Biochem. J. (2010) 425, 401–412 (Printed in Great Britain) doi:10.1042/BJ20090902 401

Lyn-mediated mitochondrial tyrosine phosphorylation is required topreserve mitochondrial integrity in early liver regenerationEnrico GRINGERI*1, Amedeo CARRARO†1, Elena TIBALDI‡, Francesco E. D’AMICO*, Mario MANCON‡, Antonio TONINELLO‡,Mario A. PAGANO‡3, Claudia VIO‡, Umberto CILLO*2 and Anna M. BRUNATI‡2

*Department of General Surgery and Organ Transplantation, Hepatobiliary and Liver Transplant Unit, University of Padova, Via Giustiniani 2, 35128 Padova, Italy, †ChirurgiaOncologica, Istituto Oncologico Veneto, Istituto di Ricovero e Cura a Carattere Scientifico, Via Gattamelata 64, 35128, Padova, Italy, and ‡Department of Biochemistry, University ofPadova, Viale G. Colombo 3, 35131 Padova, Italy

Functional alterations in mitochondria such as overproduction ofROS (reactive oxygen species) and overloading of calcium, withsubsequent change in the membrane potential, are traditionallyregarded as pro-apoptotic conditions. Although such eventsoccur in the early phases of LR (liver regeneration) after two-thirds PH (partial hepatectomy), hepatocytes do not undergoapoptosis but continue to proliferate until the mass of the liveris restored. The aim of the present study was to establish whethertyrosine phosphorylation, an emerging mechanism of regulationof mitochondrial function, participates in the response to liverinjury following PH and is involved in contrasting mitochondrialpro-apoptotic signalling. Mitochondrial tyrosine phosphorylation,negligible in the quiescent liver, was detected in the early phases

of LR with a trend similar to the events heralding mitochondrialapoptosis and was attributed to the tyrosine kinase Lyn, a memberof the Src family. Lyn was shown to accumulate in an activeform in the mitochondrial intermembrane space, where it wasfound to be associated with a multiprotein complex. Our resultshighlight a role for tyrosine phosphorylation in accompanying,and ultimately counteracting, mitochondrial events otherwiseleading to apoptosis, hence conveying information required topreserve the mitochondrial integrity during LR.

Key words: apoptosis, calcium overload, liver regeneration, Lynkinase, mitochondrial membrane potential, oxidative stress.

INTRODUCTION

Mitochondria are recognized as having a crucial role in severalcellular functions. In addition to serving as the ‘powerhouse’ ofthe cell, to supply the majority of cellular ATP, mitochondriadirectly participate in cell metabolism, Ca2+ homoeostasis, cellproliferation and death. The regulation of these processes requiresintegration of signals that converge on to the mitochondria. In thisrespect, signalling cascades generated at the cell membrane byhormones, growth factors and cytokines target mitochondria andmodulate their activity, although the molecular mechanisms arenot fully understood [1–3]. Nevertheless, compelling evidencehas been provided that reversible protein phosphorylation, themost common post-translational modification, is one of the keyregulatory mechanisms affecting most of, if not all, mitochondrialprocesses, including electron transport, the tricarboxylic acidcycle, the fatty acid β-oxidation cycle, the urea cycle, permeabilitytransition and metabolite transport [4–9]. So far, a number ofprotein kinases, as well as protein phosphatases, have been foundto be localized in all mitochondrial compartments, with mostof these also exerting a role outside mitochondria {e.g. PKA(protein kinase A), PKB (protein kinase B)/Akt and PKC (proteinkinase C), MAPKs (mitogen-activated protein kinases), GSK-3β(glycogen synthase kinase-3β), SFKs (Src family kinases) andEGFR (epidermal growth factor receptor) [10–14]}, which

suggests a mechanism of translocation into mitochondria dueto appropriate signals. Recently, we demonstrated that stimulitriggering cell proliferation brought about an elevation oftyrosine phosphorylation inside mitochondria [15]. Such post-translational modification was shown to depend on the presenceof activated SFKs, a subgroup of non-receptor tyrosine kinases,traditionally considered as ‘switch’ molecules, localized beneaththe plasma membrane and capable of coupling receptor signals todownstream signalling pathways [15].

In order to further investigate the role of mitochondrial tyrosinephosphorylation during cell proliferation, we employed LR (liverregeneration) after two-thirds PH (partial hepatectomy) whichis considered as an excellent in vivo model for studying cell-cycle progression and cell proliferation [16]. In response to thissurgical procedure, the remnant hepatocytes synchronously re-enter the cell cycle to undergo one to two rounds of replicationbefore returning to the quiescent state, eventually restoring themass and ensuring maintenance of the multiple functions of theliver [17,18]. The entry of quiescent hepatocytes into the cellcycle, corresponding to the G0-G1 transition, is largely regulatedby cytokines, such as TNFα (tumour necrosis factor α) and IL(interleukin)-6, leading to the activation of transcription factorsincluding AP-1 (activator protein-1), STAT3 (signal transducerand activator of transcription-3) and NF-κB (nuclear factorκB), followed by the transcription of genes encoding cell-cycle

Abbreviations used: AIF, apoptosis-inducing factor; ANT, adenine nucleotide translocase; BCR, B-cell receptor; Cdc2, cell division cycle 2 kinase; COX,cytochrome c oxidase; ECL, enhanced chemiluminescence; EGFR, epidermal growth factor receptor; ERK, extracellular-signal-regulated kinase; HSP90,heat-shock protein 90; LDH, lactate dehydrogenase; LR, liver regeneration; MAPK, mitogen-activated protein kinase; PARP, poly(ADP-ribose) polymerase;PTP-I, protein tyrosine phosphastase inhibitor; PH, partial hepatectomy; PKC, protein kinase C; PMCA, plasma membrane Ca2+ ATPase; PPII, polyprolinetype II helical; ROS, reactive oxygen species; RT, reverse transcriptase, SFK, Src family kinase; SH2, Src homology 2; SH3, Src homology 3; STAT3, signaltransducer and activator of transcription-3; TPP+, tetraphenylphosphonium ion; YA, activation-loop tyrosine residue (Tyr396 on Lyn); YT, C-terminal tyrosineresidue (Tyr508 on Lyn).

