The Sarcolemmal Calcium Pump Inhibits the Calcineurin/Nuclear Factor of Activated T-cell Pathway via Interaction with the Calcineurin A Catalytic Subunit

Abstract

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active calcineurin A a calcium/calmodulin-dependent The of PMCA4b is by the calcineurin the to the plasma membrane PMCA4b is We by PMCA4b of calcineurin to a calcium as a evidence a novel functional interaction between PMCA and calcineurin, a PMCA as a modulator of in mammalian cells. This the of PMCA as a and of transduction pathways. The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active calcineurin A a calcium/calmodulin-dependent The of PMCA4b is by the calcineurin the to the plasma membrane PMCA4b is We by PMCA4b of calcineurin to a calcium as a evidence a novel functional interaction between PMCA and calcineurin, a PMCA as a modulator of in mammalian cells. This the of PMCA as a and of transduction pathways. The of in signal transduction is to the of to cellular is the calcineurin calcium/calmodulin-dependent plasma membrane NFAT, factor of activated plasma membrane calcium calcium/calmodulin-dependent plasma membrane NFAT, factor of activated plasma membrane calcium is by is to and in is factor of activated T-cell (NFAT) the best characterized of calcineurin The effect of calcineurin is the of this of in to a in to of and of The of evidence in the and of a of the of calcineurin The calcineurin is a of a to catalytic calcineurin A and a calcineurin a catalytic domain the and domains the the domain the domain and the domain the of calcineurin is as the domain in the active to the domain of the domain the inhibition a the plasma membrane ATPase pump as a modulator of in the of the PMCA in and by of of the and and a and of The of of and and PMCA4b to the activity of the and calcium/calmodulin-dependent or by to cellular interaction between the of PMCA4b and domains located in the in the interaction of of PMCA a domain and the calcium/calmodulin-dependent of calcineurin, the PMCA the activity of calcineurin interaction a the this a of to its activity interaction the calcineurin A catalytic PMCA as a novel inhibitory of calcineurin in mammalian cells. We the interaction between PMCA and calcineurin is to calcineurin This the of the pathway and a PMCA as of this pathway in the and of were in and the PMCA4b and was a of and of calcineurin A was by of and the as and The was and and the of to calcineurin A was by of and the by in the the was to the and The was and the a constitutively active was to the a and the of calcineurin A was of calcineurin was by of and the and The and were by of and the and and The were and and the and of to and of the PMCA4b of the domain between and and to were by of and the and and and and The were and and the and of to and of the PMCA4b and of the between and were by of and the and and and The were and and the and of to and The of was by were in the were of the as were in the were the and of the was to the to the were and in of the were and the calcium to The were and activity was a was by of the the of the and of activity A or were and the as were by were in and the were of the as membrane were a and interaction were were and in of were to and plasma were The were and in of The were and to and cells. plasma the the plasma was of The plasma were the by to the The the were of were by were and by or PMCA4b or calcineurin A The were and as a of in PMCA4b a of in a of in or a of A in were and was a the of in were the and calcineurin A or a constitutively active the domain and the were the and by to demonstrate of PMCA calcineurin A or the constitutively active The and of the were by the of in the and demonstrate a interaction between and calcineurin A in mammalian of A the interaction domain of calcineurin A the interaction a of of calcineurin A were and by to PMCA in mammalian PMCA4b to the of calcineurin of of calcineurin calcineurin PMCA4b and of calcineurin A or were in cells. and the were as were the and the of the to were the The the in the interaction of and its the a in the interaction. The the of interaction of in the of PMCA4b to the of calcineurin were and or were the and by to demonstrate of The of were significantly were by the of the of to in PMCA demonstrating the of the interaction. or the were as of the to were by to were to the of calcineurin A is interaction to to the of PMCA4b the of PMCA4b in the interaction calcineurin the of the catalytic domain, or the of PMCA4b of PMCA4b of the of the of its catalytic domain, and the of the The to PMCA4b to of the interaction between calcineurin and the or of PMCA4b. or were in cells. was as a were calcineurin and were by of the to were by The located the catalytic of PMCA4b of of the to the of the or of PMCA4b were in cells. were as in were the to and or of PMCA4b however, to calcineurin, the interaction domain to the of were or calcineurin was to the