Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 846-852
October 31, 1991
FKBP, THE BINDING PROTEIN FOR THE IMMUNOSUPPRESS1VE DRUG, FK-506, IS NOT AN INHIBITOR OF PROTEIN KINASE C ACTIVITY John Cryan, Shirley H.Y. Hung, Gregory Wiederrecht, Nolan H. Sigal and John J. Siekierka* Department of Immunology Research Merck, Sharp and Dohme Research Laboratories P.O. Box 2000, Rahway, New Jersey Received August 20, 1991
Summary: Recently, the amino acid sequence of a 12 Kd endogenous protein inhibitor of protein kinase C (PKC-I 2) has been shown to be identical to that of the 12 KDa receptor for the immunosuppressive drug, FK-506. In view of this observation we examined the effects of recombinant and native human FKBP on protein kinase C (PKC) activity. FKBP, at molar concentrations up to 1900-fold over that of PKC, failed to inhibit PKC phosphorylation of histone H1 and failed to block the auto-phosphorylation of PKC. Interestingly, FKBP is phosphorylated by PKC in these reactions. The phosphorylation of FKBP by PKC appears to be specific since the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate the binding protein. Our results fail to support a role for FKBP as an inhibitor of protein kinase C. ®1991Academic P ..... I n c ,
The immunosuppressive agent, FK-506, is a potent inhibitor of antigen-induced expression of a limited set of early T-cell activation genes required for T-cell proliferation (1). FK-506 binds with high affinity to a 12 kDa cytoplasmic binding protein, FKBP, which is found in many organisms and tissues and is phylogenically highly conserved (2-5). FKBP is a member of a unique class of enzymes which catalyze the c i s - t r a n s isomerization of peptidyl-prolyl bonds in proteins and peptides (PPIases), a reaction believed to accelerate the rate of folding of proteins and peptides into active conformations (6). Cyclophilin, the major binding protein for the immunosuppressive agent, cyclosporin A (CsA), also exhibits PPIase *To whom correspondence should be addressed. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
846
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activity (7,8). FK-506 and CsA are potent inhibitors of their respective receptor's PPIase activity, an observation which has led to the proposal that immunosuppression is a direct consequence of PPIase inhibition (3,4,7,8). Although this mechanism is appealing, a direct relationship b e t w e e n PPIase inhibition and immunosuppression has not been established (9,10). While inhibition of PPIase activity m a y be necessary for immunosuppresson, it is clearly not sufficient, suggesting that other factors are involved in the function of FKBP and cyclophilin. Recently, it has been reported that the amino acid sequence of h u m a n FKBP is virtually identical to that for a 12 Kd inhibitor of protein kinase C (PKC-2 1 ) isolated from bovine brain (11-13). Indeed, a comparison of the sequence for bovine FKBP with bovine brain PKC-2 1 shows both sequences to be identical. From a mechanistic viewpoint, this observation is particularly intriguing because it implicates FKBP in the regulation of of protein kinase C (PKC), an enzyme known to play an important role in Tcell signal transduction (14). In view of the critical role that FKBP m a y play in T-cell activation and immunosuppression, we have investigated the effects of recombinant and native h u m a n FKBP on PKC activity. MATERIAL~ AND M E T H O D S P r o t e i n Kinase C Assays. A commercial preparation of homogeneous bovine brain protein kinase C (Promega corporation, Madison WI) was used in all experiments. Phosphorylation reactions (50 ul) utilizing the substrate histone H1 were performed according to the method of Walton et al. (15) except that phosphotidylserine (Avanti Polar Lipids, Inc. Birmingham, A1) was added at 20 ug/ml and [~32p]-ATP (ICN Biomedicals, Costa Mesa, CA.) had a specific activity of 180-300 dpm/pmol. Protein kinase