Báo cáo khoa học: Regulation of calpain B from Drosophila melanogaster by phosphorylation pot

Regulation of calpain B from Drosophila melanogaster by phosphorylation La ´ szlo ´ Kova ´ cs 1, *, Anita Alexa 2, *, Eva Klement 3 , Endre Ko ´ kai 1 ,A ´ gnes Tantos 2 , Gergo ¨ Go ´ gl 2 , Tama ´ s Sperka 4 , Katalin F. Medzihradszky 3,5 ,Jo ´ zsef To ¨ zse ´ r 4 , Viktor Dombra ´ di 1,6 and Pe ´ ter Friedrich 2 1 Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Hungary 2 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary 3 Proteomics Research Group, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary 4 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Hungary 5 Department of Pharmaceutical Chemistry, University of California at San Francisco, CA, USA 6 HAS-DU Cell Biology and Signaling Research Group, Department of Medical Chemistry, Research Center for Molecular Medicine, University of Debrecen, Hungary Keywords calcium-dependent protease; Drosophila melanogaster ; enzyme kinetics; epidermal growth factor; protein kinase Correspondence V. Dombra ´ di, Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, 98 Nagyerdei krt, Debrecen, H-4032, Hungary Fax: +36 52 412 566 Tel: +36 52 412 345 E-mail: dombradi@med.unideb.hu *These authors contributed equally to this work (Received 23 April 2008, revised 15 June 2009, accepted 6 July 2009) doi:10.1111/j.1742-4658.2009.07198.x Calpain B is one of the two catalytically competent calpain (calcium-acti- vated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombi- nant protein by protein kinase A as well as by the extracellular signal-regu- lated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca 2+ binding region of an EF-hand motif, and Ser240 close to the autocat- alytic activation site of calpain B, as being the residues phosphorylated by protein kinase A. In the transducer region of the protease, Thr747 was shown to be the target of the ERK phosphorylation. Based on the results of three different assays, we concluded that the treatment of calpain B with protein kinase A and ERK1 and ERK2 kinases increases the rate of the autoproteolytic activation of the enzyme, together with the rate of the digestion of external peptide or protein substrates. Phosphorylation also elevates the Ca 2+ sensitivity of the protease. The kinetic analysis of phos- phorylation mimicking Thr747Glu and Ser845Glu calpain B mutants con- firmed the above conclusions. Out of the three phosphorylation events tested in vitro, we verified the in vivo phosphorylation of Thr747 in epi- dermal growth factor-stimulated Drosophila S2 cells. The data obtained suggest that the activation of the ERK pathway by extracellular signals results in the phosphorylation and activation of calpain B in fruit flies. Structured digital abstract l MINT-7214239: ERK1 (uniprotkb:P40417) phosphorylates (MI:0217) CalpainB (uniprotkb: Q9VT65)byprotein kinase assay (MI:0424) l MINT-7214216, MINT-7214228: PKA (uniprotkb:P12370) phosphorylates (MI:0217) CalpainB (uniprotkb: Q9VT65)byprotein kinase assay (MI:0424) l MINT-7214325: CalpainB (uniprotkb:Q9VT65) cleaves (MI:0194) MAP2C (uniprotkb: P11137)byprotease assay (MI:0435) Abbreviations CaMKII, calcium ⁄ calmodulin-dependent protein kinase II; CID, collision-induced dissociation; EGF, epidermal growth factor; ERK, extracellular signal-regulated protein kinase; LY-AMC, N-succinyl-Leu-Tyr-7-amino-4-methyl-coumarin; MAP, microtubule-associated protein; Ni-NTA, nickel–nitrilotriacetic acid; PKA, protein kinase A. FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4959 Introduction The regulation of intracellular signaling pathways involves an intricate interplay of various enzyme sys- tems. One intriguing example is the interaction between calpains (i.e. the Ca 2+ -dependent SH-prote- ases) and protein kinases (i.e. the enzymes of protein phosphorylation). Regarding their modes of action, calpains catalyze the irreversible, limited proteolysis of their substrate proteins, whereas protein phosphoryla- tion can be reversed by the protein phosphatases via the hydrolytic elimination of the phosphate group from Ser, Thr or Tyr residues. Calpains play crucial roles in controlling various cellular processes, such as cytoskeletal remodeling, cell cycle, apoptosis and cell motility [1,2]. The ubiquitous mammalian l- and m-calpains (calpain-1: EC 3.4.22.52; calpain-2: EC 3.4.22.53) are the best-characterized members of the family. The regulation of these essential proteases still remains an open question. The micromolar to millimo- lar Ca 2+ concentrations required for the effective acti- vation of calpains in vitro is not in the physiological range. It has been suggested that the presence of addi- tional regulatory substances (e.g. phospholipids) or post-translational protein modifications (e.g. phosphor- ylation) can modulate the Ca 2+ -sensitivity of these proteases [3,4]. Phosphorylation of mammalian calpains has been intensively studied. Because recombi- nant calpains, which are devoid of phosphate groups, are fully active, it can be concluded that phosphoryla- tion is not essential for their activity. On the other hand, l- and m-calpain extracted from different tissues contain two to four phosphate group ⁄ molecule, which are distributed over eight or nine different Ser, Thr and Tyr residues [1]. The main phosphorylation sites in m-calpain are Ser50 and Ser369⁄ Thr370. In vitro and in vivo studies show that phosphorylation of Ser50 by extracellular signal-regulated