SERCA2b Activity Is Regulated by Cyclophilins in Human Platelets

Abstract

Objective-The role of cyclophilins (chaperones that are widely expressed in different cell types, including human platelets) was explored in sarcoendoplasmic calcium (Ca 2ϩ ) adenosine triphosphatase (SERCA) activity. Methods and Results-Cyclophilin inhibition by cyclosporin A (CsA) evoked a time-and concentration-dependent reduction of Ca 2ϩ uptake by SERCA2b. However, other Ca 2ϩ -adenosine triphosphatases expressed in platelets, such as SERCA3 and plasma membrane Ca 2ϩ adenosine triphophatase, remained unaltered after CsA treatment. Cypermethrin, a non-CsA-related calcineurin inhibitor, did not alter SERCA2b activity. Furthermore, SERCA2b was affected by other CsA analogues, which do not interfere with calcineurin, such as PKF-211-811-NX5 (NIM811) and sanglifehrin A. Inhibition of the immunophilin family members using FK506 (tacrolimus) did not alter SERCA2b ability to sequester Ca 2ϩ into the dense tubular system. Coimmunoprecipitation experiments confirmed that cyclophilin A associates with SERCA2b and stromal interaction molecule-1 in resting platelets. This interaction is attenuated by the physiological agonist thrombin but enhanced by treatment with CsA or sanglifehrin A. T he immunophilin family groups proteins with rotenase or peptidylprolyl cis-transisomerase activity. 1,2 These proteins have been classified according to their sensitivity to specific immunosuppressor drugs: cyclophilins (CyPs) are specific targets of cyclosporin A (CsA) [1][2][3][4] ; immunophilins (FKBPs) are sensitive to FK506 (tacrolimus) and rapamycin (sirolimus), both structurally unrelated to CsA 4 -6 ; and FCBPs are sensitive to CsA and FK506. 2 CyPs are involved in two major cellular events: (1) indirect and nonspecific regulation of serine-threonine kinase proteins, an event mediated by the formation of stable CyP/CsA complexes that interact with the regulatory center of calcineurin (CaN), PP2B, or calcium (Ca 2ϩ )/calmodulin kinase (Ca 2ϩ /CaMK) phosphatase, reducing its activity 7 ; and (2) control of cell death by apoptosis through CyP D, which participates in the formation of the mitochondrial permeability transition pore and cytochrome c release in apoptotic cells. Furthermore, CyP B suppresses apoptosis evoked by oxidative, and endoplasmic reticulum, stress. 8 -10 Immunophilins participate in Ca 2ϩ homeostasis in several cell models. However, this role has been mainly attributed to FKBPs that control the opening of Ca 2ϩ channels located in the endoplasmic reticulum, such as ryanodine or inositol 1,4,5-trisphosphate receptors. [11][12][13][14] In contrast, little is known about the role of immunophilins on Ca 2ϩ reuptake or extrusion mechanisms, and the current studies [13][14][15][16] have reported contradictory results about the role of CyPs on the activity of sarcoendoplasmic Ca 2ϩ adenosine triphosphatase (ATPase) (SERCA) or the plasma membrane Ca 2ϩ ATPase (PMCA). In the present study, we have investigated the role of CyPs in the regulation of Ca 2ϩ reuptake by SERCA. We found that CsA reduces Ca 2ϩ reuptake by...

Intracellular cyclophilin A is an important Ca2+ regulator in platelets and critically involved in arterial thrombus formation

“…However, we also found colocalization of SERCA2b and STIM1 in murine platelets, providing strong evidence that SERCA2b also belongs to the protein complex STIM1/CyPA ( Figure 4F). Interaction of SERCA2b and STIM1 takes place independently of CyPA, suggesting a role of STIM1 in the regulation of SERCA2b, as proposed previously by Rosado et al 18 CyPA deficiency leads to defective platelet adhesion and thrombus formation under flow conditions ( Figure 1F-G). To determine whether STIM1 phosphorylation is important for cell adhesion, we made use of a platelet model cell line called A5-CHO, a specific CHO cell that stably overexpresses human integrin ␣ IIb ␤ 3 , 49 and allowed these cells to spread on fibrinogen.…”

“…18 In the present study, immunoprecipitation studies also identified colocalization of CyPA and SERCA2b in murine platelets, suggesting that CyPA is also responsible for the regulation of SERCA2b in murine platelets ( Figure 4E). On thrombin stimulation, colocalization of these proteins decreased, whereas in mutant platelets, complex formation of SERCA2b and CyPA could not take place because of CyPA deficiency ( Figure 4E).…”

“…Colocalization of CyPA and STIM1 in control and CsA-treated human platelets has been demonstrated previously by Rosado et al, who demonstrated that enzymatic activity of CyPA was necessary to release STIM1 from the complex. 18 To determine the functional consequences of absent STIM1/CyPA coupling in Cypa Ϫ/Ϫ platelets, we performed phosphorylation studies to analyze STIM1 phosphorylation upon platelet activation. Immunoprecipitation experiments showed an increase in tyrosine phosphorylation of STIM1 in control platelets after thrombin stimulation for 1 and 5 minutes ( Figure 4B).…”

“…16,17 Immunophilins are known to participate in Ca 2ϩ homeostasis in different cells and are known to control Ca 2ϩ channels in the ER-like IP 3 receptors, whereas cyclophilins such as CyPA are probably involved in the regulation of sarcoplasmic/endoplasmic reticulum Ca 2ϩ ATPase 2b (SERCA2b). 18 CyPA is a ubiquitously expressed intracellular protein with a peptidyl-prolyl cis-trans isomerase activity and acts as a chaperone protein. [14][15][16] It is the major cellular target of the immunosuppressive drug cyclosporine and forms a complex with calcineurin to prevent calcineurin from regulating cytokine gene transcription.…”

“…by guest www.bloodjournal.org From A recent study from Rosado et al described an important role for cyclophilins in the regulation of platelet SERCA2b activity and provided evidence for a direct interaction of CyPA and SERCA2b by a protein-protein interaction. 18 In the presence of CsA, thrombininduced Ca 2ϩ mobilization was found to be modified, indicating a reduced SERCA2b activity without altering PMCA activity or other SERCA isoforms. 18 Consistent with these results, the reduced Ca 2ϩ release from intracellular stores of CyPA-deficient platelets shown in the present study suggests reduced Ca 2ϩ levels in stores of Cypa Ϫ/Ϫ platelets and thus defective SERCA2b activity in these cells.…”