled basic F G F precipitated by addition of 0.5 ml o f 50% TCA. An aliquot of 10/zl of 10 mg/ml bovine
[13] U,,.
O
DETECTION OF bFGF PHOSPHORYLATION
143
PKC + bFGF
1.0
I.I. e~
"6 E e f
• m
O
O
E u
O ,..-
O
E
0.5
6
O v
E
PKC
1D O O Q,. O O (= o~
(3. o3
0
20
40
Time
60
80
100
120
(rain)
FIc. 2. Stoichiometry of phosphorylation of basic FGF by protein kinase C. Recombinant human basic FGF was phosphorylated by purified PK-C in the presence of phospholipids and [y-3zP]ATP. The total amount of radioactivity incorporated into TCA-precipitable proteins was measured as a function of time as described in the text. A control incubation was done where basic FGF was omitted. The difference between the incorporations measured at the plateau of both these conditions (represented by the arrow) represents the amount of phosphate specifically incorporated into the basic FGF molecule.
serum albumin (BSA) is added as a carrier protein, and the tubes are allowed to stand in an ice bucket until the end of the assay. All precipitates are then centrifuged for l0 min in an Eppendorf microfuge. The pellets are dissolved in 0.1 ml sodium hydroxide and immediately reprecipitated with TCA. Since phosphoserine and phosphothreonine are alkali-labile, it is important to proceed quickly after the first solubilization of the pellet. After the second precipitation, the pellet of radiolabeled proteins is dissolved in sodium hydroxide and counted in a fl-counter after addition of scintillation fluid. The amount of phosphate incorporated per mole of basic FGF can be estimated from the amount of radioactivity incorporated into basic FGF at the plateau of the kinetic reaction (90-120 min, as shown on Fig. 2). Because the kinases will also autophosphorylate during the reac-
144
FIBROBLAST GROWTH FACTOR
[13]
tion, it is important to run a parallel experiment where basic FGF is omitted and to subtract the radioactivity incorporated into the kinase alone from the total amount of radioactivity incorporated (Fig. 2). Maximal incorporation of 0.7 mol phosphate/mol basic FGF was routinely obtained with PK-C (Fig. 2) and of 0.8 mol phosphate/mol basic FGF with PK-A. Taking recovery into account, this result is in agreement with the observation that a single residue in the basic FGF molecule is modified by each of these kinases. 9 In our experience, previous treatment of recombinant basic FGF with alkaline phosphatase has little effect on the amount of phosphate that can be incorporated into the molecule. By inference then, over 90% of recombinant basic FGF expressed by yeast is the dephospho form. Phosphorylation of Basic Fibroblast Growth Factor by Cells in Culture Phosphorylated forms of basic FGF have been detected in two cell lines: the human hepatoma cells SK-HEP and the bovine adrenocortical capillary endothelial cells (ACE). s The protocol that we describe in this chapter can be adapted to other cell types as well.
Reagents Ortho[32p]phosphate Phosphate-flee Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% dialyzed calf serum and 5% normal DMEM (labeling buffer) 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.1 M NaCI, 50 mM NaF, 10 ~g/ml aprotinin (RIPA buffer) Rabbit polyclonal antiserum 773 (an antiserum raised against the peptide basic FGF(1-24) coupled to bovine serum albumin (Ab 773) Recombinant human basic FGF Phosphate-buffered saline (PBS) Protein A-Sepharose, 50% (v/v) in RIPA buffer above 2% SDS, 10% glycerol, 1% fl-mercaptoethanol, 0.05% bromphenol blue, 50 mM Tris, pH 6.8 (sample buffer) Procedure. Cells are plated at a density of 6 x l 0 6 cells/10-cm diameter plate and grown for 24-48 hr in standard serum-supplemented medium. We routinely use three 10-cm plates per labeling, and, before metabolic labeling, the plates are rinsed twice with phosphate-flee labeling medium. After addition of 4 ml labeling medium containing 0.4 mCi/ml ortho[32p]phosphate, the cells are incubated overnight at 37° in a CO2 incubator. At the end of the labeling period, the cells are washed with PBS. It is important to collect and dispose of this highly radioactive
[13]
DETECTION OF bFGF PHOSPHORYLATION
145