protein kinases (ERKs) enhances calpain activity, as well as calpain- mediated physiological processes [5], whereas phos- phorylation of Ser369 ⁄ Thr370 by protein kinase A (PKA) inhibits calpain action [6]. Nicotine-induced phosphorylation by an isoform of protein kinase C, PKCi, enhances both the activity and secretion of l- and m-calpain in human lung cancer cells [7]. In the present study, we examined calpain B from Drosophila melanogaster. We chose a fly enzyme because Drosophila is a handy model organism and, out of its four calpains, three (calpain A, B and C) have been characterized in our laboratory [2]. As far as we are aware, only calpain A and B exhibit protease activity in the fruit flies. Both of them are activated at millimolar free Ca 2+ concentration. The active Dro- sophila calpains consist of a single polypeptide chain, for which the domain structure shows strong similarity to the catalytic subunits of mammalian l- and m-cal- pain. They are composed of an N-terminal regulatory domain (I), a catalytic domain (II), a C2-like domain (III) and a calmodulin-like calcium binding domain (IV). The main difference between mammalian and Drosophila calpains is represented by the length of the N-terminal domains; for example, calpain B has a 240 amino acid long N-terminal region with an extended, disordered structure [8]. Upon Ca 2+ -dependent activa- tion, this N-terminal inhibitory region is first clipped off in an autoproteolytic process. Because the bioinfor- matic analysis of the primary structure of calpain B suggested a number of potential phosphorylation sites that correspond to consensus recognition motifs of sev- eral protein kinases, we initiated the investigation of the phosphorylation of this protein under both in vitro and in vivo conditions. In the present study, we report on the identification of cAMP-dependent protein kinase (protein kinase A, PKA; EC 2.7.11.11) and mitogen-activated protein kinase (ERK1 and ERK2; EC 2.7.11.24) phosphorylation sites in calpain B and describe the effects of phosphorylation on the proteo- lytic activation and activity of the enzyme. Results and discussion Phosphorylation of calpain B in vitro The phosphorylation of any of the Drosophila calpains has not been reported yet. According to motif scan (http://scansite.mit.edu/motifscan_id.phtml) prediction, there are five putative PKA consensus sites and several ERK target sites in calpain B, when screened at a low stringency level (data not shown). The feasibility of ERK action was further supported by the presence of three ERK1 binding sites and three ERK2 binding sites in the protein. To confirm the structural predic- l MINT-7214275: ERK2 (uniprotkb:P40417-2) phosphorylates (MI:0217) CalpainB (uni- protkb: Q9VT65)byprotein kinase assay (MI:0424) l MINT-7214319: CalpainB (uniprotkb:Q9VT65) and CalpainB (uniprotkb:Q9VT65) cleave ( MI:0194)byprotease assay (MI:0435) Phosphorylation of calpain B L. Kova ´ cs et al. 4960 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS tions, purified recombinant calpain B was phosphory- lated with PKA, as well as with the two isoforms of the extracellular signal-regulated protein kinase, ERK1 and ERK2, in the presence of [ 32 P]ATP[cP] in vitro (Fig. 1). PKA incorporated 0.20 ± 0.09 (n = 5) mol PÆmol protein )1 . The phosphorylation with ERK1 was more effective, and 0.62 ± 0.27 (n = 6) mol PÆmol protein )1 was achieved within 2 h, whereas ERK2 built in 0.73 ± 0.17 (n = 7) mol PÆmol protein )1 under the same conditions (Fig. 1B). In all of the experiments, more than 95% of the total protein bound radioactivity resided in the band corresponding to the apparent molecular mass of the recombinant calpain B (Fig. 1A). For the identification of the phosphorylated amino acid residues, both the wild-type active protease and an inactive calpain B mutant were phosphorylated as described above; with the exception that nonradioac- tive ATP was used instead of [ 32 P]ATP[cP]. The inac- tive mutant was generated (aiming to avoid unwanted autoproteolytic degradation during sample handling) by replacing Cys314 with Ala in the active center of the enzyme. The active calpain B and the inactive C314A mutant were purified by SDS ⁄ PAGE after in vitro phosphorylation. The proteins were in-gel digested with trypsin and analyzed by MS. The phos- phopeptides were identified from the digests using precursor ion scanning and affinity enrichment. Phosphopeptides yield diagnostic m ⁄ z 79 (PO 3 ) ) ions in negative ion mode. The precursors of this fragment were identified in nanoLC ⁄ MS ⁄ MS experiment on a QTRAP mass spectrometer (Applied Biosystems, Fos- ter City, CA, USA). Collision-induced dissociation (CID) data acquired in positive ion mode from these precursor ions provided sufficient information for sequence and modification site assignment. These A B Fig. 1. Phosphorylation of calpain B in vitro. (A) Recombinant calpain B was phosphorylated with PKA, ERK1 and ERK2 protein kinases in the presence of [ 32 P]ATP[cP]. Samples were analyzed by SDS ⁄ PAGE followed by autoradiography. The left-hand lanes show the molecular mass standards (St) and 2 lg of unphosphorylated calpain B protein (P) stained with Coomassie brilliant blue. The molecular mass of the standards is given in kDa. The right-hand lanes present the autoradiorams of the samples taken at the indicated time points after the initia- tion of phosphorylation. (B) The phosphorylation reaction by PKA ( ), ERK1 (.) and ERK2 ( ) was