C was present at 1 ng/ul. Reactions were incubated for 5 min at 30°C and terminated by the addition of 1 ml of ice cold 25% trichloroacetic acid containing 10 mM sodium phosphate (Stop Buffer). Samples were filtered through GFC filters and washed with 50 ml of Stop Buffer. The filters were dried under a heat lamp, 10 ml of Scintiverse (Fisher Scientific Co. Springfield, N.J.) added and counted in a liquid scintillation counter. The auto-phosphorylation of PKC and phosphorylation of recombinant FKBP were assayed under the conditions described above except that assays (25 ul) contained 2 ng/ul protein kinase C, [~32p] ATP at 9,225 dpm/pmol and, where indicated, 80 ng/ul recombinant h u m a n FKBP in the presence and absence of 800 nM FK-506. In experiments using native FKPB [100 ng/ul (3)], PKC and the catalytic subunit of cAMP-dependent protein kinase [(CAT), Sigma Chemical Co. St. Louis, MO.] were present at 3 ng/ul and 10 ng/ul, respectively. [732p] ATP was present in these experiments at 22,000 dpm/pmol. Reactions were incubated for 15 min at 30oc and stopped by the addition of 25 ul of buffer containing: 125 mM TrisHC1, pH 6.9, 4% SDS, 0.025% bromophenol blue (w/v), 10% [~-mercaptoethanol and 2% glycerol (v/v). Samples were heated at 100oc for 3 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 16 % acrylamide gel using a Bio-Rad Mini Protean II 847
V o l . 180, N o . 2, 1991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
electrophoresis apparatus (Bio-Rad Laboratories, Richmond, CA). Gels were stained with Coomassie Brillant Blue, dried and subjected to autoradiography using Kodak X-omat film. R e c o m b | n n n t HumAn FKBP. The gene encoding the h u m a n FK506 binding protein was isolated from a JURKAT T-cell cDNA library (Clontech Laboratories, Inc. Palo Alto, CA.) in Xgtl0 using synthetic, degenerate, inosine-containing oligonucleotides and using hybridization conditions previously described (16). In order to express the protein in E. coli, the open reading frame of the gene was subcloned into pET3D (17) where expression can be induced with IPTG. Pttrification o f H u m a n F K B P a n d Associated Isoforms. FKBP was purified from the h u m a n T-cell line JURKAT as previously described (3). Two minor FKBP isoforms are resolved from the major cytosolic form of FKBP during the final purification step using weak cation-exchange HPLC (Siekierka et al. manuscript in preparation). RESULT~ AND DISCUSSION We first examined the effects of FKBP on the intramolecular autophosphorylation of PKC and phosphorylation of the substrate histone H1. Native and recombinant FKBP, in quantities representing up to a 1900-fold molar excess over PKC, failed to block PKC auto-phosphorylation (Fig 1A, lanes 1 & 3 and Fig 1B, lanes 1 & 2) or phosphorylation ofhistone H1 (Table I). Preincubation of PKC with FKBP and ATP prior to addition of substrate did not result in PKC inhibition (data not shown). Addition of FKBP complexed with FK-506 (formed by adding FK-506 to reaction mixtures prior to initiating the phosphorylation assays) to these assays was also without
~
A
B
i~i%~ii~
PKC ,,'CAT ,,--
1
2
~ ' ~ ' ~'~i~'~ ~'~'~"~' ~
3
4
~ PKC
FKBP 5
FKBP
1
2
3
4
5
6
Fi~ 1. Phosphorylation of native and recombinant FKBP by bovine brain PKC. Phosphorylation reactions, SDS-polyacrylamide gel electrophoresis and autoradiography, were conducted as described in Experimental Procedures. (A) FKBP purified from human JURKAT cells. Lane 1, FKBP + PKC; Lane 2, FKBP and PKC, without Ca++ and phosphytidyl serine; Lane 3, PKC alone; Lane 4, FKBP + cat; Lane 5, cat alone; Lane 6, FKBP alone. (B) Recombinant human FKBP. Lane 1, PKC alone; Lane 2, PKC + FKBP; Lane 3, FKBP alone; Lane 4, PKC, FKBP + FK-506; Lane 5, PKC + FK-506. 848
Vol. 180, No. 2, 1991
TABLE I.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effects of RecombinAnt FKBP on Histone H1 Phosphorylation by Protein l(inAge C
Additions
1. 2. 3. 4. 5.