solution using appropriate caution. The cells are then scraped with a rubber policeman into 0.5 ml RIPA buffer, collected in a screw-capped Eppendorf microtube (1.5 ml), kept on ice for 30 min, and subsequently centrifuged for 10 min in an Eppendorf microcentrifuge. 32p-Labeled basic FGF is immunoprecipitated from this supernatant. Accordingly, all operations must be carried out behind protective plexiglass shielding (2 cm thick). For the immunoprecipitation, antibasic FGF is added to the extract at an optimal concentration, in this instance at a final dilution of 1/200. Bovine serum albumin (0.5 mg/ml final concentration) is also added to the extracts in order to block nonspecific antibodies present in the rabbit antiserum. After a 2-hr incubation at 4°, 50/zl of a protein-A Sepharose suspension is added to the extracts and the incubation continued for an additional 1 hr at 4° under vigorous rotary shaking. The solution is then centrifuged for 5 min in an Eppendorf microfuge, resuspended in 0.5 ml of RIPA buffer, and washed 3 times in RIPA buffer and twice in 0.1% SDS. The final pellet is resuspended in 50/zl Laemmli 12 sample buffer, heated for 3 min at 95 °, and centrifuged, after which the supernatant is analyzed by S D S - P A G E on 0.1% SDS-15% polyacrylamide gels. After fixation, the gel is dried, and the radiolabeled, immunoprecipitated proteins are visualized by autoradiography. Remarks. In our experience, no phosphorylated forms of basic FGF are recovered from the conditioned culture medium, and phosphorylated basic FGF is always found to be cell-associated. The antibody also precipitates several phosphoproteins, but the one corresponding to phosphobasic FGF is easily identified by its absence when the precipitation is performed in the presence of an excess of cold basic FGF. 8
Biological Assays of Phosphorylated Forms of Basic Fibroblast Growth Factor The biological activity of phosphorylated basic FGF can be determined using the same conditions described for the purified mitogen. 14-~8 It is important, however, to have control preparations of basic FGF that have undergone the phosphorylation reaction without being phosphorylated 14 M. Klagsbrun, R. Sullivan, S. Smith, R. Rybka, and Y. Shing, this series, Vol. 147, p. 95. 15 D. Gospodarowicz, this series, Vol. 147, p. 106. 16 F. Esch, A. Baird, N. Ling, N. Ueno, F. Hill, L. Denoroy, R. Klepper, D. Gospodarowicz, P. B6hlen, and R. Guillemin, Proc. Natl. Acad. Sci. U.S.A. 82, 6507 (1985). 17 j. j. Feige and A. Baird, J. Biol. Chem. 263, 14023 (1988). 18 p. A. Walicke, J.-J. Feige, and A. Baird, J. Biol. Chem. 264, 4120 (1989).
146
FIBROBLAST GROWTH FACTOR
[13]
(i.e., with no enzyme or ATP). We describe here two assays among the many that are possible.
Heparin-Binding Assay The following assay is based on the strong affinity of basic FGF for heparin. 14The growth factor elutes from heparin-Sepharose columns with 1.4 and 1.8 M NaCI. The affinity of phosphorylated forms of basic FGF can be verified by loading the radiolabeled sample onto a 0.5-ml heparinSepharose column and then eluting the column with increasing concentrations of NaC1 (2 ml of 20 mM Tris-HCl, pH 7.5, containing 0, 0.15, 0.6, 1.2, 1.4, 1.6, 1.8, 2.0, and 3.0 of NaCI, respectively). Aliquots of the radiolabeled reaction mixture and of each elution step are then analyzed by S D S - P A G E on 0.1% S D S - 15% polyacrylamide gels. Autoradiography of the gel identifies the salt concentration at which 32p-labeled basic FGF elutes from the heparin-Sepharose column.
Radioreceptor Assay on BHK Cells There exist at least two binding sites for basic FGF on the surface of target cells. One site (Kd 10-10 M) corresponds to cell surface-associated heparan sulfate proteoglycans, and a second, higher affinity site (Kd 10-~1 M) corresponds to the transmembrane receptor. 19 Binding of basic FGF to heparan sulfate-proteoglycans is sensitive to high salt concentrations, whereas binding to the high affinity receptor is not. The radioreceptor assay for basic FGF is based on these observations. It is a competition assay that is routinely performed on baby hamster kidney (BHK) fibroblasts, which have a relatively high number of high-affinity receptors (20,000-40,000 receptor/cell) compared to many other cell types. 18'19
Reagents B H K cells plated in 24-well plates and grown to subconfluence in Ham's F12/DMEM (1 : 1) supplemented with 5% calf serum Binding medium: DMEM with 25 mM HEPES, 0.15% gelatin, pH 7.5 Phosphate-buffered saline (PBS) 20 mM HEPES, pH 7.5, 2 M NaCI Lysis solution: 0.1 M sodium phosphate buffer, pH 8.1, 0.5% Triton X-100 lzSI-Labeled basic FGF [100,000 counts/min (cpm)/ng] radioiodinated by the lactoperoxidase method, 2° purified by heparin-Sepharose chromatography, and stored at 4 ° 19 D. Moscatelli, J. Cell. Physiol. 131, 123 (1987). 2o D. Schubert, N. Ling, and A. Baird, J. Cell. Biol. 104, 635 (1987).
