Volume 314, number 1, 37--40 FEBS 11823 © 1992 Federation of European Biochemical Societies 00145793/9~$5.00
December 1992
Inhibition of calcineurin by cyclosporin A-cyclophilin requires calcineurin B A l i c e H a d d y , S e l e n e K . - H . S w a n s o n , T i m o t h y L. B o r n a n d F r a n k R u s n a k Section of Hematology Research, Mayo Foundation and Clinic, Rochester, M N 55905, USA Received 29 September 1992 The interaction of the imlnunosuppr¢~sivecomplex cyclosporin A-cyclophilin (CsA.CyP) with the Ca-'*/ealmodulin-dependentprotein pho~phatas~ calcineurin is investigated using a recombinat~t form of the A subunit of calelneurin (rCNA). Only in the presence of purified caleineurin B (CNB) does rCNA show the response of native calcineurin, i.e. 50% inhibition of rCNA phosphata~e activity at 6 nM human cyelophilin B and 0.6/.tM human cyclophilin A using ['~:P]caseinas substrate, yet stimulation of activity with p.nitrophenyl phosphate as snbstrate. This study demonstrates that the B subunit is necessary to confer sensitivity of c.alcincurin to CsA-CyP, Calcineuriu; Cyclosporin A; Cyclophilin; Immunosuppression
1. I N T R O D U C T I O N T h e i m m u n o s u p p r e s s a n t s eyclosporin A (CsA) a n d F K 5 0 6 inhibit T-cell activation via a c o m m o n p a t h w a y b y preventing the transcriptional activation o f the interleukin-2 gene [1-5], These drugs bind to distinct intracellular receptors, CsA to cyclophilin (CyP) [6,7] a n d F K 5 0 6 to F K 5 0 6 - b i n d i n g protein [8,9]. Recently, the Caa+/ealmodulin ( C a M ) - d e p e n d e n t protein p h o s p h a t use calcineurin (CN) was implicated as the c o m m o n p r o x i m a l target o f these d r u g receptor c o m p l e x e s [10], Several studies have since s u p p o r t e d this b y dem o n s t r a t i n g that b o t h C s A - C y p a n d F K S 0 6 - F K B P c o m p l e x e s inhibit calcineurin in vitro [10-13], a n d that overexpression o f calcineurin in the T-cell line J u r k a t resulted in an increased resistance to these drugs [14,15]. Calcineurin is a h e t e r o d i m e r c o m p r i s e d o f a 59 k D a catalytic A subunit ( C N A ) and a 19 k D a Ca""-binding B subanit (CNB), It is distinguished f r o m o t h e r serine/ t h r e o n i n e protein phosphatases by the presence o f the B subunit, which is required for maximal activity [16,17]0 and additional c a r b o x y - t e r m i n a l d o m a i n s within the A subunit t h a t c o n f e r C a M d e p e n d e n c e [18,19]. F r o m previous work, neither C a M n o r the
Correspondence address: F. Rusnak, Section of Hematology Research, Mayo Foundation and Clinic, Rochester, MN 55905, USA. Fax: (1) (507) 284 8286. Abbreviations: CaM, eaimodulin; CN, calcineurin; CNA, ¢alein~urin A; CNB, caleineurin B; CsA, cyelosporin A; CyP, cyclophilin; hCyPA, rceombhTalltt ~mman ' ~.~,.lop,,,h, ~ .~ L:,._ A; FKBP, FK59fi binding pro:e:n; hCyPB, recombinant hmnan cyclophilin B; PP, protein phosphatase; rCNA, recombinant ealeineurin A.