[~2p] Incorporated into Histone H1 (pmol)
Complete* - Ca ++, - phosphytidyl serine RcFKBP (6.9 pg) RcFKBP + FK-506 (800 nM) - PKC + RcFKBP
138.0 37.1 152.5 186.0 6.3
*PKC specific activity 1,104 units/rag (15).
effect (Fig 1B lanes 4 & 5 and Table I). These results also demonstrate t h a t FK-506 does not inhibit PKC activity suggesting t h a t the immunosuppressive effects of FK-506 are not a consequence of PKC inhibition. These results are consistent with the observations of others using CsA (18,19). Interestingly, under the conditions reported here, FKBP was phosphorylated by PKC (Fig 1A, lane 1 and Fig 1B, lane 2) but not the catalytic subunit of cAMP-dependent protein kinase (Fig 1A, lane 5). Scrutiny of the h u m a n FKBP amino acid sequence reveals a single PKC phosphorylation site ,35KKFDS*SRD 42 (16,20). These observations contrast with a recent report in which FKBP phosphorylation was not observed in stimulated and unstimulated JURKAT cells labeled with [ 32p] orthophosphate (21). While the reason for this discrepancy is not immediately apparent, it could be due to the action of protein phosphatases in vivo. The phosphorylation of FKBP by PKC may have functional significance in view of the observation t h a t PKC activation is important in T-cell activation (14). Additional experiments are underway to assess the functional relevance of FKBP phosphorylation. Cyclophilin was also examined for PKC inhibitory activity under similar conditions. Since FK-506 and CsA affect similar, if not identical, pathways in the T-cell [for a review see, (22) ], one would expect the receptors for both these immunosuppressants to possess similar activities. Cyclophilin, like FKBP, did not exhibit PKC inhibitory activity (data not shown). The possibility t h a t a modified form of FKBP functions as a PKC inhibitor was also investigated. We have separated two minor FK-506 849
V o l . 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
20000 FKBPA
15000
FKBPB
E
:KBP~!ajor
0
m
O
10000
I 5000
1 Ill
0 0
J
lllllllillll
10 20 30 40 50 60 70 80 90 100 Fraction Number
Fig 2. Separation of FKBP isoforms, FKBPA & FKBPB, from the major cytosolic form ofFKBP. FKBPA & FKBPB copurify with the major cytosolic form of FKBP and are separated during the final purification step by weak cation exchange HPLC (3). FKBPA & FKBPB elute significantly earlier than the major form of FKBP. The results shown were obtained by chromatography of 1 mg of step IV FKBP (3) using a SynChropak CM 300 column (4.6 x 250 mm, Rainin Instruments) under conditions previously described. Peaks of [3HI FK-506 binding were pooled and concentrated. Protein was determined by the Bradford assay (Bio-Rad) as in previous work (3).
binding activities (FKBP A & B) from the major cytosolic FKBP by weak cation exchange HPLC [(3,) Fig 2]. The two heat-stable, low molecular weight proteins, FKBPA & FKBPB, bind FK-506 with high affinity (Kd ~ 10-9M) and appear to be FKBP isoforms as determined by immunological cross-reactivity with anti-FKBP antibodies and N-terminal amino acid sequence data (Siekierka et al. manuscript in preparation). A minor cyclophilin isoform was previously separated from the major form using weak cation exchange HPLC (23). Partially purified F K B P A , FKBPB and FKBP major were assayed for their ability to inhibit PKC phosphorylation of histone H1, using protein concentrations similar to those described for the heat stable inhibitor of PKC, PKC I-2 (24). No inhibition of PKC activity was observed with these preparations (Table II). Taken together, our results do not confirm a PKC inhibitory role for FKBP, making it unlikely that the mechanism of action of FK-506 involves modulation of PKC activity. Although lack of effect of recombinant FKBP on PKC activity has been noted by others (25), the possibility that a minor FKBP 850
Vol. 180, No. 2, 1991
T A B L E II.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effects o f F K B P Isoforms, F K B P A & F K B P B o n H i s t o n e H1 P h o s p h o r y l a t i o n b y P r o t e i n I(inAse C
[T32p] Incorporated into Histone H1 (pmol)
Additions
2.
Complete* -Ca ++, -phosphytidyl serine
36.6 4.8
3. 4.
F ,KB,PA, 78 ug/ml , 156
42.8 35.0
5. 6.
FKBPB, 74.5 ug/ml " , 149.0
66.8 51.1
7. 8.
FKBP Major, 68,6 ug/ml " , 137.2
40.9 49.6
1.