1 These authors contributed equally to this work.2 These authors are joint senior authors.3 To whom correspondence should be addressed (email mario.pagano@unipd.it)

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regulators such as cyclin D [18]. This phase, which is reversible,primes hepatocytes to respond to growth factors, such as HGF(hepatocyte growth factor) and TGFα (transforming growthfactor α), in turn triggering signalling pathways that accountfor the up-regulation of a number of factors essential for theG1-S progression including cyclin E and cyclin A and theirrespective cyclin-dependent kinases [18]. During this series ofevents, referred to as the prereplicative phase, mitochondrialenergy metabolism is impaired with an overproduction ofROS (reactive oxygen species) and subsequent increases inthe mitochondrial GSSG/GSH ratio as well as a decrease inthe respiratory control index and in the rate of the oxidativephosphorylation [19–21]. Furthermore, accumulation of Ca2+

in mitochondria is concomitant with oxidative stress and bothof these phenomena are described as promoting mitochondrialpermeability transition in isolated mitochondria [21,22]. Despitethese biochemical events, usually regarded as pro-apoptotic,neither release of cytochrome c nor appearance of apoptotic nucleioccur, confirming that hepatocytes do not undergo apoptosisduring the prereplicative phase of LR and suggesting thatmitochondria receive intracellular signals aimed at maintainingtheir structural and functional integrity and ultimately sustainingcell proliferation.

The aim of the present study was to assess whether thesignalling pathways activated by cytokines and growth factorsduring the early phases of LR also convey signals thatmodulate mitochondrial functions by the involvement of tyrosinephosphorylation. We demonstrate that mitochondrial tyro-sine phosphorylation, negligible in the quiescent liver, increasedand displayed a trend similar to that of oxidative stress, Ca2+

overloading and transmembrane hyperpolarization in the earlyphases of LR. Moreover, a key player in the elevation ofmitochondrial tyrosine phosphorylation was shown to be thetyrosine kinase Lyn (v-yes-1 Yamaguchi sarcoma viral-relatedoncogene homologue), a member of the SFK family, whichaccumulates in an active form in mitochondria and contributesto preserve mitochondrial integrity from the damage caused byliver injury thus preventing hepatocytes from entering apoptosis.

EXPERIMENTAL

Materials

The RNeasy mini-kit was from Qiagen. Superscript II RT (reversetranscriptase) was from Invitrogen. The polymer poly(Glu-Tyr)4:1,OptiPrep and phosphatase inhibitor cocktail 1 and 2 were fromSigma–Aldrich. [γ -33P]ATP was obtained from PerkinElmer.Protease inhibitor cocktail was from Roche.

The polyclonal antibodies against phosphorylated YA

(activation-loop tyrosine residue; Tyr396 in Lyn and Tyr416 inSrc), phospho-ERK (extracellular-signal-regulated kinase), ERKand PARP [poly(ADP-ribose) polymerase] were from CellSignaling Technology. The anti-Lyn, anti-(cytochrome c), anti-(cyclin D1) and anti-(cyclin E) antibodies were from SantaCruz Biotechnology. The monoclonal anti-(phosphotyrosine)antibody was from BioSource International (clone PY-20). Anti-(phospho-STAT3) and anti-STAT3 antibodies were from UpstateBiotechnology. The anti-(β-actin) antibody was from Sigma–Aldrich (clone AC-15). The anti-aconitase, anti-AIF (apoptosis-inducing factor) and anti-LDH (lactate dehydrogenase) antibodieswere from Abcam. The anti-GM130 antibody (to label the Golgiapparatus) was from BD Transduction Laboratories, anti-calnexinantibody (to label the endoplasmic reticulum) was from Stressgenand the anti-PMCA (plasma membrane Ca2+ ATPase) antibody (tolabel the plasma membrane) was from Santa Cruz Biotechnology.

The protein kinase inhibitors and analogues PP2, PP3, SU6656,piceatannol, AG490, AG1296, AG1478 and genistein werepurchased from Calbiochem. SU11274 was obtained from Sugen.The ECL (enhanced chemiluminescence) detection system wasfrom GE Healthcare. The glutathione assay kit (for assessingGSH/GSSG ratios) was obtained from BioVision.

Partial hepatectomy

Three-month-old male Winstar rats (250 g each) were purchasedfrom Charles River Laboratories and were housed in the animalresearch facility of the Department of Biological ChemistryUniversity of Padova, Italy. They were maintained under a12 h light/12 h dark cycle and given rat chow and water. Allanimal studies were performed under animal care and usecommittee protocols approved by the State Commission onAnimal Experimentation. Rats were subjected to 70% PH [16]under isoflurane anaesthesia and allowed to recover for variousperiods. Sham-operated rats, obtained by making small mid-lineabdominal incision without the excision of the liver, were usedas a control and reported as time point 0 h in the experimentalprotocols. Sham surgeries were performed with externalized liverswhich were gently palpated to mimic the surgical stress of the PHprocedure. One set of rats was alternatively treated with increasingconcentrations of PP2, a potent and selective inhibitor for SFKs,and PP3, an inactive analogue of PP2. Such molecules weredissolved in DMSO and diluted further in normal saline (to give afinal DMSO concentration of 0.5%). PP2, PP3 or 0.5% DMSO insaline as vehicle control were injected intraperitoneally 4 h before,immediately after and 12 h following PH as described in [23]. Ratswere killed at 0, 12, 24, 36 and 48 h post-operatively; the liverswere removed, weighed and processed for distinct experimentalpurposes at the time points indicated in the text. All operationswere performed under sterile conditions.

Preparation of mitochondria

Rat liver was homogenized in isolation medium (5 mM Hepes,pH 7.4, containing 250 mM sucrose and 0.5 mM EGTA) andsubjected to centrifugation at 900 g for 5 min. The supernatantwas centrifuged at 12000 g for 10 min to precipitate crudemitochondrial pellets. The pellets were resuspended in isolationmedium containing 1 mM ATP and layered on top of adiscontinuous gradient of Ficoll diluted in isolation medium,composed of 2-ml layers of 16, 14 and 12% (w/v) Ficolland a 3-ml layer of 7 % (w/v) Ficoll. After centrifugation for30 min at 75000 g, mitochondrial pellets were suspended inisolation medium and centrifuged again for 10 min at 12000 g.The resulting pellets were suspended in isolation mediumwithout EGTA and their protein content was measured by thebiuret method, with BSA as a standard. The absence of othercontaminating subcellular compartments in our mitochondrialpreparations has been demonstrated in previous studies [14].

Subcellular fractionation

Rat liver (250 mg) was homogenized in 1 ml of fractionationmedium (5 mM Hepes, pH 7.4, 250 mM sucrose and thephosphatase inhibitor cocktail 1 and 2) by 20 strokes witha Polytron tissue homogenizer and subjected to centrifugationfor 10 min at 900 g (nuclei fraction, pellet I). The supernatantwas then subjected to ultracentrifugation for 1 h, performedat 45000 rev./min in a Beckman MLA-130 rotor, to separatecytosol from the post-nuclear particulate fraction (pellet II). The

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Lyn moves to mitochondria in early liver regeneration 403

particulate fraction was resuspended in 200 μl of the fractionationbuffer, overlaid on to a discontinuous gradient composed of30, 25, 20, 15 and 10% OptiprepTM (Accurate Chemical) anddissolved in a medium containing 50 mM Tris/HCl, pH 7.5,and the protease and phosphatase inhibitor cocktails. Afterdensity-gradient centrifugation, performed at 31000 rev./min ina Beckman SW60 Ti for 3 h at 4 ◦C, the gradient was divided into15 200-μl-aliquots, collected from the top.