also monitored by counting the radioactiv- ity incorporated into the TCA insoluble protein. The mean ± SD of five to seven independent experiments is shown. L. Kova ´ cs et al. Phosphorylation of calpain B FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4961 assignments were confirmed by phosphopeptide enrich- ment using TiO 2 affinity chromatography followed by LC ⁄ MS ⁄ MS analysis in information-dependent acqui- sition mode on an ion trap mass spectrometer. In the wild-type calpain B enzyme, two PKA-phosphorylation sites at Ser240 and Ser845 were revealed by the MS ⁄ MS spectra of the precursors at m ⁄ z 764.8 (2+) and m⁄ z 591.7 (2+), respectively. The MS ⁄ MS spec- trum of the precursor at m ⁄ z 764.8 (2+) represents the phosphopeptide 238 qNS(p)VSKGDFQSLR 250 . The phosphorylation site assignment is based on fragments observed at m ⁄ z 393.3 (b 3 ), 519.3 (y 9 2+ ), 1037.0 (y 9 ) and 569.0 (y 10 2+ )(Fig. 2A). The phosphorylation site at Ser845 was unambiguously identified from the fragment observed at m ⁄ z 512.7 (y 8 ) in the MS ⁄ MS spectrum of the precursor at m ⁄ z 591.7 (2+) corre- sponding to 842 TGS(p)IDGFHLR 852 (Fig. 2B). For the ERK2 kinase, phosphorylation at Thr747 was determined from the MS ⁄ MS spectrum of the pre- cursor at m ⁄ z 689.9 (3+) corresponding to 739 IA- PSLPPPT(p)PKEEDDPQR 756 ; the fragments at m ⁄ z 482.0 (b 5 ) and 793.5 (y 13 ) clearly indicate phosphoryla- tion at Thr747 (Fig. 2C). The same phosphorylation sites were identified in the inactive calpain B mutant, with the exception that Ser240 phosphorylation was not found. In addition, we demonstrated that ERK1 phosphorylated the same Thr747 residue as the ERK2 isoenzyme (data not shown). The sites of in vitro phos- phorylation in calpain B are summarized in Fig. 2D. Although the 3D structure of calpain B has not been solved yet, from the available atomic coordinates of m-calpain [9], the sites of phosphorylation can easily be assigned to the structural domains of the enzyme by homologous modeling. In agreement with the motif scan prediction, the PKA consensus site, Ser845, lies in domain IV within the second EF-hand motif (resi- dues 831–859). It is preceded by two basic residues, Arg841 and Arg842, that create a favorable environ- ment for PKA recognition. The second PKA site at Ser240 is at the end of domain I, close to the activat- 365.7 a 3 400.3 y 3 464.0 b 5 # 557.3 y 9 2+ 622.7 y 16 #3+ 652.0 y 17 3+ 683.3 MH 3 #3+ 744.3 y 13 -98 2+ 482.0 b 5 628.3 y 16 3+ 793.3 y 13 2+ 942.3 y 16 2+ 850.0 y 14 2+ 1114.0 y 9 393.3 b 3 297.7 PSL 503.0 y 4 374.7 y 6 *2+ 519.3 y 9 2+ 569.0 y 10 2+ 755.7 MH 2 #2+ 533.7 MH 2 -98 #2+ 463.7 y 8 -98 2+ 542.3 MH 2 -98 2+ 629.3 y 5 583.0 MH 2 #2+ 744.7 y 6 857.0 y 7 895.0 b 8 1009.0 b 9 797.0 b 8 -98 660.0 b 7 -98 579.0 b 5 650.7 y 5 x 5 A B CD x 10 x 10 x 2 x 5 x 5 x 5 822.3 y 7 879.3 b 8 1073.0 y 9 781.0 b 8 -98 288.0 y 2 425.0 y 3 512.7 y 8 2+ 227.7 b 3 -98 341.0 b 4 -98 716.0 MH 2 -98 2+ Fig. 2. Identification of the in vitro phosphorylation sites in calpain B by MS. (A) CID spectrum of the precursor at m ⁄ z 764.8 (2+) represent- ing qNS(p)VSKGDFQSLR and (B) CID spectrum of the precursor at m ⁄ z 591.7 (2+) corresponding to TGS(p)IDGFHLR, both observed in the TiO 2 enrichment of the PKA phosphorylated calpain B digest. (C) CID spectrum of the precursor at m ⁄ z 689.9 (3+) representing IAPSLPPPT(p)PKEEDDPQR detected in the TiO 2 enrichment of the ERK2 phosphorylated calpain B digest. Water loss is marked with a hash symbol (#); )98 indicates phosphoric acid loss. Nomenclature is used in accordance with Biemann [22]. The peaks used for the identification of the phosphorylation sites are marked with an arrow. (D) Summary of the in vitro phosphorylation sites (shown in bold) in calpain B. Phosphorylation of calpain B L. Kova ´ cs et al. 4962 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS ing scission site located between amino acids 224–225. Because its environment (Fig. 2D) is less preferred by the kinase, we consider this residue as a secondary site of phosphorylation. Although Ser845 appears to be the preferential PKA site within the molecule, it should be noted that both the rate and the extent of PKA phosphorylation are rather low (Fig. 1B). Thr747 was identified as the site of phosphorylation of either ERK1 or ERK2. In agreement with the consensus sequence of the two kinase isoforms, there are three consecutive Pro residues immediately at the N-terminal side of Thr747. Indeed, this site is recognized by all of the so called Pro-directed protein kinases. Thr747 is situated at the surface of the molecule between domains III and IV in an extended structural element termed ‘transducer’ [10,11]. This region of the polypep- tide was suggested to transmit the Ca 2+ signal from the Ca 2+ binding EF-hands of domain IV to the active site cleft that is situated between the IIa and IIb sub- domains. The phosphorylation of additional potential ERK consensus sites was not supported by the experi- ments. In comparison with the mammalian counterparts, none of these phosphorylation sites of calpain B have been conserved in calpain l or m and, vice versa, the known phosphorylation sites of mammalian calpains are missing from Drosophila calpain B. From the first part of our studies, we conclude that calpain B can be phosphorylated by three kinases at three different resi- dues, although the sites of phosphorylations are dis- tinct from those reported for the mammalian enzymes [5,6]. Consequently, the regulation of the Drosophila protease can be different from the well-known mam- malian calpains. Effects of phosphorylation on the activation and activity of calpain B Because the location of the phosphorylation sites suggested an effect of phosphate incorporation on the Ca 2+ regulation of calpain B, we compared the kinetic properties of the nonphosphorylated (control) and phosphorylated enzymes using three independent methods. Fluorimetric assay with a peptide substrate At high Ca 2+ concentration, a continuous assay was applied using the fluorescent dipeptide substrate N-suc- cinyl-Leu-Tyr-7-amino-4-methyl-coumarin (LY-AMC). In an earlier study [10], we determined that a 8.6 ± 0.8 mm free [Ca 2+ ] concentration was required for the half maximal activation of calpain B. Accord- ingly, at 9 and 19 mm free Ca 2+ concentrations, the enzyme works at 50% and 90% of its full activity, respectively. Under these conditions, the reaction is fast enough to reach the maximal velocity (v max ), a parameter that can be used to compare the phosphory- lated forms with the nonphosphorylated one. As an example, two calpain progress curves are presented in Fig. 3A, demonstrating the effect of PKA-treatment. Both progress curves start with a lag-phase, corre- sponding to the autoproteolytic activation; later, the sigmoid-like curves reach maximal activity. In the pres- ent study, the progress curves were fitted with a log- istic curve (see Experimental procedures), which provided the k act and v max values. The phosphorylated calpain B forms were found to be activated faster (Fig. 3B), and had a greater activity (v max )ata9mm free Ca 2+ concentration (Fig. 3C). Similar results were obtained at a 19 mm free Ca 2+ concentration (data not shown). In all of our experiments, ERK2 exerted a more pronounced effect than ERK1, in accordance with the fact that the stoichiometry of phosphorylation was somewhat higher with the former kinase. The rela- tively small effect of PKA can be attributed either to the different sites of modification or, more likely, to the lower level of phosphorylation. Activity assay with a protein substrate Microtubule-associated protein (MAP) 2c is readily digested by mammalian m-calpain as well as calpain B, even at lower Ca 2+ concentrations. The proteolytic reaction was carried out at a 350 lm free Ca 2+ con- centration and monitored by SDS ⁄ PAGE followed by the densitometric scanning of the 62 kDa intact MAP2c band ( Fig. 4A). Simple visual inspection of the results demonstrates that calpain B phosphorylated with PKA digested MAP2c faster than the nonphosph- orylated form. For quantitative evaluation of the data, we plotted the logarithm of the optical density of the MAP2c band at a given time divided by the optical density measured at the beginning of the reaction [i.e. ln(A ⁄ A 0 )] against the reaction time and determined the slope of the linear curve (Fig. 4B). Assuming that the quantity of the active enzyme is constant during the reaction, the slope gives the rate constant of the first- order reaction. We used this constant for the charac- terization of the enzyme activity and expressed the effect of phosphorylation as described above (Fig. 4C). In this independent assay, again, ERK2 increased cal- pain B activity more vigorously than the other kinases. The Ca 2+ dependence of PKA action on calpain B activity was investigated in more detail (Fig. 4D). The results obtained clearly demonstrate that the effect of L. Kova ´ cs et al. Phosphorylation of calpain B FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4963 PKA is larger at lower Ca 2+ concentrations. Because, using this method, we were unable to collect data at the very beginning of the reaction, the plots were not suited to analyze the effect of phosphorylation on the activation of calpain B. Autolysis assay To circumvent the above limitation of MAP2c diges- tion assay, we designed an alternative approach for the determination of the effects of phosphorylation on autolysis. The approach was based on our observation of a slight autocatalytic processing of calpain B in the MAP2c based assay (Fig. 4A). Under modified condi- tions, and in the absence of MAP2c, we monitored the disappearance of the 104 kDa intact calpain B band as a function of time (Fig. 5A) and used the same kinetic approach as applied before for the determination of the apparent first-order rate constant of the reaction (Fig. 5B). According to our assumption, the first auto- catalytic cleavage between amino acid residues 224– 225 is sufficient for the activation of calpain B; thus, the rate of the elimination of the 104 kDa inactive form is approximately proportional to the rate of acti- vation. Figure 5C shows that phosphorylation by either ERK2 or PKA slightly elevated the rates of autolysis in the presence of a 19 mm free Ca 2+ concen- tration. The effect was negligible when the free Ca 2+ concentration was set at 1 mm (data not shown). ERK1 was not studied in this test because it phospho- rylates the same site as ERK2, but with slightly lower efficiency. When calpain B was phosphorylated with [ 32 P]ATP[cP], the distribution of the radioactive label was monitored by autoradiography during autolysis (Fig. 5D). Although the overall rate of autolysis was comparable (i.e. the rate constants were k ERK2 = 0.039 s )1 and k PKA = 0.034 s )1 , respectively), the gen- eral picture was quite different for the two kinases tested. The radioactive phosphate incorporated