[13]
DETECTION OF bFGF PHOSPHORYLATION
147
Phospho-basic FGF, prepared as described above except that no [y32p]ATP is added to the reaction mixture; it is phosphorylated either by PK-C or by PK-A (for controls, it is important to prepare "unphospho-FGF" under identical conditions, but in the absence of enzyme or ATP) Procedure. B H K cells are rinsed once with 0.5 ml binding buffer and incubated for 2 hr at 4 ° with 0.3 ml binding medium, containing 1-2 × 105 cpm 125I-labeled basic FGF and various concentrations of phosphorylated or unphospho-basic FGF. At the end of the incubation, cells are washed twice with PBS and twice with 2 M NaC1 in 20 mM HEPES, pH 7.5. The salt washes are collected, counted, and used to measure 125I-labeled basic FGF bound to glycosaminoglycans. The 125I-labeled basic FGF that is bound to the high-affinity receptor is then collected by solubilization of the cells in the lysis buffer and counted for radioactivity on a y-counter. We routinely observe that PK-A-phosphorylated basic FGF is more potent than unphosphorylated basic FGF at displacing the binding of 125I-labeled basic FGF to its high-affinity receptor. In contrast, PK-C-phosphorylated basic FGF is equipotent. 8,9 No difference has been observed between these different forms in the binding to cell surface heparan sulfate proteoglycans (salt washes). 8 Summary and Perspectives Phosphorylation of secreted factors by intact cells is a recent observation which has been reported for atrial natriuretic factor 21 and acidic 22 and basic FGF. 8'9 Several growth factors have been reported to be substrates for purified protein kinases. 8'23 Thus, the techniques described in this chapter may be successfully adapted to other growth factors and should lead to a better understanding of the possible role of phosphorylation in the regulation of their biological activity. Acknowledgments We are grateful for the skillful secretarial work of Sonia Lidy and Denise Higgins. This work was supported by National Institutes of Health Grants HD-09690 and DK-18811, The Whittier Institute Angiogenesis Research Program, the Institut National de la Sant6 et de la Recherche M6dicale (INSERM), and the Commissariat ~ l'Energie Atomique (DRF-G).
21 j. Rittenhouse, L. Moberly, H. Ahmed, and F. Marcus, J. Biol. Chem. 263, 3778 (1988). 22 F. Mascarelli, D. Raulais, and Y. Courtois, EMBO J. 8, 420 (1989). 23 H. F. Kung, I. Calvert, E. Bekesi, F. R. Khan, K. P. Huang, S. Oroszlan, L. E. Henderson, T. D. Copeland, R. C. Sowder, and S. J. Wei, Mol. Cell, Biochem. 89, 29 (1989).
F I B R O B L A S T G R O W T H FACTOR
[13]
41,42 Evidently, one of the most exciting targets of the current biological research is analysis of the HBGF ligand-receptor systems, which may be involved in a number of important biological processes. laboratories
4I p. L. Lee, D. E. Johnson, L. S. Cousens, V. A. Fried, and L. T. Williams, Science 245, 57 (1989). 42 y . Hattori and M. Terada, unpublished results, 1990.
