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Protein Kinase CK2-Dependent Phosphorylation of the Human Regulators of Calcineurin Reveals a Novel Mechanism Regulating the Calcineurin–Nfatc Signaling Pathway

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Protein Kinase CK2-Dependent Phosphorylation of the Human Regulators of Calcineurin Reveals a Novel Mechanism Regulating the Calcineurin–Nfatc Signaling Pathway CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1833 (2013) 2311–2321 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr Protein kinase CK2-dependent phosphorylation of the human Regulators of Calcineurin reveals a novel mechanism regulating the calcineurin–NFATc signaling pathway Sergio Martínez-Høyer a, Álvaro Aranguren-Ibáñez a, Javier García-García b, Eva Serrano-Candelas a, Jordi Vilardell c, Virginia Nunes e, Fernando Aguado d, Baldo Oliva b, Emilio Itarte c, Mercè Pérez-Riba a,⁎ a Cellular Signaling group, Cancer and Molecular Genetics Program, Bellvitge Biomedical Research Institute — IDIBELL, L'Hospitalet de Llobregat, 08908 Barcelona, Catalonia, Spain b Structural Bioinformatics Goup (GRIB-IMIM), Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona Research Park of Biomedicine (PRBB), 08003 Barcelona, Catalonia, Spain c Unitat de Bioquímica de Biociències, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain d Department of Cell Biology, University of Barcelona, Barcelona, Spain e Laboratorio de Genética Molecular, IDIBELL, 2U-730 (CIBERER), and Departament de Ciències Fisiològiques II, Facultad de Medicina, Universitat de Barcelona, Barcelona, Spain article info abstract Article history: Cyclosporine A and FK506 produce immunosuppression by blocking calcineurin phosphatase activity and Received 14 December 2012 consequently activation of cytosolic Nuclear Factor of Activated T-cell (NFATc) transcription factor. Due to Received in revised form 21 May 2013 the chronic toxicity associated with their administration, the development of more specific immunosuppres- Accepted 22 May 2013 sants is currently an important unmet medical need. In this context, an immunosuppressant peptide derived Available online 1 June 2013 from the CIC motif of the human Regulators of Calcineurin (RCAN) proteins has been shown to inhibit NFATc signaling without affecting general phosphatase activity of calcineurin. Here we show that protein kinase CK2 Keywords: Calcineurin–NFATc signaling phosphorylates a conserved serine residue within the CIC motif of vertebrate RCANs, which increases its af- RCAN finity for calcineurin and consequently its inhibition of NFATc-dependent gene expression in activated PXIXIT T-cells. Molecular modeling studies have led us to identify a positively charged interaction site on the surface CK2 of calcineurin where the phosphorylated serine residue of the CIC motif would normally locate. Finally, we Immunosuppression have also identified RCAN3 as a new phosphoprotein with multiple phosphorylation sites. Therefore, our Phosphorylation findings reveal for the first time a novel molecular mechanism underlying the regulation of calcineurin– NFATc signaling by means of phosphorylation of the CIC motif of RCAN proteins. The knowledge of how RCAN proteins modulate the calcineurin–NFATc pathway paves the way for the development of potent novel selective immunosuppressant drugs. © 2013 Published by Elsevier B.V. 1. Introduction transcription factors, they trigger the expression of genes involved in diverse biological processes including T-cell activation, cardiac hy- The highly conserved serine/threonine phosphatase calcineurin pertrophy, bone remodeling or angiogenesis among others [3]. The (Cn, also known as PPP3, formerly PP2B) is a cellular sensor enzyme importance of this pathway came to light with the discovery that that plays a pivotal role in transducing Ca2+ signals into cellular re- the most widely used current immunosuppressant drugs, cyclospor- sponses [1]. The activation of Cn, which is ubiquitously expressed, ine A (CsA) and FK506, elicited their clinical response by inhibiting results in the dephosphorylation of its substrates, including the cyto- Cn enzymatic activity and therefore also activation of NFATc [4]. solic members of the Nuclear Factor of Activated T-cell (NFATc) fam- However, further studies revealed that the inhibition of Cn by these ily of transcription factors [2]. Once dephosphorylated, NFATc drugs inhibits dephosphorylation of all Cn substrates and is associat- proteins translocate to the nucleus where, in cooperation with other ed to severe undesirable side effects [5]. The Regulators of Calcineurin (RCAN; previously called DSCR, fi Abbreviations: Cn, calcineurin; CnA, calcineurin catalytic subunit A; CIC sequence, MCIP or calcipressin (CALP)) family of proteins are speci c endoge- RCAN (formerly calcipressin) inhibitor of calcineurin sequence; CK2, protein