Immunoprecipitation

Liver tissue and mitochondria were homogenized in a Douncehomogenizer (20 strokes) in buffer A (20 mM Tris/HCl, pH 7.4,containing 1 mM EDTA, phosphatase inhibitor cocktail 1 and2, and the protease inhibitor cocktails), 1% (v/v) Triton X-100was added and the lysate was incubated for 1 h at 4 ◦C. Aftercentrifugation at 20000 g for 30 min at 4 ◦C, supernatants wereincubated overnight in the presence of anti-Lyn antibody andthereafter with protein A–Sepharose beads for 2 h at 4 ◦C. Theprotein–bead complex was centrifuged, washed and solubilizedas detailed below.

Proteinase K treatment

Purified mitochondria were treated with 50 ng/ml proteinase K inisolation medium without EGTA (see the section on preparationof mitochondria) in the absence or presence of 0.5% Triton X-100 at room temperature (25 ◦C) for 30 min. The reaction wasstopped by the addition of protease inhibitor cocktail and wasthen analysed by Western blotting with anti-Lyn, anti-aconitaseand anti-AIF antibodies.

Mitochondrial subfractionation

To separate the mitochondrial membranes from the solublefractions, 5 mg of mitochondria, suspended in 5 mM Hepes,pH 7.4, containing 250 mM sucrose and 0.5 mM EGTA, weresonicated in an MSE Sonicator and subjected to eight freeze–thaw cycles. Mitochondrial suspensions were then subjected toultracentrifugation performed at 45000 rev./min for 30 min at4 ◦C in a Beckman MLA-130 rotor to obtain membrane pelletsand supernatant fractions.

Digitonin treatment

Purified mitochondria (1 mg/ml) were incubated with increasingconcentrations of digitonin (from 0.1 to 0.6 mg/ml) for 30 minat 4 ◦C, after which the samples were centrifuged at 22800 g for20 min. Supernatant (S) and pellet (P) fractions were subjectedto SDS/PAGE (10 % gels) and Western blot analysis with theappropriate antibody.

Fractionation by centrifugation on glycerol gradient

Soluble protein (150 μg) from rat liver at 24 h of regenerationwere loaded on to a 3.9 ml linear glycerol gradient (10–40%) in 25 mM Hepes, pH 7.4, containing 1 mM EDTA. Thesamples were subjected to density-gradient centrifugation for18 h at 31000 rev./min in a Beckman SW60Ti rotor at 4 ◦C,and divided into 18 equal aliquots of 200 μl each collected fromthe top. Thyroglobulin (669 kDa), apoferritin (443kDa), alcoholdehydrogenase (150 kDa) and glutamate dehydrogenase (62 kDa)were used as standards for estimating the molecular mass of theprotein complexes.

Western blot analysis

Samples from total homogenates (rat liver, brain and spleen),different cell fractions (rat liver) and immunoprecipitates, wererapidly solubilized in 62 mM Tris/HCl buffer, pH 6.8, containing5% (v/v) glycerol, 0.5% 2-mercaptoethanol and 0.5% SDS, andsubjected to SDS/PAGE (10% gels) before being transferred on tonitrocellulose membranes by electroblotting. After treatment with3% (w/v) BSA at 4 ◦C overnight, membranes were incubated withthe primary antibodies for 2 h and, after washing, with secondaryhorseradish peroxidase-conjugated polyclonal antibody for 1 h.Immunoblots were developed using the ECL detection system,captured using a Kodak Image Station 2000R and visualized usingthe Kodak 1D Image software. Loading controls were performedby reprobing membranes with appropriate primary antibodiesafter stripping twice in 0.1 M glycine, pH 2.5, 0.5 M NaCl, 0.1%Tween 20, 1% 2-mercaptoethanol and 0.1% NaN3, for 10 mineach.

Determination of mitochondrial calcium content

Purified mitochondria were solubilized to give a concentration of1 mg/ml in a solution comprising 1 mM EDTA, 0.1 % NaCl and1% sodium deoxycholate. The calcium content in the resultingsolution was estimated by atomic absorption using a PerkinElmerAnalyst 100 spectrophotometer.

Measurements of GSH and GSSG

GSH and GSSG were measured in isolated mitochondria usingthe Glutathione Assay Kit (GSH, GSSG and Total).

Determination of mitochondrial membrane potential (��m)

Mitochondrial membrane potential (��m) was estimated from thedistribution of the TPP+ (tetraphenylphosphonium ion) measuredacross the mitochondrial membrane with a selective electrodeprepared in our laboratory, as described previously [14].

Phosphorylation assays

Tyrosine phosphorylation was assayed on samples from totalhomogenate, mitochondrial lysates and OptiprepTM fractions byincubating 15 μg of liver homogenate or liver mitochondriaat 30 ◦C for 10 min in 30 μl of reaction medium {50 mMTris/HCl, pH 7.5, containing 10 mM MnCl2 and 20 μM[γ -33P]ATP (3 × 106 c.p.m. per nmol)} with either 1 mg/ml ofthe random poly(Glu-Tyr)4:1 polymer or 200 μM Cdc2 (celldivision cycle 2 kinase) peptide substrate (a gift from ProfessorOriano Marin, University of Padova, Italy) as the exogenoussubstrates, and in the absence and presence of specific tyrosinekinase inhibitors (2 μM SU11274, 20 μM AG1478, 20 μMAG1296, 20 μM AG490, 7.5 μM PP2, 7.5 μM SU6656, 15 μMpiceatannol or 20 μM genistein). After incubation, samples wereanalysed by SDS/PAGE (10 % gels) and revealed using a PackardCyclone PhosphorImager.

RT–PCR analysis of Lyn, Src and β-actin transcripts

Total RNA from regenerating rat livers at different time-points after PH were isolated using the Qiagen column kit,then 2 μg of RNA from each sample was reverse-transcribedand the cDNA samples were divided and amplified usingspecific primers (20 pmol/tube). Primers were as follows: sense

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5′-GGCTGAAGCCTCTGTCATGACGCA-3′ and antisense 5′-GCGTCTACACTACAGGCGTGACAG-3′ for Lyn; sense 5′- GG-CTGAAGCCTCTGTCATGACGCA-3′ and antisense 5′-GTCG-AGGACTTCGGAACTCTCTC-3′ for Src; sense 5′-GTGGG-GCGCCCCAGGCACCA-3′ and antisense 5′-GAAATC GT-GCGTGACATTAAGGAG-3′ for β-actin. The reaction mixtureconsisted of 1.5 mM MgCl2, 500 mM KCl, 200 μM dNTP mix

and 2.5 units of Taq polymerase. Reaction conditions were 30 sof denaturation at 94 ◦C, 30 s of annealing at 60 ◦C and 40 s ofextension at 72 ◦C for 30 cycles for Src and Lyn and 25 cyclesfor β-actin, followed by a final extension of 7 min at 72 ◦C.Aliquots of PCR products were separated by electrophoresison 2% (w/v) agarose gels and visualized by ethidium bromidestaining.