by PKA was eliminated very quickly from the 104 kDa band, and most of the total radioactivity (more than 85%) accumulated in the 75 kDa fragment after the first steps of autolysis. According to the activation model of calpain B [12], this fragment represents the C-terminal portion of the protein. Thus, the distribu- tion of radioactivity is in agreement with our previous result indicating that the PKA phosphorylation sites of calpain B reside inside the 75 kDa fragment. Regard- ing the ERK2 phosphorylation, approximately 75% of the radioactivity incorporated by the kinase dis- appeared within less than 30 s from the gel. Only 21–26% of 32 P remained in the 75 kDa C-terminal proteolytic fragment. The most likely explanation is A B C Fig. 3. Effect of phosphorylation on calpain B activity as measured with a peptide substrate. The progress curve of calpain activity measurement with the fluorimetric assay (see Experimental proce- dures) in the presence of a 9 m M free Ca 2+ concentration is shown as an example (A). Calpain B was phosphorylated by PKA (broken line) for 60 min to the stoichiometry of 0.21 mol PÆmol protein )1 ,or treated under the same conditions without the kinase (solid line) in a control experiment. The fluorescent signal was recorded after the addition of the enzyme to the reaction mixture. From the progress curves, the rate of activation (k act ) as well as the maximal activity (v max ) of calpain B was calculated. The average k act for the non- phosphorylated form was 8.5 · 10 3 M )1 Æs )1 at a 9 mM free Ca 2+ concentration, whereas the average v max was 10 )8 M )1 Æs )1 , and the k cat , which can be determined from the v max , was 1.5 · 10 )2 s )1 . The increase in the rate of activation (B) and in the activity (C) upon phosphorylation with PKA, ERK1 and ERK2 is given as the percent- age of the unphosphorylated controls. The extent of phosphoryla- tion was 0.19 ± 0.03, 0.63 ± 0.18 and 0.75 ± 0.18 mol PÆmol protein )1 for the three kinases, respectively. The mean ± SD of three or four independent experiments is shown. Phosphorylation of calpain B L. Kova ´ cs et al. 4964 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS that, beside Thr747, ERK2 effectively phosphorylated another residue(s) in the unstructured N-terminal regu- latory domain that was very quickly degraded during the autoproteolytic activation process. Site-directed mutagenesis mimicking phosphorylation of calpain B Although the direct phosphorylation of calpain B with the selected protein kinases provides realistic results, the explanation of the data is complicated by the low stoichiometry of phosphorylation in the case of PKA, and by the existence of multiple phosphorylation sites in all cases. To assess the contribution of a well estab- lished site to the regulation of calpain B, we adopted the approach of Smith et al. [13] and replaced the target sites of the kinases with Glu by site-directed mutagenesis. The negative Glu side-chain mimics the effect of phosphorylation. In addition to being specific, site-directed mutagenesis has two additional advanta- ges: the modifications are stoichiometric and perma- nent. The mutated forms of calpain B carrying the point-mutations T747E and S845E were expressed and purified in exactly the same way as the wild-type recombinant protein. The yield and the purity of the three calpain B variants was the same (Fig. 6A). The effect of the phosphorylation mimicking mutations on calpain B activity was first analyzed by the fluorimetric assay (Fig. 6B). Both mutations activated the protease in a wide range of free Ca 2+ concentrations. The mathematical analysis of the Ca 2+ response curves is given in Table 1. The data can be fitted well to sig- moid curves and the parameters of the curves provide an excellent tool for the characterization of the Ca 2+ dependence of the three enzyme forms. On the basis of the [Ca 2+ ] 1 ⁄ 2 and dx parameters, we conclude that the mutants are activated at lower Ca 2+ concentrations, but are less sensitive to changes in the Ca 2+ concentra- tion than the wild-type enzyme (Table 1). Although phosphorylation-mimicking mutations can enhance the Ca 2+ sensitivity of calpain B, the values of [Ca 2+ ] 1 ⁄ 2 are still far above the physiological range. Both muta- tions raise the maximal activity of calpain B, and this activity enhancement is greater at low Ca 2+ (Table 1); thus, the effect of phosphorylation could be more pro- nounced at Ca 2+ concentrations that are closer to the physiological conditions. The effects of the mutations on the autocatalysis of calpain B were tested using two independent methods. The elimination of the intact 104 kDa protein band A B C D Fig. 4. Effect of phosphorylation on calpain B activity as measured with MAP2c substrate. The digestion of MAP2c by calpain B (see Experi- mental procedures) at a 50 l M free Ca 2+ concentration is shown as an example (A). The time-course of proteolysis with calpain B that had been either phosphorylated by PKA (0.21 mol PÆmol protein )1 ), or not phosphorylated (control) was monitored by SDS ⁄ PAGE. The arrow points towards the calpain B bands. The densities of the intact MAP2c bands (indicated by an arrowhead) were estimated by densitometry and the apparent first-order rate constants of MAP2c digestion were determined for both the phosphorylated ( ) and nonphosphorylated (h) protease (B). The percentage increase in the reaction rate upon phosphorylation by PKA, ERK1 and ERK2 was calculated as