were a A and by calcineurin was the or domains of PMCA4b were in the were as a The PMCA4b were the to of interaction the domain the of PMCA4b calcineurin, a of this was and The of the as by the of calcineurin demonstrate the of the catalytic of PMCA4b is the interaction between calcineurin and to the activity of calcineurin We a functional of the interaction as a modulator of calcineurin activity, by the effect of the transcriptional activity of NFAT, of the best characterized calcineurin were the and and the calcium to calcineurin and transcriptional activity was in the significantly of the PMCA the activity of calcineurin in mammalian significantly transcriptional PMCA4b the of in to the calcium of or and of the were in cells. were activity is as the of the in cells. to 60% inhibition of the activity as a of co-expression of PMCA4b. of of PMCA4b and the PMCA4b interaction domain the inhibitory effect of PMCA4b. of the and of was of the in mammalian cells. were activity is as the of the in cells. to 60% inhibition of the activity as a of co-expression of is reversed by the co-expression of the of the interaction between the and the inhibition of calcineurin activity, the interaction by of the of PMCA4b ectopic PMCA4b was to the activity of calcineurin, demonstrating the functional significance of the interaction. were and the of transcriptional by was by co-expression of to a of assessed the effect of PMCA the activity of a constitutively active of calcineurin A is to in a calcium/calmodulin-independent were the and or The constitutively active calcineurin A activity in the presence of the of however, was to this constitutively active calcineurin A of activity is unable to the of in to a constitutively active of calcineurin of a a constitutively active of calcineurin A or its was of the or and of the in mammalian cells. were and activity is as the of the in the and was by of the as and of of Endogenous the to the effect of PMCA4b calcineurin interaction the of calcineurin in the plasma to effect of PMCA4b the calcineurin of a of the plasma and a of the the presence of and plasma calcineurin were a of calcineurin and of calcineurin in the plasma membrane was of calcineurin A and of plasma membrane calcineurin by a in calcineurin the plasma membrane in the presence of ectopic PMCA4b in the a in calcineurin was in the the calcineurin the to the plasma were or A of the plasma and a of the were by to the effect of PMCA4b calcineurin is to in the plasma membrane and the the presence of ectopic is a of calcineurin in the and of calcineurin in the plasma membrane the of calcineurin by as of calcineurin in cells. calcineurin in plasma membrane and calcineurin in the of as a of co-expression of PMCA4b of its effect the of calcineurin represents a crucial of cellular and this a novel the plasma membrane pump, as of calcineurin A and PMCA in mammalian cells. PMCA to calcineurin A and a constitutively active A and and the interaction domain to located the catalytic domain of calcineurin interaction of PMCA4b the catalytic of calcineurin A is to the calcineurin interaction or inhibitory effect by to the catalytic interaction of PMCA of calcineurin or to calcineurin is The interaction of PMCA calcineurin A a the and to calcineurin a the domain the of calcineurin, the of interaction calcineurin the This is in to the PMCA domain, to to of calcineurin and of the PMCA4b is to a of of PMCA4b the of a to This inhibition the effect of PMCA4b We this functional effect is by the of calcineurin by PMCA4b to a cellular the of the activity of the This is by the of inhibition PMCA4b is its interaction the of PMCA4b to confer inhibitory effect constitutively active calcineurin A this functional interaction is domain of PMCA the interaction calcineurin was to the located the catalytic of PMCA4b between domains and the of PMCA4b the interaction domains in the and to a is in the The presence of this in the inhibitory to calcineurin, and of the of PMCA4b the to in The this inhibition is this to in the PMCA The of this in the of the PMCA of calcineurin A PMCA interaction is calcineurin and PMCA There is of calcineurin to the plasma in and a of the calcineurin in a to PMCA the a in PMCA4b in the of calcineurin the to the plasma membrane the and PMCA is a a is by the effect of PMCA4b calcineurin, calcineurin interaction the PMCA catalytic domain the activity of the the between the of PMCA4b and the of calcineurin, as as the is a and the of this We this in of PMCA signal transduction as calcineurin and a its as a of signal transduction the of PMCA characterized this is in of of a functional interaction between PMCA and the This interaction to inhibition of the pathway the factor and to a in the of the calcineurin and to of and to a in PMCA as the of PMCA as of The of to its the of the and and the transcriptional of of calcineurin or PMCA in inhibition of the calcineurin and to a in the