[13] P h o s p h o r y l a t i o n a n d I d e n t i f i c a t i o n o f P h o s p h o r y l a t e d F o r m s o f Basic F i b r o b l a s t G r o w t h F a c t o r B y JEAN-JACQUES F E I G E a n d A N D R E W BAIRD
Introduction Basic fibroblast growth factor (FGF) is a potent mitogen for endothelial cells and for a wide variety of mesoderm- and neuroectoderm-derived cells. 1-4 It has recently been suggested that the tight association (Kd --l0 nM) of basic FGF with the basement membrane regulates its bioavailability and controls its interaction with its high-affinity (Kd 10-20 pM) receptors. 5-7 It has thus been important to determine whether posttranslational changes in basic FGF participate in the regulation of its activity or modulate its extracellular and intracellular localization. Although many mechanisms might exist, we have studied the potential role of protein phosphorylation. Careful analysis of the primary structure of basic FGF revealed the presence of consensus sequences for the phosphorylation of basic FGF by both protein kinase C and cyclic AMP-dependent protein kinases (Table I). 8'9 Accordingly, we established that basic FGF is a substrate for these kinases and is synthesized as a phosphoprotein by bovine capillary endoJ W. H. Burgess and T. Maciag, Annu. Rev. Biochem. 58, 575 (1989). 2 D. Gospodarowicz, N. Ferrara, L. Schweigerer, and G. Neufeld, Endocr. Rev. 8, 95 (1987). 3 A. Baird and P. B6hlen, in "Handbook of Experimental Pharmacology" (M. B. Sporn and A. B. Roberts, eds.), p. 163. Academic Press, New York, 1990. 4 A. Baird, F. Esch, P. Morm~de, N. Ueno, N. Ling, P. B6hlen, S. Y. Ying, W. Wehrenberg, and R. Guillemin, Recent Prog. Horm. Res. 42, 143 (1986). 5 A. Baird and N. Ling, Biochem. Biophys. Res. Commun. 142, 428 (1987). 6 j. Folkman and M. Klagsbrun, Science 2.35, 442 (1987). 7 A. Baird and P. A. Waiicke, Br. Med. J. 45, 438 (1989). 8 j._j. Feige and A. Baird, Proc. Natl. Acad. Sci. U.S.A. 86, 3174 (1989). 9 j. j. Feige, J. D. Bradley, K. Fryburg, J. Farris, L. C. Cousens, P. Barr, and A. Baird, J. Cell Biol. 109, 3105 (1989).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[13]
DETECTION OF b F G F PHOSPHORYLATION
139
:> ,<
L~ .<
~
o
× Z
. ~
0
~:~
. ~ : ~,,~
~
~.~.~=
C
m 0 I<
I-
~"
~z
•
~:o,~-~._
~
~
~.
~
r.D
~z ~ ~ i
~
~'-~
.~ ~ -~ ~ ,~ ~. ~ ~ .r. .~
.1 e~
~1 z
0
~I
"~
. ¢~ ~ . -
~ L ~
Z
~
~'~
~ ~,~.-~
"~ ~
0
~,~OX
~
~
~
.
e
>,
<
~,
~.~
.~ 0
0
<,
<,
¢~-
..
c~
.< IZ
L~ L~
I....
0
~'~
~:~
0
~
~
..~< •
~
~
..
~
,.~ ~:~--
~o ~ ~ ~o' ~. - ~ • . ~. ~~ ~ ~ ~ ~ : z ~ z ~ = ~ , .
u~
~.~;
~
140
FIBROBLAST GROWTH FACTOR
[13]
thelial cells and human hepatoma cells in culture. 8 Although the posttranslational modification of basic FGF, which is modulated by components of the extracellular matrix, 9 has no known physiological function, it has been postulated to play a role in the control of basic FGF bioavailability. 7-9 The methods which were used to demonstrate that basic FGF is phosphorylated are the subject of this chapter. Source of Basic Fibroblast Growth Factor and Protein Kinases Basic FGF has been purified from a variety of sources, and recombinant forms have now been made available. We use routinely recombinant human basic FGF expressed in yeast or Escherichia coli. Stock solutions (10 mg/ml in 100 mM Tris-HC1, pH 7.4, buffer) are stored as aliquots at - 8 0 ° in Eppendorf microtubes and thawed only once to maintain stability and to avoid adsorption of the growth factor to the plastic tubes. Even then, irreversible polymerization of basic FGF to a 35-kDa species can be observed and must be monitored carefully by S D S - P A G E under reducing conditions. Protein kinase C (PK-C) is purified from bovine brain according to the procedure outlined by Walton et al. Jo The catalytic subunit of cyclic AMP-dependent protein kinase (PK-A) is purified from porcine skeletal muscle according to Zoller et al. ~j The specific activity of both enzyme preparations is 1000 units/mg protein, where 1 unit is defined as 1 nmol phosphate incorporated per minute into histones H~ (PK-C) or histones HII A (PK-A). Phosphorylation of Basic Fibroblast Growth Factor in Vitro by Purified Kinases Principle. The phosphorylation of recombinant human basic FGF by purified PK-C and PK-A is based on the ability of these enzymes to catalyze the transfer of the T-phosphate of ATP into specific amino acids (serine, threonine) of the substrate (see Table I). The growth factor can thus be radiolabeled with [7-32p]ATP and then separated from unreacted [T-32p]ATP by S D S - P A G E or by trichloroacetic acid (TCA) precipitation. Reagents
10 mM ATP in 10 mM Tris-HCl, pH 7.4 [T-32p]ATP (3000 Ci/mmol), 10 mCi/ml 5 mM CaClz i0 G. M. Walton, P. J. Bertics, L. G. Hudson, T. S. Vedvick, and G. N. Gill, Anal. Biochem. 161, 425 (1987). t1 M. J. Zoller, A. R. Kerlavage, and S. S. Taylor, J. Biol. Chem. 254, 2408 (1979).