in Fig. 3. The proteolytic reactions were carried out in the presence of a 350 l M free Ca 2+ concentration, and the average first-order rate constant was 2.2 · 10 )3 Æs )1 for the nonphosphorylated protease. The average stoichiometry of phosphorylation was the same as that in Fig. 3. The mean ± SD of seven to eleven independent experiments is shown (C). The effect of PKA-mediated phosphorylation on calpain B activity was also measured as a function of Ca 2+ concentration (D). The free Ca 2+ concentration is given in molÆdm )3 , and the mean ± SD for three to five experiments is shown. The stoichiometry of calpain B phosphorylation was 0.19 ± 0.03 mol PÆmol protein )1 in this experiment. L. Kova ´ cs et al. Phosphorylation of calpain B FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4965 was determined by SDS ⁄ PAGE and densitometry (Fig. 6C) as described above (Figs 5A,B). The data obtained by this approach are in agreement with the k act values calculated from the fluorimetric progress curves. Both approaches resulted in comparable data indicating that the T747E mutation causes a larger (and the S845E mutation a small but reproducible) increase in the activation of calpain B (Fig. 6D). It is readily apparent that the Glu mutations and the incorporation of a phosphate into the Thr or Ser side- chains are not fully equivalent modification; neverthe- less, the mutations tested here confirm our previous results obtained with the phosphorylated proteins, at least in qualitative terms. In vivo phosphorylation of calpain B To determine the possible physiological significance of our in vitro findings, we investigated whether calpain B was phosphorylated in the S2 Drosophila cell-line. First, we isolated the protein by immunoprecipitation and SDS⁄ PAGE from the untreated cells (Fig. 7A) and analyzed the putative phosphorylation sites by MS. None of the phosphopeptides presented in Fig. 2 D were detected; thus, we concluded that calpain B was not phosphorylated in resting cells. The same result was obtained when the cells were treated with the phosphatase inhibitor calyculin A. Next, we inves- tigated whether calpain B become phosphorylated upon the stimulation of the cells. When epidermal growth factor (EGF) was used to activate the MAP kinase ⁄ ERK pathway and the dephosphorylation of proteins was prevented by calyculin A, we noted that, on SDS ⁄ PAGE, the calpain B band was split into a doublet of two closely migrating bands, suggesting a postsynthetic modification of the protein (Fig. 7A). MS revealed that two residues (Thr747 and Ser240) were phosphorylated in the EGF-treated cells. The ion chromatograms corresponding to the m ⁄ z 690 and 765 values are shown in Fig. 7B. The identity of the two phosphopeptides was confirmed by MS ⁄ MS experi- ments, which gave mass spectra very similar to those shown in Figs 2A,C. The phosphopeptide peaks were missing from the ion chromatograms of the untreated sample, despite the fact that, in the tryptic digest, many unphosphorylated calpain B peptides were detected with higher relative abundance than in the +EGF sample. Thus, we demonstrated that the main ERK1 ⁄ ERK2 site, Thr747, was phosphorylated in vivo upon EGF stimulation. No additional ERK sites expected from the motif scan prediction and from the in vitro phosphorylation experiment (Fig. 5D) were verified in vivo. Surprisingly, Ser240, a putative PKA A B C D Fig. 5. Effect of phosphorylation on the autolysis of calpain B. The autolysis of recombinant calpain B without phosphorylation (h)or after treatment with ERK2 (m) was tested at a 19 m M free Ca 2+ concentration. The process was monitored by SDS ⁄ PAGE (A) and the rate constants for the disappearance of the 104 kDa band were determined (B). The increase of autolysis rate caused by phosphor- ylation with ERK2 and PKA is shown in (C) as the mean ± SD of three to four independent experiments. The average first-order rate constant of autolysis was 0.028 s )1 for the nonphosphorylated cal- pain B. The stoichiometry of phosphorylation was 0.77 and 0.13 mol PÆmol protein )1 for ERK2 and PKA, respectively. (D) After phosphorylation of the recombinant protein with [ 32 P]ATP[cP], the autolysis of radioactive calpain B was analyzed by SDS ⁄ PAGE and Coomassie staining (upper panel) followed by autoradiography (lower panel). The arrows point towards the intact calpain B bands. Phosphorylation of calpain B L. Kova ´ cs et al. 4966 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS site, was also phosphorylated in the same experiment, despite the fact that the treatment of S2 cell with fors- kolin and calyculin A was not sufficient to induce in vivo calpain B phosphorylation (data not shown). Obviously, the activation of the MAP kinase signaling was more effective than the stimulation of the PKA pathway alone. A positive interaction between the two signaling pathways cannot be excluded, but it is not clear why the other, more potent in vitro PKA site (i.e. Ser845) remained unaffected. One likely explanation is that Ser240 was phosphorylated not by PKA, but by another kinase in the living cells. In this respect, it is important to note that the stoichiometry of in vitro PKA phosphorylation has always been rather low and, according to the results of the motif scan, the peptide sequence 233 ATSARQNSVSKGDFQ 247 , containing Ser240, is a preferred target of the calcium ⁄ calmodu- lin-dependent protein kinase II (CaMKII kinase; EC 2.7.11.17) as well. Thus, it is possible