Figure 1 Protein tyrosine phosphorylation increases in isolated mitochondria from rat regenerating liver after PH

(A) Mitochondrial lysates from rat livers after PH at different time points were assayed by Western blot analysis with anti-phosphotyrosine antibody (pTyr). The membranes were reprobed withanti-aconitase antibody as a loading control. The positions of molecular-mass-markers in kDa are indicated on the left. (B) Total cell lysates from rat livers after PH at different time points were assayedby Western blot analysis with anti-(cyclin D1), anti-(cyclin E1), anti-(phospho-STAT3) (P-STAT 3) and anti-(phospho-ERK1/2) (P-ERK1/2) antibodies. The resulting bands underwent densitometricanalysis (arbitrary units). The membranes were reprobed with anti-β-actin, anti-STAT3 and anti-ERK1/2 antibodies as loading controls. (C) Mitochondrial lysates from rat livers after PH at differenttime points were tested for glutathione redox status (GSSG/GSH, top panel), Ca2+ content (middle panel) and membrane potential (��m, bottom panel) by monitoring the distribution of TPP+

across the mitochondrial membrane, as described in the Experimental section. (D) Mitochondrial lysates (upper panel) and cytosol (lower panel) from rat livers after PH at different time points wereassayed by Western blot analysis with the anti-(cytochrome c) antibody. The resulting bands underwent densitometric analysis (arbitrary units). The membranes were reprobed with anti-aconitase andanti-β-actin antibodies as loading controls. (E) Total cell lysates from rat livers after PH at different time points were assayed by Western blot analysis with the anti-PARP antibody. The membraneswere reprobed with anti-β-actin antibody as a loading control. All results are shown as means +− S.D. and are representative of 3–6 rats per time point after PH. Wb, Western blot.

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Lyn moves to mitochondria in early liver regeneration 405

Figure 2 Effect of inhibitors on tyrosine kinase activities of total cell andmitochondrial lysates 24 h after PH

Proteins from (A) total cell and (B) mitochondrial lysates (100 μg) were tested for tyrosinekinase (PTK) activity in the absence (Ctrl) and presence of SU11274 (2 μM), AG1478 (20 μM),AG1296 (20 μM), AG490 (20 μM), PP2 (7.5 μM), SU6656 (7.5 μM), piceatannol (15 μM)or genistein (20 μM) respectively by using the non-specific random polymer poly(Glu-Tyr)4:1

as a substrate. Results are representative of three experiments performed in triplicate and arepresented as means +− S.D.; *P< 0.05; and **P< 0.01 compared with the control experiment.

Statistical analysis

The bands on Western blots were quantified by densitometricanalysis. Results are presented as means +− S.D. and comparedusing one-way ANOVA followed by the Bonferroni correctionspost-test. A P-value of <0.05 was considered as statisticallysignificant. All statistics were performed using GraphPad Prism(version 4) statistical software.

RESULTS

Protein tyrosine phosphorylation increases in isolatedmitochondria from regenerating rat liver after PH

To establish whether mitochondrial tyrosine phosphorylation wasimplicated in the transient alterations of mitochondrial functionusually observed during the early phase of LR after PH [20–23],we purified mitochondria from the liver of sham-operated andpartially hepatectomized rats as described previously [14]. Asshown in Figure 1(A), mitochondrial tyrosine phosphorylationincreased in proteins with a wide range of molecular massesin a time-dependent manner. The maximal level of tyrosinephosphorylation was observed between 24 and 36 h after PH,subsequently declining to the basal value by 48 h. The timeinterval at which this mitochondrial tyrosine phosphorylation wasmaximal reflected progression from the G1-phase into the S-phaseof the cell cycle, as assessed by monitoring the expression of bothcyclin D1, whose up-regulation indicates a G0-G1 transition, andof cyclin E, which is involved in the G1-S transition, in the totalcell lysate (Figure 1B) [24,25]. We similarly detected phospho-STAT3 and phospho-ERK 1/2 (Figure 1B), which are markers thatcharacterize the cell cycle re-entry resulting from the cytokine-and growth-factor-mediated response to liver injury [26–28].

Figure 3 Lyn is the predominant SFK expressed in the liver

(A) Protein from total cell lysate (50 μg) from rat spleen, brain and liver were assayed byWestern blot analysis with antibodies against each the indicated SFKs. The resulting bandswere subjected to densitometric analysis (arbitrary units). Results are representative of threeexperiments performed in triplicate and are presented as means +− S.D. Wb, Western blot.(B) The level of mRNAs coding for Src and Lyn from rat liver after PH at different timepoints was analysed by RT–PCR. The level of mRNA for β-actin was measured as an internalcontrol.

Within the same time frame, we measured a few parameters ofmitochondrial function which are affected during LR, namely theglutathione redox status (the GSSG/GSH ratio), the concentrationof Ca2+ and the mitochondrial transmembrane potential (��m)[20–23]. In accordance with previous reports, GSSG/GSH, anindex of mitochondrial oxidative stress [29] and the concentrationof Ca2+, a key regulator of multiple mitochondrial functions actingat several levels [30], were increased approx. 5-fold and 2-foldrespectively at 24 h after PH, thereafter gradually decreasing, butnot totally returning to the starting level by 48 h (Figure 1C,top and middle panels). The maximal hyperpolarization of ��m

occurred after 24 h and subsequently declined to the basal value(Figure 1C, bottom panel). We also observed that cytochromec was not released into the cytosol (Figure 1D) and that PARPcleavage did not occur (Figure 1E), thus underscoring the absenceof any apoptotic event [21,22].