*PKC specific activity; 600 units/mg (15). isoform present in the naturally derived material was responsible for the inhibitory effect has been specifically addressed in this report. In addition, the novel observation that FKBP is a substrate for PKC is also inconsistent with FKBP being an endogenous protein inhibitor of PKC. The PKC inhibitory activity in the preparation of Mozier et al. (13) m a y be due to an isoform of FKBP not isolated by the methods used here or due to another polypeptide unrelated to FKBP. REFERENCES .
Tocci, M.J., Matkovich, D.A., Collier, K.A., Kwok, P., Dumont, F., Lin, S., Degudicibus, S., Siekierka, J.J., Chin, J., and Hutchinson, N. (1989) J. Immunol. 143, 718-726.
.
Siekierka, J.J., Staruch, M.J., Hung, S.H.Y., and Sigal, N.H. (1989) J. Immunol. 143, 1580-1583.
.
Siekierka, J.J., Hung, S.H.Y., Poe, M. Lin, C.S., and Sigal, N.H. (1989) Nature 341,755-757.
.
Harding, M.W., Galat, A., Uehling, D.E., and Schreiber, S.L. (1989) Nature 341, 758-760.
.
Siekierka, J.J., Wiederrecht, G., Greulich, H., Boulton, D., Hung, S.H.Y., Cryan, J., Hodges, P.J., and Sigal, N.H. (1990) J. Biol. Chem. 2 6 5 , 21011-21015.
.
7.
Fischer, G., and Schmid. (1990) Biochemistry 29, 2205-2212. Takahashi, N., Hagano, T., and Suzuki, M. (1989) Nature 337, 473-475. 851
Vol. 180, No. 2, 1991
.
.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fischer, G., Wittme~n-Liebold, B., Lang, K., Kiethaber, T., and Schmidt, F.X. (1989) Nature 337, 476-478. Bierer, B.E., Mattila, P.S., Standaert, R.F. Herzenberg, L.A., Burakoff, S.J., Crabtree, G., and Schreiber, S.L. (1990) Proc. Natl. Acad. Sci. USA. 87, 9231-9235.
10.
Sigal, N.H., Dumont, F. Durette, P., Siekierka, J.J., Peterson, L. Rich, D.H., Dunlap, B.E., Staruch, M.J., Melino, M.R., Koprak, S.L., Williams, D., Witzel, B., and Pisano, J.M. (1991) J. Exp. Med. 173, 619-628.
11.
Goebl, M.G. (1991) Cell 64, 1051-1052.
12.
Tropschug, M., and Hofmann, R. (1991) Nature 351, 195.
13.
Mozier, N.M., Zurcher-Neely, H.A., Guido, D.M., Mathews, W.R, Heinrikson, R.L., Fraser, E.D., Walsh, M.P., and Pearson, J.D. (1990) Eur. J. Biochem. 194, 19-23.
14.
Berry, N., and Nishizuka, Y. (1990) Eur. J. Biochem. 189, 205-214.
15.
Walton, G.M., Bertics, P.J., Hudson, L.G., Vedvick, T.S., and Gordon N. Gill. (1987)Anal. Biochem. 161,425-437.
16.
Wiederrecht, G., Brizuela, L., Elliston, K., Sigal, N.H., and Siekierka, J.J. (1991) Proc. Natl. Acad. Sci. USA. 88, 1029-1033.
17.
Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89.
18.
Bijsterbosch, M.K., and Klaus, G.G.B. (1985) Immunology 56, 435-440.
19.
Fidelus, R.K., and Laughter, A.H. (1986) Transplantation 41, 187-192.
20.
Kemp, B.E. and Pearson, R.B. (1990) Trends in Biol. Sci, 15, 342-346.
21.
Fretz, H., Albers, M.W., Galat, A., Standaert, R.F., Lane, W.F., Burakoff, S.J., Bierer, B.E., and Schreiber, S.L. (1991) J. Am. Chem. Soc. 113, 1409-1411.
22.
Sigal, N.H., Siekierka, J.J., and Dumont, F.J. (1990) Biochem. Pharm. 40, 2201-2208.
23.
Harding, M.W., Handchumacher, R.E., and Speicher, D.W. (1986) J. Biol. Chem. 261, 8547-8555.
24.
McDonald, J.R., and Walsh, M.P. (1986) Biochem. Soc. Trans. 14, 585-586.
25.