kinase CK2; nous modulators of the Cn–NFATc signaling axis [6,7]. This protein NFATc, cytosolic Nuclear Factor of Activated T-cells; RCAN, Regulators of Calcineurin; family, defined by several highly conserved motifs in Eukarya such CsA, cyclosporin A; Io, Ionomycin; PMA, Phorbol 12-miristate 13-acetate as the FLISPP motif (FLISPPASPP sequence), is conserved from yeast ⁎ Corresponding author at: Human Molecular Genetics Laboratory, Cancer and Molecular to human and in vertebrates constitutes a highly conserved subfamily Genetics Program, Bellvitge Biomedical Research Institute — IDIBELL. L'Hospitalet de Llobregat, Barcelona 08908, Spain. Tel.: +34 932607427; fax: +34 932607414. comprised of three proteins: RCAN1, RCAN2 and RCAN3 [8,9], each of E-mail address: [email protected] (M. Pérez-Riba). which is expressed as different isoforms [9]. The specificity towards 0167-4889/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.bbamcr.2013.05.021 2312 S. Martínez-Høyer et al. / Biochimica et Biophysica Acta 1833 (2013) 2311–2321 NFATc signaling modulation by these proteins is explained by two from Invitrogen. The anti-calcineurin A antibody was purchased from conserved Cn-interacting motifs shared by both NFATc and RCAN pro- BD Biosciences, the anti-CK2α (H-286 clone) from Santa Cruz, and the teins: the LXXP (LXVP in NFATc proteins) and PXIXIT motifs [10–14]. anti-HA (Clone 12CA5) and monoclonal anti-α-tubulin (P-6074) were The PXIXIT motif has been described as the major Cn-anchoring site of from Sigma. The protease and phosphatase inhibitor cocktails were NFATc proteins [15]. The crystal structure of the synthetic VIVIT peptide from Calbiochem. derived from the PXIXIT sequence of NFATc [16] in complex with Cn, helped to clarify how this sequence interacts in a hydrophobic cleft 2.2. Plasmid constructs and site-directed mutagenesis formed by strands β14 and β11 of Cn [17]. Variations in the consensus PXIXIT sequence modulate the binding affinity for Cn of the different The different pGEX5X–RCAN3, pGEX6P–CnA (human CnAα PXIXIT-containing proteins and therefore the biological consequence amino acids 2–347), HA–RCAN3 (NP_038469.1) and HA–RCAN1 of such interaction [18–20]. RCAN proteins possess a PSVVVH sequence (NP_004405.3) plasmid constructs used for this work have been de- which has been shown to behave as a PXIXIT motif [12–14]. This PXIXIT scribed previously [8]. RCAN2-3 cDNA (NM_001251973.1) was a gift sequence, together with a C-terminal ELHA sequence, comprise the from Dr. X. Cao and was subcloned as an N-terminal HA-tagged fu- RCAN inhibitor of Cn (CIC) motif, conserved in all vertebrate RCANs sion in pcDNA3.1-HA vector using BamHI restriction sites. The [8,21]. We and others have shown that in RCANs, the CIC motif needs Flag–hCnAα and GST–NFATc2 expression plasmids were kindly pro- both the ELHA and PXIXIT sequences to bind with high affinity to Cn vided by Dr. J. M. Redondo (CNIC, Madrid, Spain), the 3xNFAT-luc and inhibit downstream NFATc signaling [12,14]. plasmid construct by Dr. J. Aramburu (Universitat Pompeu Fabra, Both inhibitory and facilitative roles on the Cn–NFATc signaling Barcelona, Spain), and the pRL-null plasmid, which lacks a promoter pathway have been associated with the RCANs, depending on the spe- driving Renilla expression, from Dr. C. Marín (Universidad de cific tissue or cell type and expression level [22–24]. In addition, RCAN León, León, Spain). The EGFPc–RCAN3178–203 construct has been proteins are also known to be regulated by post-translational modifica- previously described [12]. The RCAN3178–210 protein fragment was tion including phosphorylation. For instance, phosphorylation at the PCR-amplified from the HA–RCAN3 plasmid using specificprimers conserved FLISPP motif of RCAN1 by GSK3β, primed by phosphorylation and subcloned as an EGFPc-fusion protein using BamHI restriction by either BMK, MAPK or DYRK1A, facilitates Cn–NFATc signaling sites. Oligonucleotide sequences are listed in Supplementary Table 1. [25–28]. Also, phosphorylation of RCAN1 by TAK1 has been related to a facilitative role towards the Cn–NFATc signaling pathway [29].On 2.3. Recombinant protein expression and purification the other hand, phosphorylated RCAN1 at both serine residues of the FLISPP motif has been shown to be a better inhibitor of NFATc activity GST fusion proteins were transformed in BL21-CodonPlus® strain. Sol- [26]. In addition, phosphorylation of RCAN1 has also been associated uble extracts were produced and GST fusion protein was purified using to an increased protein half life and therefore and enhanced inhibition Glutathione Sepharose 4B beads (GE Healthcare) as described previously of Cn–NFATc signaling [28,30]. [21]. Free CnA was obtained by Prescission protease (GE Healthcare) incu- Protein kinase CK2 is a ubiquitous enzyme with pleiotropic
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