transcriptional to evidence a novel functional interaction between PMCA and calcineurin, a PMCA as and modulator of in mammalian cells. The of the of calcineurin and its in cellular of this interaction in to of a and The of in signal transduction is to the of to cellular is the calcineurin calcium/calmodulin-dependent plasma membrane NFAT, factor of activated plasma membrane calcium calcium/calmodulin-dependent plasma membrane NFAT, factor of activated plasma membrane calcium is by is to and in is The factor of activated T-cell (NFAT) the best characterized of calcineurin The effect of calcineurin is the of this of in to a in to of and of The of evidence in the and of a of the of calcineurin The calcineurin is a of a to catalytic calcineurin A and a calcineurin a catalytic domain the and domains the the domain the domain and the domain the of calcineurin is as the domain in the active to the domain of the domain the inhibition We a the plasma membrane ATPase pump as a modulator of in the of the PMCA in and by of of the and and a and of The of of and and PMCA4b to the activity of the and calcium/calmodulin-dependent or by to cellular interaction between the of PMCA4b and domains located in the in the interaction of of PMCA a domain and the calcium/calmodulin-dependent of calcineurin, the PMCA the activity of calcineurin interaction a the this a of to its activity interaction the calcineurin A catalytic PMCA as a novel inhibitory of calcineurin in mammalian cells. We the interaction between PMCA and calcineurin is to calcineurin This the of the pathway and a PMCA as of this pathway in the and of were in and the PMCA4b and was a of and of calcineurin A was by of and the as and The was and and the of to calcineurin A was by of and the by in the the was to the and The was and the a constitutively active was to the a and the of calcineurin A was of calcineurin was by of and the and The and were by of and the and and The were and and the and of to and of the PMCA4b of the domain between and and to were by of and the and and and and The were and and the and of to and of the PMCA4b and of the between and were by of and the and and and The were and and the and of to and The of was by were in the were of the as were in the were the and of the was to the to the were and in of the were and the calcium to The were and activity was a was by of the the of the and of activity A or were and the as were by were in and the were of the as membrane were a and interaction were were and in of were to and plasma were The were and in of The were and to and cells. plasma the the plasma was of The plasma were the by to the The the were of were by were and by or PMCA4b or calcineurin A The were and as a of in PMCA4b a of in a of in or a of A in were and was a were in and the PMCA4b and was a of and The of calcineurin A was by of and the as and The was and and the of to The calcineurin A was by of and the by in the the was to the and The was and the a constitutively active was to the a and the of calcineurin A was The of calcineurin was by of and the and The and were by of and the and and The were and and the and of to and of the PMCA4b of the domain between and and to were by of and the and and and and The were and and the and of to and of the PMCA4b and of the between and were by of and the and and and The were and and the and of to and The of was by were in the were of the as were in the were the and of the was to the to the were and in of the were and the calcium to The were and activity was a was by of the the of the and of activity A or were and the as were by were in and the were of the as membrane were a and interaction were were and in of were to and plasma were The were and in of The were and to and cells. plasma the the plasma was of The plasma were the by to the The the were of were by were and by or PMCA4b or calcineurin A The were and as a of in PMCA4b a of in a of in or a of A in were and was a the of in were the and calcineurin A or a constitutively active the domain and the were the and by to demonstrate of PMCA calcineurin A or the constitutively active The and of the were by the of in the and demonstrate a interaction between and calcineurin A in mammalian of A the interaction domain of calcineurin A the interaction a of of calcineurin A were and by to PMCA in mammalian were and or were the and by to demonstrate of The of were significantly were by the of the of to in PMCA demonstrating the of the interaction. or the were as of the to were by to were to the of calcineurin A is interaction to to the of PMCA4b the of PMCA4b in the interaction calcineurin the of the catalytic domain, or the of PMCA4b of PMCA4b of the of the of its catalytic domain, and the of the The to PMCA4b to of the interaction between calcineurin and the or of PMCA4b. or were in cells. was as a were calcineurin and were by of the to were by The located the catalytic of PMCA4b of of the to the of the or of