[13]
DETECTIONOF bFGF PHOSPHORYLATION
141
3 M MgC12 160/xg/ml phosphatidylserine and 3.2/xg/ml dioctanoylglycerol in 100 mM Tris-HCl, pH 7.4 [phospholipids are first dissolved in chloroform/methanol under nitrogen and solubilized into 100 mM Tris buffer (pH 7.4) using sonication at 4° for 10 min] 5 × sample buffer [10% sodium dodecyl sulfate (SDS), 50% glycerol (v/v), 5% mercaptoethanol (v/v), 0.25% bromphenol blue (w/v), 250 mM Tris, pH 6.8] 50% TCA 1 N NaOH Using these stock solutions, a radioactive ATP mix is prepared containing the following: (1) for PK-C, 100/xCi/ml [7-32p]ATP, 2 × 10 -5 M ATP, 1.2 mM CaClz, 20 mM MgCl2 ; (2) for PK-A, 100/xCi/ml [y-32p]ATP, 2 × l0 -5 M ATP, 20 mM MgC12. Procedure. When performing the phosphorylation assays, it is important to include the appropriate controls. They include omitting the kinase, its activator (phospholipids for PK-C), or the substrate. The final assay volume is 20/zl and contains 2/zl basic FGF (0.25 mg/ml), 5/zl of the phospholipid mixture, and 3 /zl of PK-C (0.02 units) or PK-A (0.06 units). All the reagents are kept on ice while the assay is being set up, and the reaction is started by adding the ATP mix (10/xl), vortexing, and placing the Eppendorf microtube in a 30° water bath. The reaction is stopped by adding of 5 /zl of a 5 × Laemmli's sample buffer, and the phospho-FGF is analyzed by polyacrylamide gel electrophoresis. After heating at 95 ° for 3 min, the sample is loaded onto a 0.8-mm thick 0.1% SDS- 15% polyacrylamide gel (8 × 10 cm, Idea Scientific, Corvallis, OR), and phosphoproteins are electrophoresed according to Laemmli 12 and visualized by autoradiography of the dried gel. An example of this result is shown in Fig. 1A, where basic FGF phosphorylated by purified PK-C is shown. Identification of Phosphoamino Acids Phosphoamino acids are identified in phosphorylated basic FGF using the procedure of Cooper et al. 13Briefly, 3zP-labeled basic FGF is extracted from the polyacrylamide gel in 50 mM ammonium bicarbonate (pH 7.3-7.6) supplemented with 0.1% SDS and I% mercaptoethanol. After precipitation with 50% TCA, the pellet is dissolved in 6 N HCI, and the protein is hydrolyzed for 60 min at 110°. 3Zp-Labeled amino acids are then I2 U. K. Laemmli, Nature (London) 227, 680 (1970). 13 j. A. Cooper, B. M. Sefton, and T. Hunter, this series, Vol. 99, p. 387.
142
FIBROBLAST GROWTH FACTOR
1 2
[13]
34 m
43-
261814-
b
6-
A
IB
FIG. 1. Detection of phosphorylated human basic FGF. (A) In this example, recombinant human basic FGF was phosphorylated for l0 min by purified PK-C and [y-3zp]ATPas described in the text. The 32p-labeled proteins were separated by SDS-PAGEand visualized by autoradiography. Lane 1, PK-C, no phospholipid, no basic FGF; lane 2, PK-C, phospholipids, no basic FGF; lane 3, PK-C, no phospholipid, basic FGF; lane 4, PK-C, phospholipids, basic FGF. (B) Phosphoamino acid analysis was performed on the 18-kDaband from lane 4 in (A) (corresponding to 32P-labeled basic FGF), which was excised from the gel and hydrolyzed in 6 N HC1 as described in the text. The radiolabeled amino acids were mixed with standard phosphoamino acids, analyzed by two-dimensional electrophoresis on cellulose plates, and visualized by autoradiography. The asterisk indicates the position of sample loading. The position of phosphoamino acids is indicated as follows: P-ser, phosphoserine; P-thr, phosphothreonine; P-tyr, phosphotyrosine.