SFKs are major agents in mitochondrial tyrosine phosphorylation

We next tried to identify the tyrosine protein kinase(s) responsiblefor the tyrosine phosphorylation detected in mitochondria duringLR at 24 h after PH. Liver homogenates and lysates from highlypurified mitochondria, obtained from both sham-operated andpartially hepatectomized rats, were tested for tyrosine kinaseactivity on a generic poly(Glu-Tyr)4:1 substrate in the absence

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Figure 4 Lyn localizes to mitochondria in an activated form

Comparable aliquots of (A) total cell and (B) mitochondrial lysates from rat liver after PH at different time points were assayed for in vitro Lyn activity on the Src-specific peptide substrate Cdc2-(6–20)and by Western blotting for Lyn. The membranes were reprobed with anti-β-actin and anti-aconitase antibodies respectively as loading controls. (C) Total cell and (D) mitochondrial lysates from ratliver after PH at different time points were immunoprecipitated (IP) with anti-Lyn antibody. The immunoprecipitates were assayed with anti-(phospho-YA) antibody (P-YA) and, after stripping, withanti-Lyn antibody. The Figure is representative of experiments performed in triplicate. Wb, Western blot.

and presence of various specific tyrosine kinase inhibitors.In particular, we used selective inhibitors of both receptortyrosine kinases [SU11274, AG1478 or AG1296 against MET,EGFR and PDGFR (platelet-derived growth factor receptor)respectively] [31–33], non-receptor tyrosine kinases [AG490, PP2or, alternatively, SU6656 and piceatannol against JAK (Januskinase), SFKs and SYK (spleen tyrosine kinase) respectively][34–36] and genistein as a more generic compound active againstboth groups [37]. All the inhibitors assayed apart from AG1296and the Syk inhibitor significantly affected the tyrosine kinaseactivity in total cell lysates (Figure 2A), whereas only PP2 andSU6656, specific inhibitors of the SFKs, as well as the generalinhibitor genistein, proved to be effective in inhibiting the tyrosinekinase activity in the mitochondrial lysate of regenerating livers(Figure 2B). These results corroborate the notion that SFKs arethe major agents in mitochondrial tyrosine phosphorylation.

Lyn is the predominant SFK expressed in the liver

SFKs comprise eight non-receptor protein tyrosine kinases whichare characterised by a common domain structure and groupedinto two subfamilies on the basis of their amino acid sequence,namely the Src-related enzymes (Src, Yes, Fyn and Fgr) and theLyn-related enzymes (Lyn, Hck, Lck and Blk) [38,39]. To date,the roles of these enzymes in the liver have not been extensivelystudied; the majority of such work has been performed whenthe enzymes have been implicated in carcinogenesis and, morerecently, in LR [40–42]. To assess which member of the SFKswas responsible for the tyrosine kinase activity in mitochondriaafter PH, we compared the protein level of SFKs in liver, spleenand brain (the latter two organs are known to abundantly expressSFKs [40]). Equal amounts of protein lysate (50 μg) from ratliver, spleen and brain were analysed by Western blotting withspecific antibodies against SFKs and the resulting bands werequantified by densitometric analysis. As shown in Figure 3(A),the Src-related SFKs displayed a lower protein level in the

liver than in spleen and brain, whereas the Lyn-related SFKswere undetectable, with the exception of Lyn, which was thepredominant SFK expressed in the liver in comparison with theother two organs. This result was confirmed by RT–PCR, whichrevealed that the mRNA levels of Lyn were 5–6-fold higherthan those of Src in the quiescent liver. Notably, Lyn expressionremained unchanged over time even after PH, whereas the levelsof Src were significantly lower compared with Lyn itself, althoughincreased when compared with the basal value (Figure 3B).

Lyn accumulates in mitochondria in an active form in early LR

As Lyn was the predominant SFK in the liver, we next investigatedwhether activity and protein level of Lyn in mitochondria differedin regenerating liver compared with quiescent liver. Therefore wemonitored SFK activity in both mitochondrial and total cell lysatesby using the SFK-specific Cdc2-(6–20) peptide as a substrate.In total cell lysates SFK activity increased by 12 h after PHand remained substantially unchanged until 48 h (Figure 4A). Incontrast, mitochondrial SFK activity was negligible at 12 h afterPH, but had risen dramatically by 24 h, subsequently declining toa basal level by 48 h (Figure 4B). A Western blot analysis of totalcell lysate with the anti-Lyn antibody (Figure 4A) demonstratedthat the protein level of Lyn was not significantly altered duringthe regenerating process, confirming the results obtained by RT–PCR; conversely, Lyn was undetectable in mitochondria until 12 hafter PH, subsequently increasing to a peak between 24 and 36 hand thereafter declining to the level reached at 12 h (Figure 4B).As the activity and protein level of Lyn were proportionally relatedto each other in the mitochondrial lysate, but not in the total celllysate (compare Figures 4A and 4B), we evaluated the activationstate of Lyn by Western blot analysis using an antibody againstthe phosphorylated YA (Tyr396) residue, which is suggestive of anactivated form of Lyn [39]. Lyn immunoprecipitated from eitherthe total cell lysate or the mitochondrial lysate of the regeneratingliver was subjected to Western blot analysis and revealed that

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Figure 5 Subcellular localization of Lyn during LR after PH

Rat liver homogenate (250 μg) at (A) 0, (B) 12 and (C) 24 h after PH was subjected todifferential centrifugation to separate the particulate fraction from nuclei and cytosol. Theparticulate fraction was fractionated further by centrifugation on a discontinuous OptiPrepTM

gradient to separate the cellular organelles as described in the Experimental section. Aliquotsof the resulting fractions were assayed for Lyn activity using the Src-specific peptide substrateCdc2-(6–20) and analysed by immunoblotting with anti-Lyn antibody. (D) Aliquots of thesame fractions underwent immunoblotting with the following organelle-specific antibodies:anti-PMCA antibody (plasma membrane), anti-GM130 antibody (Golgi apparatus), anti-calnexinantibody (endoplasmic reticulum) and anti-aconitase antibody (mitochondria). The Figure isrepresentative of experiments performed in triplicate. Wb, Western blot.

the activation state of Lyn followed a profile similar to that ofthe kinase activity (compare Figures 4A and 4B with Figures 4Cand 4D respectively). Lyn appeared to be transiently localizedto mitochondria in an active form during the early phases of LRafter PH (Figure 4D). Notably, Src, Yes, Fyn and Fgr were notdetected by Western blot analysis in this experiment (results notshown). To verify this Lyn distribution change during LR, the post-nuclear particulate obtained from either regenerating or quiescentliver at 12 and 24 h after PH was centrifuged on a discontinuousOptiPrepTM gradient to separate the subcellular fractions. In thequiescent liver Lyn was abundantly represented on the plasmamembrane in an inactive form (Figure 5A), at 12 h after PHno change in the protein distribution of Lyn was observed, butthe protein appeared to be activated in this cellular compartment(Figure 5B). At 24 h after PH, we observed an increase inthe SFK activity in the mitochondrial fractions which wasconcomitant with both the appearance of Lyn in the mitochondrialfractions and a decrease in the protein level of Lyn in theplasma membrane fractions (Figure 5C). These results suggestthat Lyn, in response to liver injury, translocates and conveysinformation from the plasma membranes directly to mitochondriaduring LR.