Albers, M.W., Liu, J., and Schreiber, S.L. (1991) Nature 351,527.
852
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 846-852
October 31, 1991
FKBP, THE BINDING PROTEIN FOR THE IMMUNOSUPPRESS1VE DRUG, FK-506, IS NOT AN INHIBITOR OF PROTEIN KINASE C ACTIVITY John Cryan, Shirley H.Y. Hung, Gregory Wiederrecht, Nolan H. Sigal and John J. Siekierka* Department of Immunology Research Merck, Sharp and Dohme Research Laboratories P.O. Box 2000, Rahway, New Jersey Received August 20, 1991
Summary: Recently, the amino acid sequence of a 12 Kd endogenous protein inhibitor of protein kinase C (PKC-I 2) has been shown to be identical to that of the 12 KDa receptor for the immunosuppressive drug, FK-506. In view of this observation we examined the effects of recombinant and native human FKBP on protein kinase C (PKC) activity. FKBP, at molar concentrations up to 1900-fold over that of PKC, failed to inhibit PKC phosphorylation of histone H1 and failed to block the auto-phosphorylation of PKC. Interestingly, FKBP is phosphorylated by PKC in these reactions. The phosphorylation of FKBP by PKC appears to be specific since the catalytic subunit of cAMP-dependent protein kinase fails to phosphorylate the binding protein. Our results fail to support a role for FKBP as an inhibitor of protein kinase C. ®1991Academic P ..... I n c ,
The immunosuppressive agent, FK-506, is a potent inhibitor of antigen-induced expression of a limited set of early T-cell activation genes required for T-cell proliferation (1). FK-506 binds with high affinity to a 12 kDa cytoplasmic binding protein, FKBP, which is found in many organisms and tissues and is phylogenically highly conserved (2-5). FKBP is a member of a unique class of enzymes which catalyze the c i s - t r a n s isomerization of peptidyl-prolyl bonds in proteins and peptides (PPIases), a reaction believed to accelerate the rate of folding of proteins and peptides into active conformations (6). Cyclophilin, the major binding protein for the immunosuppressive agent, cyclosporin A (CsA), also exhibits PPIase *To whom correspondence should be addressed. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
846
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activity (7,8). FK-506 and CsA are potent inhibitors of their respective receptor's PPIase activity, an observation which has led to the proposal that immunosuppression is a direct consequence of PPIase inhibition (3,4,7,8). Although this mechanism is appealing, a direct relationship b e t w e e n PPIase inhibition and immunosuppression has not been established (9,10). While inhibition of PPIase activity m a y be necessary for immunosuppresson, it is clearly not sufficient, suggesting that other factors are involved in the function of FKBP and cyclophilin. Recently, it has been reported that the amino acid sequence of h u m a n FKBP is virtually identical to that for a 12 Kd inhibitor of protein kinase C (PKC-2 1 ) isolated from bovine brain (11-13). Indeed, a comparison of the sequence for bovine FKBP with bovine brain PKC-2 1 shows both sequences to be identical. From a mechanistic viewpoint, this observation is particularly intriguing because it implicates FKBP in the regulation of of protein kinase C (PKC), an enzyme known to play an important role in Tcell signal transduction (14). In view of the critical role that FKBP m a y play in T-cell activation and immunosuppression, we have investigated the effects of recombinant and native h u m a n FKBP on PKC activity. MATERIAL~ AND M E T H O D S P r o t e i n Kinase C Assays. A commercial preparation of homogeneous bovine brain protein kinase C (Promega corporation, Madison WI) was used in all experiments. Phosphorylation reactions (50 ul) utilizing the substrate histone H1 were performed according to the method of Walton et al. (15) except that phosphotidylserine (Avanti Polar Lipids, Inc. Birmingham, A1) was added at 20 ug/ml and [~32p]-ATP (ICN Biomedicals, Costa Mesa, CA.) had a specific