mixed with standard p h o s p h o a m i n o acids and are subsequently separated by two-dimensional electrophoresis on cellulose thin-layer plates. The first dimension is run for 20 min at 1.5 kV in p H 1.9 buffer, and the second dimension is run for 16 min at 1.3 kV in pH 3.5 buffer. 13Standard phosphoamino acids are revealed by ninhydrin staining and used to identify the radiolabeled amino acids, which are visualized by autoradiography (Fig. 1B).
Kinetic Analyses of Phosphorylation When the stoichiometry of phosphorylation is to be determined, the reaction volume is scaled up to 200/zl with the final reagent concentrations kept unchanged, except for the concentration of ATP which is raised to 5 × 10 - 4 M. At appropriate time intervals, 20-/zl aliquots are removed from the reaction tube and the radiolabeled basic F G F precipitated by addition of 0.5 ml o f 50% TCA. An aliquot of 10/zl of 10 mg/ml bovine
[13] U,,.
O
DETECTION OF bFGF PHOSPHORYLATION
143
PKC + bFGF
1.0
I.I. e~
"6 E e f
• m
O
O
E u
O ,..-
O
E
0.5
6
O v
E
PKC
1D O O Q,. O O (= o~
(3. o3
0
20
40
Time
60
80
100
120
(rain)
FIc. 2. Stoichiometry of phosphorylation of basic FGF by protein kinase C. Recombinant human basic FGF was phosphorylated by purified PK-C in the presence of phospholipids and [y-3zP]ATP. The total amount of radioactivity incorporated into TCA-precipitable proteins was measured as a function of time as described in the text. A control incubation was done where basic FGF was omitted. The difference between the incorporations measured at the plateau of both these conditions (represented by the arrow) represents the amount of phosphate specifically incorporated into the basic FGF molecule.
serum albumin (BSA) is added as a carrier protein, and the tubes are allowed to stand in an ice bucket until the end of the assay. All precipitates are then centrifuged for l0 min in an Eppendorf microfuge. The pellets are dissolved in 0.1 ml sodium hydroxide and immediately reprecipitated with TCA. Since phosphoserine and phosphothreonine are alkali-labile, it is important to proceed quickly after the first solubilization of the pellet. After the second precipitation, the pellet of radiolabeled proteins is dissolved in sodium hydroxide and counted in a fl-counter after addition of scintillation fluid. The amount of phosphate incorporated per mole of basic FGF can be estimated from the amount of radioactivity incorporated into basic FGF at the plateau of the kinetic reaction (90-120 min, as shown on Fig. 2). Because the kinases will also autophosphorylate during the reac-
144
FIBROBLAST GROWTH FACTOR
[13]
tion, it is important to run a parallel experiment where basic FGF is omitted and to subtract the radioactivity incorporated into the kinase alone from the total amount of radioactivity incorporated (Fig. 2). Maximal incorporation of 0.7 mol phosphate/mol basic FGF was routinely obtained with PK-C (Fig. 2) and of 0.8 mol phosphate/mol basic FGF with PK-A. Taking recovery into account, this result is in agreement with the observation that a single residue in the basic FGF molecule is modified by each of these kinases. 9 In our experience, previous treatment of recombinant basic FGF with alkaline phosphatase has little effect on the amount of phosphate that can be incorporated into the molecule. By inference then, over 90% of recombinant basic FGF expressed by yeast is the dephospho form. Phosphorylation of Basic Fibroblast Growth Factor by Cells in Culture Phosphorylated forms of basic FGF have been detected in two cell lines: the human hepatoma cells SK-HEP and the bovine adrenocortical capillary endothelial cells (ACE). s The protocol that we describe in this chapter can be adapted to other cell types as well.