Lyn is detected in a complex in the intermembrane space ofmitochondria

To establish the mitochondrial subcompartment in which Lyn waslocalized, mitochondria isolated from regenerating liver at 24 hafter PH were treated with proteinase K. As shown in Figure 6(A),Lyn, AIF, a structural component of the inner mitochondrialmembrane, and aconitase, a mitochondrial matrix protein, werenot affected by the proteinase K treatment, which was onlyeffective on the three proteins after total solubilization with TritonX-100. Following the separation of mitochondrial membranes andthe soluble fraction, Lyn, as well as aconitase, were predominantlylocalized in the soluble fraction, whereas AIF was in themembrane fraction (Figure 6B). Digitonin treatment was also usedin order to selectively permeabilize only the outer mitochondrialmembrane [43], causing a partial release of Lyn at a concentrationof the detergent as low as 0.10 mg/ml and full release atconcentrations above 0.20 mg/ml (Figure 6C). In contrast AIFand aconitase were only nearly totally released into the solublefraction at concentrations of digitonin as high as 0.60 mg/ml.Hence we conclude that Lyn is localized to the intermembranespace of mitochondria as a soluble protein, as described previouslyin the mitochondria of rat brain [14]. It is known that Lyn is boundto the plasma membrane by myristoylation of its N-terminal [5], soin order to attempt to explain how Lyn was subsequently targetedto mitochondria, the soluble submitocondrial fraction was run ona glycerol gradient. Lyn was detected by Western blot analysis at amolecular mass of 230 kDa, suggesting it is part of a multiproteincomplex (Figure 6D).

Lyn-mediated tyrosine phosphorylation preserves mitochondrialintegrity

It is widely known that disruption of the mitochondrial membranepotential (��m) is often a consequence of oxidative stressand elevation of the Ca2+ concentration in mitochondria, withsubsequent functional impairment, and the activation of theapoptosis pathways. In Figure 1(C), we demonstrated that anincreased GSSG/GSH ratio and Ca2+ overload were associatedwith a hyperpolarization, rather than a fall, of the inner membranepotential. Given that the trend of these functional parametersmeasured over time displayed a pattern analogous to that oftyrosine phosphorylation (Figure 1A), we examined whethertyrosine phosphorylation had a role in the behaviour of themitochondrial membrane potential during LR. For this purpose,��m of mitochondria purified from regenerating liver werecompared with those from quiescent liver at 24 h after PH,a time point that corresponded to the peak of both tyrosinephosphorylation and the other parameters (Figures 1A and 1C).Notably, mitochondria were isolated and maintained either ina medium containing PTP-Is (protein tyrosine phosphastaseinhibitors), consistent with the experimental protocol adoptedthroughout the present study to which was necessary to emphasizethe transient phosphotyrosine signal, or devoid of PTP-Is so asnot to rule out a possible role of phosphatases to counteract thetyrosine kinase activity. Interestingly, the presence of PTP-Is didnot change the yield of mitochondria from quiescent liver (resultsnot shown), the mitochondrial tyrosine phosphorylation pattern,which was virtually absent (Figure 7A, compare lanes 1 and lane2), or the ��m pattern (Figure 7B, left-hand panel). Mitochondriafrom regenerating liver in the absence of PTP-I displayed asignificantly lower value of ��m than those from quiescent liver,with the ��m finally collapsing after the addition of ADP, whenthe mitochondrial capability to perform oxidative phosphorylationwas tested. Conversely, mitochondria from regenerating liver

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408 E. Gringeri and others

Figure 6 Lyn is detected as a soluble protein in the intermembrane space of mitochondria

(A) Mitochondria purified from rat liver at 24 h after PH were incubated in the absence (−) or presence (+) of proteinase K and Triton X-100 (Tx-100) as indicated. Aliquots of mitochondrial lysatewere subjected to Western blot analysis with anti-Lyn, anti-AIF and anti-aconitase antibodies respectively. (B) Intact mitochondria, mitochondrial membranes and soluble fractions obtained from ratlivers at 24 h after PH were subjected to Western blot analysis with anti-Lyn, anti-AIF and anti-aconitase antibodies respectively. (C) Mitochondria purified from rat liver 24 h after PH were incubatedin the presence of increasing concentrations of digitonin. After each treatment the samples were centrifuged to separate pellet (P) from soluble fraction (S). Aliquots of such fractions after differentialdigitonin treatment were subjected to Western blot analysis with anti-Lyn, anti-AIF and anti-aconitase antibodies respectively. (D) The soluble fraction of mitochondria purified from rat liver 24 h afterPH was subjected to density-gradient centrifugation, as described in the Experimental section, and analysed by Western blotting with the anti-Lyn antibody. The Figure is representative of experimentsperformed in triplicate. Downward arrows show the position of the molecular-mass standards on glycerol gradients on parallel gradient runs which are used to estimate the molecular mass of theprotein complexes (glutamate dehydrogenase at 62 kDa; alcohol dehydrogenase at 150 kDa; apoferritin at 443 kDa; thyroglobulin at 669 kDa). Wb, Western blot.

isolated in the presence of PTP-I showed a remarkable elevation ofboth tyrosine phosphorylation (Figure 7A, lane 3) and membranepolarization (Figure 7B, right-hand panel) not only whencompared with mitochondria from regenerating liver isolatedwithout PTP-I, but also when compared with those from quiescentliver. Moreover, the experimental conditions determined by thepresence of PTP-I highlighted a conserved efficiency in oxidativephosphorylation, as confirmed by the transient effect on ��m

upon addition of ADP, although the recovery of ��m was slowerin mitochondria from regenerating liver compared with those fromquiescent liver (Figure 7B, compare the right-hand and left-handpanels), suggesting some residual activity that disappears withina few minutes (results not shown).

We next wanted establish how tyrosine phosphorylation,namely that dependent on SFKs, had on impact on themitochondrial role in the early phases of LR. Therefore aspecific SFK inhibitor, PP2, was administered to rats undergoingPH. The effect of PP2 was assessed by investigating a fewextra- and intra-mitochondrial events at 24 h after PH, whenthe tyrosine phosphorylation level was highest. Mitochondrialtyrosine phosphorylation, activation of Lyn and ��m were alldecreased in PP2-treated rats in a dose-dependent manner whencompared with rats treated with vehicle control (Figures 8A–C).Interestingly, inhibition of Lyn (Figure 8B, upper panel) appearedto directly prevent the accumulation of Lyn itself in mitochondria(Figure 8B, lower panel) and consequently accounted for the fall inmitochondrial tyrosine phosphorylation illustrated in Figure 8(A).The collapse of ��m, caused by the increasing doses of PP2(Figure 8C), was a further finding which indicated that themitochondrial tyrosine phosphorylation was a determining factorin preserving the functional integrity of mitochondria in the early

phases of LR. Notably, inhibition of SFK activity by PP2 resultedin a lower yield in intact mitochondria purified from rat liver(results not shown), as a result of disruption of mitochondrialmembranes documented by the presence of cytochrome c andaconitase in the cytosolic fraction (Figure 8D). PP3, an inactiveanalogue of PP2, at concentrations identical with those of PP2 orsaline was incapable of affecting the mitochondrial events underinvestigation (results not shown).