activity of 180-300 dpm/pmol. Protein kinase C was present at 1 ng/ul. Reactions were incubated for 5 min at 30°C and terminated by the addition of 1 ml of ice cold 25% trichloroacetic acid containing 10 mM sodium phosphate (Stop Buffer). Samples were filtered through GFC filters and washed with 50 ml of Stop Buffer. The filters were dried under a heat lamp, 10 ml of Scintiverse (Fisher Scientific Co. Springfield, N.J.) added and counted in a liquid scintillation counter. The auto-phosphorylation of PKC and phosphorylation of recombinant FKBP were assayed under the conditions described above except that assays (25 ul) contained 2 ng/ul protein kinase C, [~32p] ATP at 9,225 dpm/pmol and, where indicated, 80 ng/ul recombinant h u m a n FKBP in the presence and absence of 800 nM FK-506. In experiments using native FKPB [100 ng/ul (3)], PKC and the catalytic subunit of cAMP-dependent protein kinase [(CAT), Sigma Chemical Co. St. Louis, MO.] were present at 3 ng/ul and 10 ng/ul, respectively. [732p] ATP was present in these experiments at 22,000 dpm/pmol. Reactions were incubated for 15 min at 30oc and stopped by the addition of 25 ul of buffer containing: 125 mM TrisHC1, pH 6.9, 4% SDS, 0.025% bromophenol blue (w/v), 10% [~-mercaptoethanol and 2% glycerol (v/v). Samples were heated at 100oc for 3 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 16 % acrylamide gel using a Bio-Rad Mini Protean II 847
V o l . 180, N o . 2, 1991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
electrophoresis apparatus (Bio-Rad Laboratories, Richmond, CA). Gels were stained with Coomassie Brillant Blue, dried and subjected to autoradiography using Kodak X-omat film. R e c o m b | n n n t HumAn FKBP. The gene encoding the h u m a n FK506 binding protein was isolated from a JURKAT T-cell cDNA library (Clontech Laboratories, Inc. Palo Alto, CA.) in Xgtl0 using synthetic, degenerate, inosine-containing oligonucleotides and using hybridization conditions previously described (16). In order to express the protein in E. coli, the open reading frame of the gene was subcloned into pET3D (17) where expression can be induced with IPTG. Pttrification o f H u m a n F K B P a n d Associated Isoforms. FKBP was purified from the h u m a n T-cell line JURKAT as previously described (3). Two minor FKBP isoforms are resolved from the major cytosolic form of FKBP during the final purification step using weak cation-exchange HPLC (Siekierka et al. manuscript in preparation). RESULT~ AND DISCUSSION We first examined the effects of FKBP on the intramolecular autophosphorylation of PKC and phosphorylation of the substrate histone H1. Native and recombinant FKBP, in quantities representing up to a 1900-fold molar excess over PKC, failed to block PKC auto-phosphorylation (Fig 1A, lanes 1 & 3 and Fig 1B, lanes 1 & 2) or phosphorylation ofhistone H1 (Table I). Preincubation of PKC with FKBP and ATP prior to addition of substrate did not result in PKC inhibition (data not shown). Addition of FKBP complexed with FK-506 (formed by adding FK-506 to reaction mixtures prior to initiating the phosphorylation assays) to these assays was also without
~
A
B
i~i%~ii~
PKC ,,'CAT ,,--
1
2
~ ' ~ ' ~'~i~'~ ~'~'~"~' ~
3
4
~ PKC
FKBP 5
FKBP
1
2
3
4
5
6
Fi~ 1. Phosphorylation of native and recombinant FKBP by bovine brain PKC. Phosphorylation reactions, SDS-polyacrylamide gel electrophoresis and autoradiography, were conducted as described in Experimental Procedures. (A) FKBP purified from human JURKAT cells. Lane 1, FKBP + PKC; Lane 2, FKBP and PKC, without Ca++ and phosphytidyl serine; Lane 3, PKC alone; Lane 4, FKBP + cat; Lane 5, cat alone; Lane 6, FKBP alone. (B) Recombinant human FKBP. Lane 1, PKC alone; Lane 2, PKC + FKBP; Lane 3, FKBP alone; Lane 4, PKC, FKBP + FK-506; Lane 5, PKC + FK-506. 848
Vol. 180, No. 2, 1991
TABLE I.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effects of RecombinAnt FKBP on Histone H1 Phosphorylation by Protein l(inAge C
Additions
1. 2. 3. 4. 5.