Reagents Ortho[32p]phosphate Phosphate-flee Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% dialyzed calf serum and 5% normal DMEM (labeling buffer) 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.1 M NaCI, 50 mM NaF, 10 ~g/ml aprotinin (RIPA buffer) Rabbit polyclonal antiserum 773 (an antiserum raised against the peptide basic FGF(1-24) coupled to bovine serum albumin (Ab 773) Recombinant human basic FGF Phosphate-buffered saline (PBS) Protein A-Sepharose, 50% (v/v) in RIPA buffer above 2% SDS, 10% glycerol, 1% fl-mercaptoethanol, 0.05% bromphenol blue, 50 mM Tris, pH 6.8 (sample buffer) Procedure. Cells are plated at a density of 6 x l 0 6 cells/10-cm diameter plate and grown for 24-48 hr in standard serum-supplemented medium. We routinely use three 10-cm plates per labeling, and, before metabolic labeling, the plates are rinsed twice with phosphate-flee labeling medium. After addition of 4 ml labeling medium containing 0.4 mCi/ml ortho[32p]phosphate, the cells are incubated overnight at 37° in a CO2 incubator. At the end of the labeling period, the cells are washed with PBS. It is important to collect and dispose of this highly radioactive
[13]
DETECTION OF bFGF PHOSPHORYLATION
145
solution using appropriate caution. The cells are then scraped with a rubber policeman into 0.5 ml RIPA buffer, collected in a screw-capped Eppendorf microtube (1.5 ml), kept on ice for 30 min, and subsequently centrifuged for 10 min in an Eppendorf microcentrifuge. 32p-Labeled basic FGF is immunoprecipitated from this supernatant. Accordingly, all operations must be carried out behind protective plexiglass shielding (2 cm thick). For the immunoprecipitation, antibasic FGF is added to the extract at an optimal concentration, in this instance at a final dilution of 1/200. Bovine serum albumin (0.5 mg/ml final concentration) is also added to the extracts in order to block nonspecific antibodies present in the rabbit antiserum. After a 2-hr incubation at 4°, 50/zl of a protein-A Sepharose suspension is added to the extracts and the incubation continued for an additional 1 hr at 4° under vigorous rotary shaking. The solution is then centrifuged for 5 min in an Eppendorf microfuge, resuspended in 0.5 ml of RIPA buffer, and washed 3 times in RIPA buffer and twice in 0.1% SDS. The final pellet is resuspended in 50/zl Laemmli 12 sample buffer, heated for 3 min at 95 °, and centrifuged, after which the supernatant is analyzed by S D S - P A G E on 0.1% SDS-15% polyacrylamide gels. After fixation, the gel is dried, and the radiolabeled, immunoprecipitated proteins are visualized by autoradiography. Remarks. In our experience, no phosphorylated forms of basic FGF are recovered from the conditioned culture medium, and phosphorylated basic FGF is always found to be cell-associated. The antibody also precipitates several phosphoproteins, but the one corresponding to phosphobasic FGF is easily identified by its absence when the precipitation is performed in the presence of an excess of cold basic FGF. 8
Biological Assays of Phosphorylated Forms of Basic Fibroblast Growth Factor The biological activity of phosphorylated basic FGF can be determined using the same conditions described for the purified mitogen. 14-~8 It is important, however, to have control preparations of basic FGF that have undergone the phosphorylation reaction without being phosphorylated 14 M. Klagsbrun, R. Sullivan, S. Smith, R. Rybka, and Y. Shing, this series, Vol. 147, p. 95. 15 D. Gospodarowicz, this series, Vol. 147, p. 106. 16 F. Esch, A. Baird, N. Ling, N. Ueno, F. Hill, L. Denoroy, R. Klepper, D. Gospodarowicz, P. B6hlen, and R. Guillemin, Proc. Natl. Acad. Sci. U.S.A. 82, 6507 (1985). 17 j. j. Feige and A. Baird, J. Biol. Chem. 263, 14023 (1988). 18 p. A. Walicke, J.-J. Feige, and A. Baird, J. Biol. Chem. 264, 4120 (1989).
146
FIBROBLAST GROWTH FACTOR
[13]
(i.e., with no enzyme or ATP). We describe here two assays among the many that are possible.
Heparin-Binding Assay The following assay is based on the strong affinity of basic FGF for heparin. 14The growth factor elutes from heparin-Sepharose columns with 1.4 and 1.8 M NaCI. The affinity of phosphorylated forms of basic FGF can be verified by loading the radiolabeled sample onto a 0.5-ml heparinSepharose column and then eluting the column with increasing concentrations of NaC1 (2 ml of 20 mM Tris-HCl, pH 7.5, containing 0, 0.15, 0.6, 1.2, 1.4, 1.6, 1.8, 2.0, and 3.0 of NaCI, respectively). Aliquots of the radiolabeled reaction mixture and of each elution step are then analyzed by S D S - P A G E on 0.1% S D S - 15% polyacrylamide gels. Autoradiography of the gel identifies the salt concentration at which 32p-labeled basic FGF elutes from the heparin-Sepharose column.