Publisl;ed by £lsevier Science Publishers B. V,
C a M - b i n d i n g and a u t o i n h i b i t o r y d o m a i n s are necessary for inhibition by C s A - C y P [l 1,12]. In this r e p o r t the role o f C N B in the interaction o f C N with C s A - C y P is investigated t h r o u g h the use o f a r e c o m b i n a n t f o r m o f C N A . It is f o u n d that the B subunit is required to c o n f e r sensitivity to C s A - C y P complexes. 2. E X P E R I M E N T A L 2.1. En'-yme preparaffon Native CN was prepared from bovine brain as described previously [20]. The eDNA gene for the tz isoform o1" rat CNA [21,22] was obtained as a gift from Drs. B. Perrino and T. Soderling. Recombinant CNA (rCNA) was overexpressed in g. coil using a T7 sy~rem and purified by a method similar to that for native bovine brain CN [20] (Haddy, A,, Swanson, S.K.-H. and Rusnak, F., in preparation). CNB was purified from native bovine brain CN by gel filtration chromatography in the presence of SDS [17] and was essentially devoid of phosphatase activity (
December 1992
Inhibition of calcineurin by cyclosporin A-cyclophilin requires calcineurin B A l i c e H a d d y , S e l e n e K . - H . S w a n s o n , T i m o t h y L. B o r n a n d F r a n k R u s n a k Section of Hematology Research, Mayo Foundation and Clinic, Rochester, M N 55905, USA Received 29 September 1992 The interaction of the imlnunosuppr¢~sivecomplex cyclosporin A-cyclophilin (CsA.CyP) with the Ca-'*/ealmodulin-dependentprotein pho~phatas~ calcineurin is investigated using a recombinat~t form of the A subunit of calelneurin (rCNA). Only in the presence of purified caleineurin B (CNB) does rCNA show the response of native calcineurin, i.e. 50% inhibition of rCNA phosphata~e activity at 6 nM human cyelophilin B and 0.6/.tM human cyclophilin A using ['~:P]caseinas substrate, yet stimulation of activity with p.nitrophenyl phosphate as snbstrate. This study demonstrates that the B subunit is necessary to confer sensitivity of c.alcincurin to CsA-CyP, Calcineuriu; Cyclosporin A; Cyclophilin; Immunosuppression
1. I N T R O D U C T I O N T h e i m m u n o s u p p r e s s a n t s eyclosporin A (CsA) a n d F K 5 0 6 inhibit T-cell activation via a c o m m o n p a t h w a y b y preventing the transcriptional activation o f the interleukin-2 gene [1-5], These drugs bind to distinct intracellular receptors, CsA to cyclophilin (CyP) [6,7] a n d F K 5 0 6 to F K 5 0 6 - b i n d i n g protein [8,9]. Recently, the Caa+/ealmodulin ( C a M ) - d e p e n d e n t protein p h o s p h a t use calcineurin (CN) was implicated as the c o m m o n p r o x i m a l target o f these d r u g receptor c o m p l e x e s [10], Several studies have since s u p p o r t e d this b y dem o n s t r a t i n g that b o t h C s A - C y p a n d F K S 0 6 - F K B P c o m p l e x e s inhibit calcineurin in vitro [10-13], a n d that overexpression o f calcineurin in the T-cell line J u r k a t resulted in an increased resistance to these drugs [14,15]. Calcineurin is a h e t e r o d i m e r c o m p r i s e d o f a 59 k D a catalytic A subunit ( C N A ) and a 19 k D a Ca""-binding B subanit (CNB), It is distinguished f r o m o t h e r serine/ t h r e o n i n e protein phosphatases by the presence o f the B subunit, which is required for maximal activity [16,17]0 and additional c a r b o x y - t e r m i n a l d o m a i n s within the A subunit t h a t c o n f e r C a M d e p e n d e n c e [18,19]. F r o m previous work, neither C a M n o r the
Correspondence address: F. Rusnak, Section of Hematology Research, Mayo Foundation and Clinic, Rochester, MN 55905, USA. Fax: (1) (507) 284 8286. Abbreviations: CaM, eaimodulin; CN, calcineurin; CNA, ¢alein~urin A; CNB, caleineurin B; CsA, cyelosporin A; CyP, cyclophilin; hCyPA, rceombhTalltt ~mman ' ~.~,.lop,,,h, ~ .~ L:,._ A; FKBP, FK59fi binding pro:e:n; hCyPB, recombinant hmnan cyclophilin B; PP, protein phosphatase; rCNA, recombinant ealeineurin A.
Publisl;ed by £lsevier Science Publishers B. V,
C a M - b i n d i n g and a u t o i n h i b i t o r y d o m a i n s are necessary for inhibition by C s A - C y P [l 1,12]. In this r e p o r t the role o f C N B in the interaction o f C N with C s A - C y P is investigated t h r o u g h the use o f a r e c o m b i n a n t f o r m o f C N A . It is f o u n d that the B subunit is required to c o n f e r sensitivity to C s A - C y P complexes. 2. E X P E R I M E N T A L 2.1. En'-yme preparaffon Native CN was prepared from bovine brain as described previously [20]. The eDNA gene for the tz isoform o1" rat CNA [21,22] was obtained as a gift from Drs. B. Perrino and T. Soderling. Recombinant CNA (rCNA) was overexpressed in g. coil using a T7 sy~rem and purified by a method similar to that for native bovine brain CN [20] (Haddy, A,, Swanson, S.K.-H. and Rusnak, F., in preparation). CNB was purified from native bovine brain CN by gel filtration chromatography in the presence of SDS [17] and was essentially devoid of phosphatase activity (