PMCA4b were in cells. were as in were the to and or of PMCA4b however, to calcineurin, the interaction domain to the of were or calcineurin was to the were a A and by calcineurin was the or domains of PMCA4b were in the were as a The PMCA4b were the to of interaction the domain the of PMCA4b calcineurin, a of this was and The of the as by the of calcineurin demonstrate the of the catalytic of PMCA4b is the interaction between calcineurin and to the activity of calcineurin We a functional of the interaction as a modulator of calcineurin activity, by the effect of the transcriptional activity of NFAT, of the best characterized calcineurin were the and and the calcium to calcineurin and transcriptional activity was in the significantly of the PMCA the activity of calcineurin in mammalian significantly transcriptional PMCA4b the of in to the calcium of or and of the were in cells. were activity is as the of the in cells. to 60% inhibition of the activity as a of co-expression of PMCA4b. of of PMCA4b and the PMCA4b interaction domain the inhibitory effect of PMCA4b. of the and of was of the in mammalian cells. were activity is as the of the in cells. to 60% inhibition of the activity as a of co-expression of is reversed by the co-expression of the of the interaction between the and the inhibition of calcineurin activity, the interaction by of the of PMCA4b ectopic PMCA4b was to the activity of calcineurin, demonstrating the functional significance of the interaction. were and the of transcriptional by was by co-expression of to a of assessed the effect of PMCA the activity of a constitutively active of calcineurin A is to in a calcium/calmodulin-independent were the and or The constitutively active calcineurin A activity in the presence of the of however, was to this constitutively active calcineurin A of activity is unable to the of in to a constitutively active of calcineurin of a a constitutively active of calcineurin A or its was of the or and of the in mammalian cells. were and activity is as the of the in the and was by of the as and of of Endogenous the to the effect of PMCA4b calcineurin interaction the of calcineurin in the plasma to effect of PMCA4b the calcineurin of a of the plasma and a of the the presence of and plasma calcineurin were a of calcineurin and of calcineurin in the plasma membrane was of calcineurin A and of plasma membrane calcineurin by a in calcineurin the plasma membrane in the presence of ectopic PMCA4b in the a in calcineurin was in the the calcineurin the to the plasma were or A of the plasma and a of the were by to the effect of PMCA4b calcineurin is to in the plasma membrane and the the presence of ectopic is a of calcineurin in the and of calcineurin in the plasma membrane the of calcineurin by as of calcineurin in cells. calcineurin in plasma membrane and calcineurin in the of as a of co-expression of PMCA4b of PMCA the of in were the and calcineurin A or a constitutively active the domain and the were the and by to demonstrate of PMCA calcineurin A or the constitutively active The and of the were by the of in the and demonstrate a interaction between and calcineurin A in mammalian cells. The of A the interaction domain of calcineurin A the interaction a of of calcineurin A were and by to PMCA in mammalian cells. were and or were the and by to demonstrate of The of were significantly were by the of the of to in PMCA demonstrating the of the interaction. or the were as of the to were by to were to the of calcineurin A is interaction to to the interaction. The of PMCA4b the of PMCA4b in the interaction calcineurin the of the catalytic domain, or the of PMCA4b were or calcineurin was to the were a A and by calcineurin was the or domains of PMCA4b were in the were as a The PMCA4b were the to of interaction the domain the of PMCA4b calcineurin, a of this was and The of the as by the of calcineurin demonstrate the of the catalytic of PMCA4b is the interaction PMCA4b between calcineurin and to the activity of calcineurin We a functional of the interaction as a modulator of calcineurin activity, by the effect of the transcriptional activity of NFAT, of the best characterized calcineurin were the and and the calcium to calcineurin and transcriptional activity was in the significantly of the PMCA the activity of calcineurin in mammalian cells. the of the interaction between the and the inhibition of calcineurin activity, the interaction by of the of PMCA4b ectopic PMCA4b was to the activity of calcineurin, demonstrating the functional significance of the interaction. were and the of transcriptional by was by co-expression of PMCA4b to a of assessed the effect of PMCA the activity of a constitutively active of calcineurin A is to in a calcium/calmodulin-independent were the and or The constitutively active calcineurin A activity in the presence of the of however, was to this constitutively active calcineurin A of activity PMCA4b