that, after EGF treatment, the EGF induced Ca 2+ influx activated the latter kinase [14], which in turn incorporated the phos- phate into Ser240 in a cAMP-independent manner. To make things even more complicated, a motif scan test indicates that CaMKII can also phosphorylate the PKA site Ser845, but with lower efficiency. However, modification at the latter site was not observed in any Table 1. Parameters characterizing the Ca 2+ concentration dependence of enzyme activity. [Ca 2+ ] 1 ⁄ 2 denotes the Ca 2+ concentration which corresponds to half-maximal activity of the enzyme, whereas parameter dx is the width of the logistic function characterizing the depen- dence of the activity on the logarithm of the Ca 2+ concentration, lg[Ca 2+ ]. It is a dimensionless quantity and gives the sensitivity of enzyme activity to small changes in the ion concentration around the value [Ca 2+ ] 1 ⁄ 2 . Greater values of dx correspond to a lower sensitivity to changes in Ca 2+ concentration. Calpain B [Ca 2+ ] 1 ⁄ 2 (mM) A max dx Activity ratio at high [Ca 2+ ] Activity ratio at low [Ca 2+ ] Wild-type 6.4 ± 0.4 110 ± 5 0.34 ± 0.02 – – T747E 5.5 ± 0.8 150 ± 12 0.46 ± 0.03 1.44 ± 0.15 1.66 ± 0.2 S845E 5.5 ± 0.8 170 ± 10 0.44 ± 0.03 1.86 ± 0.25 2.3 ± 0.15 A B C D Fig. 6. The effects of phosphorylation mimicking mutations on calpain B. Thr747 (ERK site) and Ser845 (PKA site) were substituted with Glu in calpain B by site-directed mutagenesis. The purified mutated proteins T747E and S845E behaved as the wild-type (Wt) recombinant protein on SDS ⁄ PAGE (A). St, standards; molecular mass values are given in kDa. The Ca 2+ -dependent activity of the wild-type (h) as well as the T747E ( ) and S845E ( ) mutant calpain B was determined with the fluorescent LY-AMC substrate (B). The maximal activity of the wild-type enzyme was taken as 100%. The mean ± SD of eight independent experiments is shown. The autolysis of the native (h) and mutated ( ), ( ) calpain B was monitored by SDS ⁄ PAGE (C). The effect of the mutations on the activation of the protease was estimated from the progress curves, as in Fig. 3 (white columns), or from the SDS ⁄ PAGE patterns, as in Fig. 5 (black columns), in the presence of a 19 m M free Ca 2+ concentration (D). The mean ± SD of three to seven experiments is shown. L. Kova ´ cs et al. Phosphorylation of calpain B FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS 4967 of our in vivo experiments. Taking these arguments together, the phosphorylation of Ser240 can take place in living cells and may contribute to the regulation of the enzyme, although, most probably, this reaction is not catalyzed by PKA. Additional experiments are required for the clarification of the role for this site. On the other hand, it is clear that Thr747 can be phos- phorylated in vitro and in vivo by the ERK enzymes. The extracellular signal regulated modification of cal- pain B, as identified in the present study, represents a physiologically relevant regulatory tool. Conclusions The identification of the in vitro phosphorylation sites comprised the first step towards a better understanding of the regulation of calpain B. According to homolo- gous modeling, the amino acid residues phosphory- lated by PKA and the ERK isoforms are situated in sensitive regions of the molecule and can be involved, either directly or indirectly, in the regulation of enzyme activity. We demonstrated that the postsynthetic modi- fication of these sites increases the rate of autocatalytic activation, as well as the activity and Ca 2+ sensitivity of recombinant calpain B. Although the changes were moderate, they could contribute to the modulation of regulatory networks in a more significant way. Struc- tural predictions and in vitro experiments with recom- binant proteins reveal biochemically feasible mechanisms that are not necessarily operating in a living organism. We found that the phosphorylation of Thr747 in the transducer region of the protease occurs in EGF-stimulated S2 cells. The results obtained in the present study suggest that the activation of the MAP kinase ⁄ ERK pathway by extracellular signals, among many diverse changes, results in the phosphorylation and activation of calpain B in D. melanogaster.We suggest that the regulation of calpain B in fruit flies must be different from that in mammalian counter- parts. Although calpains catalyze the same proteolytic reaction, the position and function of the critical phos- phorylation sites have not been conserved. Indeed, instead of the Thr747 residue that is phosphorylated in Drosophila calpain B, Asp524 or Glu524 are found in the corresponding position in different mammalian m-calpain enzymes [1], suggesting that a natural drift in the protein sequence mimics the effect of phosphor- ylation in mammals. This example confirms that evolu- tion operates at the level of regulation. Without understanding the small structural alterations affecting the regulatory potential of a protein, it would be pre- mature to suggest functional equivalence based on overall structural similarities. A B Fig. 7. Phosphorylation of calpain B in vivo.S2Drosophila cells were incubated in the presence of 10 n M EGF and 100 nM calyculin A (+EGF) or in the absence of these additions (–EGF). Calpain B was partially purified from the treated and the untreated cells and was analyzed by SDS ⁄ PAGE (A). Two hundred nanograms of recombinant calpain B was used as a control (C). The mass of the standards (St) is given in kDa on