[~2p] Incorporated into Histone H1 (pmol)
Complete* - Ca ++, - phosphytidyl serine RcFKBP (6.9 pg) RcFKBP + FK-506 (800 nM) - PKC + RcFKBP
138.0 37.1 152.5 186.0 6.3
*PKC specific activity 1,104 units/rag (15).
effect (Fig 1B lanes 4 & 5 and Table I). These results also demonstrate t h a t FK-506 does not inhibit PKC activity suggesting t h a t the immunosuppressive effects of FK-506 are not a consequence of PKC inhibition. These results are consistent with the observations of others using CsA (18,19). Interestingly, under the conditions reported here, FKBP was phosphorylated by PKC (Fig 1A, lane 1 and Fig 1B, lane 2) but not the catalytic subunit of cAMP-dependent protein kinase (Fig 1A, lane 5). Scrutiny of the h u m a n FKBP amino acid sequence reveals a single PKC phosphorylation site ,35KKFDS*SRD 42 (16,20). These observations contrast with a recent report in which FKBP phosphorylation was not observed in stimulated and unstimulated JURKAT cells labeled with [ 32p] orthophosphate (21). While the reason for this discrepancy is not immediately apparent, it could be due to the action of protein phosphatases in vivo. The phosphorylation of FKBP by PKC may have functional significance in view of the observation t h a t PKC activation is important in T-cell activation (14). Additional experiments are underway to assess the functional relevance of FKBP phosphorylation. Cyclophilin was also examined for PKC inhibitory activity under similar conditions. Since FK-506 and CsA affect similar, if not identical, pathways in the T-cell [for a review see, (22) ], one would expect the receptors for both these immunosuppressants to possess similar activities. Cyclophilin, like FKBP, did not exhibit PKC inhibitory activity (data not shown). The possibility t h a t a modified form of FKBP functions as a PKC inhibitor was also investigated. We have separated two minor FK-506 849
V o l . 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
20000 FKBPA
15000
FKBPB
E
:KBP~!ajor
0
m
O
10000
I 5000
1 Ill
0 0
J
lllllllillll
10 20 30 40 50 60 70 80 90 100 Fraction Number
Fig 2. Separation of FKBP isoforms, FKBPA & FKBPB, from the major cytosolic form ofFKBP. FKBPA & FKBPB copurify with the major cytosolic form of FKBP and are separated during the final purification step by weak cation exchange HPLC (3). FKBPA & FKBPB elute significantly earlier than the major form of FKBP. The results shown were obtained by chromatography of 1 mg of step IV FKBP (3) using a SynChropak CM 300 column (4.6 x 250 mm, Rainin Instruments) under conditions previously described. Peaks of [3HI FK-506 binding were pooled and concentrated. Protein was determined by the Bradford assay (Bio-Rad) as in previous work (3).
binding activities (FKBP A & B) from the major cytosolic FKBP by weak cation exchange HPLC [(3,) Fig 2]. The two heat-stable, low molecular weight proteins, FKBPA & FKBPB, bind FK-506 with high affinity (Kd ~ 10-9M) and appear to be FKBP isoforms as determined by immunological cross-reactivity with anti-FKBP antibodies and N-terminal amino acid sequence data (Siekierka et al. manuscript in preparation). A minor cyclophilin isoform was previously separated from the major form using weak cation exchange HPLC (23). Partially purified F K B P A , FKBPB and FKBP major were assayed for their ability to inhibit PKC phosphorylation of histone H1, using protein concentrations similar to those described for the heat stable inhibitor of PKC, PKC I-2 (24). No inhibition of PKC activity was observed with these preparations (Table II). Taken together, our results do not confirm a PKC inhibitory role for FKBP, making it unlikely that the mechanism of action of FK-506 involves modulation of PKC activity. Although lack of effect of recombinant FKBP on PKC activity has been noted by others (25), the possibility that a minor FKBP 850
Vol. 180, No. 2, 1991
T A B L E II.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effects o f F K B P Isoforms, F K B P A & F K B P B o n H i s t o n e H1 P h o s p h o r y l a t i o n b y P r o t e i n I(inAse C
[T32p] Incorporated into Histone H1 (pmol)
Additions
2.
Complete* -Ca ++, -phosphytidyl serine
36.6 4.8
3. 4.
F ,KB,PA, 78 ug/ml , 156
42.8 35.0
5. 6.
FKBPB, 74.5 ug/ml " , 149.0
66.8 51.1
7. 8.
FKBP Major, 68,6 ug/ml " , 137.2
40.9 49.6
1.
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