Endogenous the to the effect of PMCA4b calcineurin interaction the of calcineurin in the plasma to effect of PMCA4b the calcineurin of a of the plasma and a of the the presence of and plasma calcineurin were a of calcineurin and of calcineurin in the plasma membrane was of calcineurin A and of plasma membrane calcineurin by a in calcineurin the plasma membrane in the presence of ectopic PMCA4b in the a in calcineurin was in the the its effect the of calcineurin represents a crucial of cellular and this a novel the plasma membrane pump, as of calcineurin A and PMCA in mammalian cells. PMCA to calcineurin A and a constitutively active A and and the interaction domain to located the catalytic domain of calcineurin interaction of PMCA4b the catalytic of calcineurin A is to the calcineurin interaction or inhibitory effect by to the catalytic interaction of PMCA of calcineurin or to calcineurin is The interaction of PMCA calcineurin A a the and to calcineurin a the domain the of calcineurin, the of interaction calcineurin the This is in to the PMCA domain, to to of calcineurin and of the PMCA4b is to a of of PMCA4b the of a to This inhibition the effect of PMCA4b We this functional effect is by the of calcineurin by PMCA4b to a cellular the of the activity of the This is by the of inhibition PMCA4b is its interaction the of PMCA4b to confer inhibitory effect constitutively active calcineurin A this functional interaction is domain of PMCA the interaction calcineurin was to the located the catalytic of PMCA4b between domains and the of PMCA4b the interaction domains in the and to a is in the The presence of this in the inhibitory to calcineurin, and of the of PMCA4b the to in The this inhibition is this to in the PMCA The of this in the of the PMCA of calcineurin A PMCA interaction is calcineurin and PMCA There is of calcineurin to the plasma in and a of the calcineurin in a to PMCA the a in PMCA4b in the of calcineurin the to the plasma membrane the and PMCA is a a is by the effect of PMCA4b calcineurin, calcineurin interaction the PMCA catalytic domain the activity of the the between the of PMCA4b and the of calcineurin, as as the is a and the of this We this in of PMCA signal transduction as calcineurin and a its as a of signal transduction the of PMCA characterized this is in of of a functional interaction between PMCA and the This interaction to inhibition of the pathway the factor and to a in the of the calcineurin and to of and to a in PMCA as evidence a novel functional interaction between PMCA and calcineurin, a PMCA as and modulator of in mammalian cells. The of the of calcineurin and its in cellular of this interaction in to of a and its effect the of calcineurin represents a crucial of cellular and this a novel the plasma membrane pump, as of calcineurin A and PMCA in mammalian cells. PMCA to calcineurin A and a constitutively active A and and the interaction domain to located the catalytic domain of calcineurin The interaction of PMCA4b the catalytic of calcineurin A is to the calcineurin interaction or inhibitory effect by to the catalytic interaction of PMCA of calcineurin or to calcineurin is The interaction of PMCA calcineurin A a the and to calcineurin a the domain the of calcineurin, the of interaction calcineurin the This is in to the PMCA domain, to to of calcineurin and of the PMCA4b is to a of of PMCA4b the of a to This inhibition the effect of PMCA4b We this functional effect is by the of calcineurin by PMCA4b to a cellular the of the activity of the This is by the of inhibition PMCA4b is its interaction the of PMCA4b to confer inhibitory effect constitutively active calcineurin A this functional interaction is The domain of PMCA the interaction calcineurin was to the located the catalytic of PMCA4b between domains and the of PMCA4b the interaction domains in the and to a is in the The presence of this in the inhibitory to calcineurin, and of the of PMCA4b the to in The this inhibition is this to in the PMCA The of this in the of the PMCA of calcineurin A PMCA This interaction is calcineurin and PMCA There is of calcineurin to the plasma in and a of the calcineurin in a to PMCA the a in PMCA4b in the of calcineurin the to the plasma membrane the and PMCA is a a is by the effect of PMCA4b calcineurin, calcineurin interaction the PMCA catalytic domain the activity of the the between the of PMCA4b and the of calcineurin, as as the is a and the of this We this in The of PMCA signal transduction as calcineurin and a its as a of signal transduction the of PMCA characterized this is in of of a functional interaction between PMCA and the This interaction to inhibition of the pathway the factor and to a in the of the calcineurin and to of and to a in PMCA as evidence a novel functional interaction between PMCA and calcineurin, a PMCA as and modulator of in mammalian cells. The of the of calcineurin and its in cellular of this interaction in to of a and