DISCUSSION

During the prereplicative phase of LR, mitochondria undergofunctional changes that are traditionally considered as pro-apoptotic, such as Ca2+ overload and excessive production ofROS, but these events do not lead to the activation of the deathcascade. In the present study, we provide evidence that thetyrosine kinase Lyn, a SFK, accumulates in an active form inthe intermembrane space of mitochondria and contributes to thepreservation of the overall integrity of these organelles in the earlyphases of LR by phosphorylating a wide range of substrates.

SFKs are non-receptor tyrosine kinases that are co-expressedin multiple combinations in various cell types. They serveas molecular switches and are involved in the integrationand transmission of diverse signals generated by cell-surfacereceptors in response to a large number of extracellular stimuli,such as growth factors and cytokines. Thereby the SFKsregulate a variety of cellular events, including cell growthand proliferation, cell adhesion and migration, differentiation,survival and death [38,39]. SFKs are ordinarily maintained in aclosed inactive conformation through two major intramolecularinhibitory interactions, namely binding of the phosphorylated YT

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Lyn moves to mitochondria in early liver regeneration 409

Figure 7 Effect of tyrosine phosphatase inhibitors on mitochondrialmembrane potential (��m)

Mitochondria were purified from equivalent amounts of both quiescent and regenerating ratliver at 24 h after PH in the presence or absence of PTP-Is. (A) Mitochondrial aliquots fromboth quiescent (lanes 1 and 2) and regenerating (lanes 3 and 4) rat liver at 24 h after PH inthe presence (+) or absence (−) of PTP-Is were subjected to Western blot analysis with theanti-phosphotyrosine (pTyr) antibody and the membranes were reprobed with anti-aconitaseantibody as a loading control. The positions of molecular-mass markers are indicated on theright. Wb, Western blot. (B) ��m was measured as described in [14] on mitochondrial aliquotsfrom both quiescent (left-hand panel) and regenerating (right-hand panel) rat liver at 24 h afterPH in the presence (+) or absence (−) of PTP-Is and incubated in standard medium. Thedownward arrows indicate the time-point at which ADP was added (to a final concentration of200 μM). The Figure is representative of experiments performed in triplicate on three samplesfrom both quiescent and regenerating livers. �E, electrode potential. The scale bar indicates atime of 1 s.

(C-terminal tyrosine residue, Tyr508 in Lyn) to the SH2 (Srchomology 2) domain and interaction of a PPII (polyprolinetype II helical) motif in the SH2-kinase linker with the SH3 (Srchomology 3) domain [44,45]. The activation of SFKs involvesdisruption of these inhibitory interactions through multiplemechanisms, such as dephosphorylation of YT, displacement ofthe tail from the SH2 domain, displacement of the PPII motif fromthe SH3 domain and is characterized by autophosphorylation ofthe specific YA (Tyr396 in Lyn) in the activation-loop.

Although SFKs are generally believed to have redundantfunctions, emerging evidence indicates that each SFK membermay possess unique roles in conferring signalling specificitydue to their ability to phosphorylate particular substrates, theirspatial compartmentalization in membrane microdomains andtheir subcellular distribution [39,46]. Although most of ourknowledge of SFKs has been gained by studies on haematopoietictissue, the presence and the role of SFKs and their substrates havebeen highlighted in other tissues and organs, including the liverunder normal and pathological conditions [40–42].

The results in the present paper show that, among the SFKsthat are co-expressed in the quiescent liver, Lyn is quantitativelypredominant. Moreover, although its expression remainsunchanged during LR, Lyn undergoes spatial-temporal changes inthe distribution and activity profiles, as observed in the subcellularmembrane fractions from liver homogenate. In particular, weobserved an increase in the protein level of Lyn in the active formin mitochondria, with a concurrent decrease in levels at the plasmamembrane, where the activation of the tyrosine kinase occurs inresponse to stimuli initiating the LR following PH. At the time ofthe rise in SFK activity in the total liver lysate, no SFK memberother than Lyn was detected in mitochondria either in the quiescentliver or in the regenerating liver (results not shown), indicatingthat Lyn is the only SFK member to be imported into mitochondriaduring LR. These results from the present study are also consistentwith findings on the role of Lyn in the immune system, where it,so far, has been studied in a greater depth than in the liver. In theimmune system Lyn, in addition to functions redundant with otherSFKs, proves irreplaceable in the regulation of specific signallingpathways. For instance, Lyn not only contributes to initiationof the signalling pathways upon ligation of the BCR (B-cellreceptor), redundantly with Blk and Fyn [47], but also negativelyregulates BCR signalling by phosphorylating inhibitory receptors,displaying a unique ability to modulate the negative feedbackcontrol mechanism of signalling.

Activation of Lyn at the plasma membrane is a prerequisitefor its accumulation in mitochondria, as demonstrated by theadministration of the PP2 inhibitor (Figure 8B), which is specificfor SFKs in general, to rats in the early phases of LR. Thisfinding allows us to classify Lyn into the group of kinases (whichincludes EGFR, PKC and Akt) that are activated at the plasmamembrane in response to external stimuli and are imported intomitochondria, where they impinge on mitochondrial function,thereby influencing cell survival [48–50]. Recently, it has beendemonstrated that the level of activated Akt in mitochondria isdependent on the chaperoning activity of HSP90 (heat-shockprotein 90) and that Akt import is involved in preservingmitochondrial integrity [51]. As we have recently demonstrated,albeit under pathological conditions, that Lyn is stabilized inan active form when part of a multiprotein complex along withHSP90 [52], we propose that the mechanism of import of Lyn may,similarly to Akt, rely upon the interaction with this chaperone,although further investigation is needed to verify this hypothesis.

Irrespective of how Lyn translocates into mitochondria, Lynturns out to be responsible for the phosphorylation of numerousproteins inside these organelles during the prereplicative phaseof LR. First, the temporal pattern of the level of activatedLyn in mitochondria (Figurse 4B and 4D) is analogous tothat of mitochondrial tyrosine phosphorylation (Figure 1A) andsecondly, administration of PP2 to rats undergoing PH results in adecrease in the protein level of Lyn in mitochondria that parallelsthe fall in mitochondrial tyrosine phosphorylation.