Radioreceptor Assay on BHK Cells There exist at least two binding sites for basic FGF on the surface of target cells. One site (Kd 10-10 M) corresponds to cell surface-associated heparan sulfate proteoglycans, and a second, higher affinity site (Kd 10-~1 M) corresponds to the transmembrane receptor. 19 Binding of basic FGF to heparan sulfate-proteoglycans is sensitive to high salt concentrations, whereas binding to the high affinity receptor is not. The radioreceptor assay for basic FGF is based on these observations. It is a competition assay that is routinely performed on baby hamster kidney (BHK) fibroblasts, which have a relatively high number of high-affinity receptors (20,000-40,000 receptor/cell) compared to many other cell types. 18'19
Reagents B H K cells plated in 24-well plates and grown to subconfluence in Ham's F12/DMEM (1 : 1) supplemented with 5% calf serum Binding medium: DMEM with 25 mM HEPES, 0.15% gelatin, pH 7.5 Phosphate-buffered saline (PBS) 20 mM HEPES, pH 7.5, 2 M NaCI Lysis solution: 0.1 M sodium phosphate buffer, pH 8.1, 0.5% Triton X-100 lzSI-Labeled basic FGF [100,000 counts/min (cpm)/ng] radioiodinated by the lactoperoxidase method, 2° purified by heparin-Sepharose chromatography, and stored at 4 ° 19 D. Moscatelli, J. Cell. Physiol. 131, 123 (1987). 2o D. Schubert, N. Ling, and A. Baird, J. Cell. Biol. 104, 635 (1987).
[13]
DETECTION OF bFGF PHOSPHORYLATION
147
Phospho-basic FGF, prepared as described above except that no [y32p]ATP is added to the reaction mixture; it is phosphorylated either by PK-C or by PK-A (for controls, it is important to prepare "unphospho-FGF" under identical conditions, but in the absence of enzyme or ATP) Procedure. B H K cells are rinsed once with 0.5 ml binding buffer and incubated for 2 hr at 4 ° with 0.3 ml binding medium, containing 1-2 × 105 cpm 125I-labeled basic FGF and various concentrations of phosphorylated or unphospho-basic FGF. At the end of the incubation, cells are washed twice with PBS and twice with 2 M NaC1 in 20 mM HEPES, pH 7.5. The salt washes are collected, counted, and used to measure 125I-labeled basic FGF bound to glycosaminoglycans. The 125I-labeled basic FGF that is bound to the high-affinity receptor is then collected by solubilization of the cells in the lysis buffer and counted for radioactivity on a y-counter. We routinely observe that PK-A-phosphorylated basic FGF is more potent than unphosphorylated basic FGF at displacing the binding of 125I-labeled basic FGF to its high-affinity receptor. In contrast, PK-C-phosphorylated basic FGF is equipotent. 8,9 No difference has been observed between these different forms in the binding to cell surface heparan sulfate proteoglycans (salt washes). 8 Summary and Perspectives Phosphorylation of secreted factors by intact cells is a recent observation which has been reported for atrial natriuretic factor 21 and acidic 22 and basic FGF. 8'9 Several growth factors have been reported to be substrates for purified protein kinases. 8'23 Thus, the techniques described in this chapter may be successfully adapted to other growth factors and should lead to a better understanding of the possible role of phosphorylation in the regulation of their biological activity. Acknowledgments We are grateful for the skillful secretarial work of Sonia Lidy and Denise Higgins. This work was supported by National Institutes of Health Grants HD-09690 and DK-18811, The Whittier Institute Angiogenesis Research Program, the Institut National de la Sant6 et de la Recherche M6dicale (INSERM), and the Commissariat ~ l'Energie Atomique (DRF-G).
21 j. Rittenhouse, L. Moberly, H. Ahmed, and F. Marcus, J. Biol. Chem. 263, 3778 (1988). 22 F. Mascarelli, D. Raulais, and Y. Courtois, EMBO J. 8, 420 (1989). 23 H. F. Kung, I. Calvert, E. Bekesi, F. R. Khan, K. P. Huang, S. Oroszlan, L. E. Henderson, T. D. Copeland, R. C. Sowder, and S. J. Wei, Mol. Cell, Biochem. 89, 29 (1989).