the left-hand side. The excessive band at around 52 kDa indicates the presence of immunoglobulins. Arrows indicate a single band (–EGF) or a doublet of bands (+EGF) that corresponds to the molecular mass of calpain B. These bands were excised, digested with trypsin and analyzed by MS. The extracted ion chromatograms of calpain B phosphopeptides after TiO 2 enrichment are shown in (B). m ⁄ z 690 corresponds to the IAPSLPPPT(p)PKEEDDPQR peptide, whereas m ⁄ z 765 represents the qNS(p)VSKGDFQSLR peptide. The retention time (RT) is given (min) above the peaks. Phosphorylation of calpain B L. Kova ´ cs et al. 4968 FEBS Journal 276 (2009) 4959–4972 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... presence of < /b> a 9 mm free Ca2+ concentration, in a final volume of < /b> 50 lL The reaction was initiated by the addition of < /b> CaCl2 Aliquots were withdrawn from the mixture at regular time intervals and were investigated by SDS ⁄ PAGE and densitometry of < /b> the 104 kDa intact calpain < /b> B band, as described above In a few experiments, the autolysis of < /b> radioactive [32P]-labeled calpain < /b> B was also investigated by SDS... Activation of < /b> m -calpain < /b> (calpain < /b> II) by epidermal growth factor is limited by protein kinase A phosphorylation of < /b> m -calpain < /b> Mol Cell Biol 22, 2716–2727 7 Xu L & Deng X (2006) Protein kinase Ci promotes nicotine-induced migration and invasion of < /b> cancer cells via phosphorylation of < /b> l- and m -calpain < /b> J Biol Chem 281, 4457–4466 8 Jekely G & Friedrich P (1999) Characterization of < /b> two recombinant Drosophila calpains... Coomassie brilliant blue R250, the intensity of < /b> residual intact MAP2c was determined by densitometry using a BioRad Fluor-SÔ densitometer (Bio-Rad, Hercules, CA, USA) and multi-analyst software, version 1.1 Autolysis assay The autocatalytic cleavage of < /b> calpain < /b> B was monitored in a similar setup but in the absence of < /b> MAP2c Twenty-five micrograms of < /b> calpain < /b> B was subjected to self-proteolysis at 37 °C in a buffer... procedure [19] Briefly, to avoid nonspecific binding, the supernatant was pre-cleared with 100 lL of < /b> Protein A Sepharose (Sigma) with gentle rotation of < /b> the mixture for 3–4 h at 4 °C followed by centrifugation at 15 800 g for 10 min Meanwhile, 50 lL of < /b> Protein A Sepharose was incubated with 40 lg of < /b> affinity-purified calpain < /b> B antibody [12] in ‘lysis buffer’ for 3–4 h at 4 °C and the calpain < /b> B antibody-coupled... at 15 800 g for 10 min, the sample was analyzed by SDS ⁄ PAGE [18] and western blotting In the latter experiment, anti -calpain < /b> B sera [12] was used together with an anti-rabbit secondary sera (Sigma) for the unambiguous identification of < /b> the calpain < /b> B band Database search MS ⁄ MS data were searched against the NCBI 20060603 and SwissProt 51.5 protein databases using the mascot (version 2.1, http://www.matrixscience.com)... (2003) The calpain < /b> system Physiol Rev 83, 731–801 2 Friedrich P, Tompa P & Farkas A (2004) The calpainsystem of < /b> Drosophila melanogaster: coming of < /b> age Bioessays 26, 1088–1096 ´ 3 Friedrich P & Bozoky Z (2005) Digestive versus regulatory proteases: on calpain < /b> action in vivo Biol Chem 386, 609–612 4 Shao H, Chou J, Baty CJ, Burke NA, Watkins SC, Stolz DB & Wells A (2006) Spatial localization of < /b> m -calpain.< /b> .. the plasma membrane by phosphoinositide biphosphate binding during epidermal growth factor receptor-mediated activation Mol Cell Biol 26, 5481–5496 5 Glading A, Bodnar RJ, Reynolds IJ, Shihara H, Satish L, Potter DA, Blair HC & Wells A (2004) Epidermal growth factor activates m -calpain < /b> (calpain < /b> II), at least in part by extracellular signal-regulated kinase-mediated phosphorylation Mol Cell Biol 24, 2499–2512... Sepharose was collected by centrifugation at 15 800 g for 5 min at 4 °C The pre-cleared supernatant was mixed with the calpain < /b> B antibody-coupled Protein A Sepharose and was incubated overnight at 4 °C with gentle rotation The resin was washed three times with 200 lL of < /b> ‘lysis buffer’ and then the immune complex was released by boiling in 200 lL of < /b> SDS sample buffer After clearing by centrifugation at... homolog, CALPB J Biol Chem 274, 23893–23900 9 Reverter D, Sorimachi H & Bode W (2001) The structure of < /b> calcium-free human m calpain < /b> Implications for calcium activation and function Trends Cardiovasc Med 11, 222–229 4972 ´ 10 Alexa A, Bozoky Z, Farkas A, Tompa P & Friedrich P (2004) Contribution of < /b> distinct structural elements to activation of < /b> calpain < /b> by Ca2+ ions J Biol Chem 279, 20118–20126 ´ 11 Bozoky... microgram quantities of < /b> protein utilizing the principle of < /b> protein-dye binding Anal Biochem 72, 248–254 17 Witt JJ & Roskoski R Jr (1975) Rapid protein kinase assay using phosphocellulose-paper absorption Anal Biochem 66, 253–258 18 Laemmli UK (1970) Cleavage of < /b> structural proteins during the assembly of < /b> the head of < /b> bacteriophage T4 Nature 227, 680–685 ´ ´ ´ ´ ´ 19 Kiss A, Lontay B, Becsi B, Markasz L, Olah . phosphorylates (MI:0217) CalpainB (uni- protkb: Q9VT65)byprotein kinase assay (MI:0424) l MINT-7214319: CalpainB (uniprotkb:Q9VT65) and CalpainB (uniprotkb:Q9VT65) cleave ( MI:0194)byprotease. calpains are missing from Drosophila calpain B. From the first part of our studies, we conclude that calpain B can be phosphorylated by three kinases at