In support of the role of tyrosine phosphorylation inmitochondria, studies have revealed that several proteinscontaining phosphotyrosine are found to be strategically localizedin the various mitochondrial compartments and that they arefunctionally affected by the post-translational modification [6].For instance, the efficiency of the electron transport chain isenhanced when the subunit II of COX (cytochrome c oxidase)is phosphorylated by Src, whereas it is reduced when Tyr304 ofsubunit I is phosphorylated by a still unknown tyrosine kinasedependent on cAMP-mediated signalling [4,6]. Cytochrome chas also been shown to be regulated by the phosphorylation ofTyr97, resulting in inhibition of respiration in the reaction withCOX. In addition, tyrosine phosphorylation is proposed to prevent

c© The Authors Journal compilation c© 2010 Biochemical Society

410 E. Gringeri and others

Figure 8 Effects of PP2 treatment of rats on LR at 24 h after PH

(A) Mitochondrial lysates obtained at 24 h after PH from liver of rats treated with either increasing concentrations of PP2 or vehicle control were assayed by Western blotting with the anti-phosphotyrosine(pTyr) antibody. The membranes were reprobed with the anti-aconitase antibody as loading control. The positions of molecular-mass markers are indicated on the left. (B) Total cell (upper panel)and mitochondrial (lower panel) lysates obtained at 24 h after PH from livers of rats treated with either increasing concentrations of PP2 or vehicle control were immunoprecipitated (Ip) with theanti-Lyn antibody, the immunoprecipitates were assayed with the anti-(phospho-YA) (P-YA) antibody and, after stripping, with the anti-Lyn antibody. (C) ��m was measured as described in [14] onmitochondria obtained at 24 h after PH from liver of rats treated with either increasing concentrations of PP2 or vehicle control and incubated in standard medium. The downward arrows indicate thetime point at which ADP was added (to a final concentration of 200 μM). �E, electrode potential. The scale bar indicates a time of 1 s. (D) Cytosol obtained at 24 h after PH from liver of rats treatedwith either increasing concentrations of PP2 or vehicle control were assayed by Western blot analysis with anti-(cytochrome c), anti-aconitase and anti-LDH antibodies respectively. The Figure isrepresentative of experiments performed in triplicate. Wb, Western blot.

the interaction of cytochrome c with Apaf1 (apoptotic peptidaseactivating factor 1), which would otherwise lead to apoptosomeformation and inhibit the peroxidase activity responsible forcardiolipin oxidation (the two latter events are required forthe release of cytochrome c into the cytosol together withother pro-apoptotic factors) [6]. Tyrosine phosphorylation mayalso have a role in the regulation of ANT (adenine nucleotidetranslocase), which catalyses the ADP/ATP exchange across theinner mitochondrial membrane and is implicated in the formationor regulation of the mitochondrial permeability transition pore.We have recently identified two tyrosine residues in ANT, Tyr190

and Tyr194, which are phosphorylated by SFKs in mitochondriafrom rat brain [9]. As these tyrosine residues are localized in the

cavity where nucleotides bind for translocation, phosphorylationmight represent a further mode of regulation of ANT activity, inaddition to the activating mechanism dependent on oxidation ofthiol groups. A recent study showed that phosphorylation of theseresidues is critical for mitochondrial bioenergetics and also asso-ciated with cardioprotection in a model of ischaemia/reperfusioninjury [53]. This latter finding is in agreement with our resultsand highlights a role for tyrosine phosphorylation in preservingthe mitochondrial function during the early stage of LR despitethe occurrence of possible pro-apoptotic processes.

Notably, tyrosine phosphorylation was emphasized only byadding PTP-Is into the medium used to isolate mitochondria(Figure 7A, lane 3), in order to prevent the dephosphorylation of

c© The Authors Journal compilation c© 2010 Biochemical Society

Lyn moves to mitochondria in early liver regeneration 411

mitochondrial proteins that regularly occurs during such proced-ures. The role of tyrosine phosphorylation in liver mitochondriaunder regenerating conditions was also highlighted by theevidence that the absence of PTP-Is is accompanied by a very low��m, in sharp contrast with the remarkable rise in ��m observedwhen PTP-Is were used (Figure 7, right-hand panel). It is alsonoteworthy that the ��m profiles of mitochondria from quiescentliver were identical regardless of the presence of PTP-I (Figure 7B,left-hand panel). Another striking feature that further highlightsthe importance of tyrosine phosphorylation in the processesdescribed in the present study is how, by sustaining and evenincreasing ��m, ATP synthesis is efficiently preserved, whichexperimentally follows the addition of ADP to mitochondriaisolated in the presence of PTP-I in a very similar manner as in theresting state (Figure 7B, right- compared with the left-hand panel).On the other hand, mitochondria from regenerating liver withoutsuch PTP-I treatment displayed not only a much lower ��m

but also an ultimate collapse after addition of ATP, suggestiveof irreversible damage to the inner mitochondrial membrane.Treatment with PP2 in rats undergoing PH and subsequent LRcaused effects similar to those observed in the absence of PTP-Ison isolated mitochondria, in addition to an increased fragilityof mitochondrial membranes as evidenced by the release ofcytochrome c and aconitase in the cytosol (Figure 8D).

In summary, these results are consistent with a role for Lyn-mediated tyrosine phosphorylation in protecting the structuraland consequently functional integrity, as well as the bioenergeticcompetence, of mitochondria during the early phases of LR.This is probably achieved by buffering the potentially harmfuleffects of ROS and Ca2+ overload which would otherwise lead tothe activation of death signalling. In this respect, these findingswarrant further efforts to broaden our knowledge on the rolesof mitochondrial tyrosine-phosphorylated proteins and how theirfunction is affected by such post-translational modification.

AUTHOR CONTRIBUTION

Enrico Gringeri performed the PH on rats, some of in vitro the assays, analysed the dataand wrote parts of the manuscript. Amedeo Carraro performed PH on rats, analysed thedata and wrote parts of the manuscript. Elena Tibaldi performed the majority of the invitro research, analysed the data and wrote parts of the manuscript. Francesco D’Amicoperformed PH on rats and some of the in vitro assays. Mario Pagano reviewed all ofthe results, participated in analysis of results and wrote the manuscript. Mario Manconperformed some of the in vitro research. Antonio Toninello performed some of the invitro research and assisted in preparation of the manuscript. Claudia Vio, performedsome of the in vitro research. Umberto Cillo provided intellectual support for the LRand PH work and assisted in preparation of the manuscript. Anna Brunati designed theresearch, reviewed all of the results, participated in analysis of results and wrote themanuscript.

FUNDING

This work was supported by the University of Padova [grant Progetto di Ateneo 2005 toA.M.B.].

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Received 15 June 2009/5 October 2009; accepted 16 October 2009Published as BJ Immediate Publication 16 October 2009, doi:10.1042/BJ20090902

c© The Authors Journal compilation c© 2010 Biochemical Society