Adrian T. Saurin

Widespread sulfenic acid formation in tissues in response to hydrogen peroxide

A principal product of the reaction between a protein cysteinyl thiol and hydrogen peroxide is a protein sulfenic acid. Because protein sulfenic acid formation is reversible, it provides a mechanism whereby changes in cellular hydrogen peroxide concentration may directly control protein function. We have developed methods for the detection and purification of proteins oxidized in this way. The methodology is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin–maleimide. Arsenite-dependent signal generation was fully blocked by pretreatment with dimedone, consistent with its reactivity with sulfenic acids to form a covalent adduct that is nonreducible by thiols. The biotin tag facilitates the detection of protein sulfenic acids on Western blots probed with streptavidin–horseradish peroxidase and also their purification by streptavidin–agarose. We have characterized protein sulfenic acid formation in isolated hearts subjected to hydrogen peroxide treatment. We have also purified and identified a number of the proteins that are oxidized in this way by using a proteomic approach. Using Western immunoblotting we demonstrated that a highly significant proportion of some individual proteins (68% of total in one case) form the sulfenic derivative. We conclude that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue, and are elevated in response to hydrogen peroxide. These approaches may find widespread utility in the study of oxidative stress, particularly because hydrogen peroxide is used extensively in models of disease or redox signaling.

The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes

Activation of protein kinase C (PKC) and more recently mitogen‐activated protein kinases (MAPKs) have been associated with the cardioprotective effect of ischemic preconditioning. We examined the interplay between these kinases in a characterized model of ischemic preconditioning in cultured rat neonatal ventricular cardiocytes where ectopic expression of active PKC‐δ results in protection. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning by brief simulated ischemia/reoxygenation. Overexpression of active PKC‐δ, rather than augmenting, completely abolished this activation. We therefore determined whether a similar process occurred during lethal prolonged simulated ischemia. In contrast to ischemia, brief, lethal‐simulated ischemia activated only p38 (2.8±0.45 vs. basal, P<0.01), which was attenuated by expression of active PKC‐δ or by preconditioning (0.48 ±0.1 vs. ischemia, P<0.01). To determine whether reduced p38 activation was the cause or an effect of protection, we used SB203580, a p38 inhibitor. SB203580 reduced ischemic injury (CK release 38.0± 3.1%, LDH release 77.3±4.0%, and MTT bioreduction 127.1 ±4.8% of control, n=20, P<0.05). To determine whether p38 activation was isoform selective, myocytes were infected with adenoviruses encoding wild‐type p38a or p38β. Transfected p38a and β show differential activation (P<0.001) during sustained simulated ischemia, with p38a remaining activated (1.48±0.36 vs. basal) but p38β deactivated (0.36± 0.1 vs. basal, P<0.01). Prior preconditioning prevented the activation of p38a (0.65±0.11 vs. ischemia, P<0.05). Moreover, cells expressing a dominant negative p38α, which prevented ischemic p38 activation, were resistant to lethal simulated ischemia (CK release 82.9±3.9% and MTT bioreduction 130.2±6.5% of control, n=8, P<0.05). Thus, inhibition of p38α activation during ischemia reduces injury and may contribute to preconditioning‐induced cardioprotection in this model.—Saurin, A. T., Martin, J. L., Heads, R. J., Foley, C., Mockridge, J. W., Wright, M. J., Wang, Y., Marber, M. S. The role of differential activation of p38‐mitogen‐activated protein kinase in preconditioned ventricular myocytes. FASEB J. 14, 2237–2246 (2000)

Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts

OBJECTIVE Activation of protein kinase C (PKC) isoforms is associated with the cardioprotective effect of early ischaemic preconditioning (IP). PKC consists of at least 10 different isoforms, encoded by separate genes, which mediate distinct physiological functions. Although the PKC-epsilon isoform has been implicated in preconditioning, uncertainty remains. We investigated whether preconditioning still occurs in a mouse line lacking cardiac PKC-epsilon protein due to a targeted disruption within the pkc-epsilon allele. METHODS The isolated buffer-perfused hearts from knockout mice lacking PKC-epsilon (-/-) and sibling heterozygous mice (+/-), with a normal PKC-epsilon complement, were preconditioned by 4 x 4 min ischaemia/6 min reperfusion. Hearts then underwent 45 min of global ischaemia followed by 1.5 h of reperfusion. RESULTS In PKC-epsilon (+/-) hearts ischaemic preconditioning reduced infarction volume as a percentage of myocardial volume (24.3+/-4.5 vs. 41.3+/-4.7%, P<0.001). In contrast, in PKC-epsilon (-/-) hearts preconditioning failed to diminish infarction (36.4+/-2.9 vs. 38.8+/-4.5%). Surprisingly however, although preconditioning did not reduce infarct size, it did enhance contractile recovery in PKC-epsilon (-/-) mice (43.1+/-3.9 vs. 24.9+/-5.1%, P<0.05), similar to the level seen in PKC-epsilon (+/-) hearts (35.2+/-3.9 vs. 20.9+/-5.0%, P<0.05). CONCLUSIONS These data suggest that PKC-epsilon is essential for the reduction in infarction that follows early ischaemic preconditioning, but is not associated with the improvement in functional recovery.

Altered cleavage and localization of PINK1 to aggresomes in the presence of proteasomal stress

Following our identification of PTEN‐induced putative kinase 1 (PINK1) gene mutations in PARK6‐linked Parkinsons disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG‐132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis.

Diverse Mechanisms of Myocardial p38 Mitogen-Activated Protein Kinase Activation Evidence for MKK-Independent Activation by a TAB1-Associated Mechanism Contributing to Injury During Myocardial Ischemia

Abstract— The ischemic activation of p38&agr; mitogen-activated protein kinase (p38&agr;-MAPK) is thought to contribute to myocardial injury. Under other circumstances, activation is through dual phosphorylation by MAPK kinase 3 (MKK3). Therefore, the mkk3−/− murine heart should be protected during ischemia. In retrogradely perfused mkk3−/− and mkk3+/+ mouse hearts subjected to 30 minutes of global ischemia and 120 minutes of reperfusion, infarction/risk volume was similar (50±5 versus 51±4, P =0.93, respectively), as was intraischemic p38-MAPK phosphorylation (10 minutes ischemia as percent basal, 608±224 versus 384±104, P =0.43, respectively). This occurred despite undetectable activation of MKK3/6 in mkk3−/− hearts. However, tumor necrosis factor (TNF)-induced p38-MAPK phosphorylation was markedly diminished in mkk3−/− vs mkk3+/+ hearts (percent basal, 127±23 versus 540±267, respectively, P =0.04), suggesting an MKK-independent activation mechanism by ischemia. Hence, we examined p38-MAPK activation by TAB1-associated autophosphorylation. In wild-type mice and mkk3−/− mice, the p38-MAPK catalytic site inhibitor SB203580 (1 &mgr;mol/L) diminished phosphorylation during ischemia versus control (10 minutes ischemia as percent basal, 143±2 versus 436±96, P =0.003, and 122±25 versus 623±176, P =0.05, respectively) and reduced infarction volume (infarction/risk volume, 57±5 versus 36±3, P <0.001, and 50±5 versus 29±3, P =0.003, respectively) but did not alter TNF-induced activation, although in homogenates of ischemic hearts but not TNF-exposed hearts, p38-MAPK was associated with TAB1. Furthermore, adenovirally expressed wild-type and drug-resistant p38&agr;-MAPK, lacking the SB203580 binding site, was phosphorylated when H9c2 myoblasts were subjected to simulated ischemia. However, SB203580 (1 &mgr;mol/L) did not prevent the phosphorylation of resistant p38&agr;-MAPK. These findings suggest the ischemic activation of p38-MAPK contributing to myocardial injury is by TAB1-associated autophosphorylation.

Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis.

PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity

The protein kinase C (PKC) Ser/Thr kinases account for ∼2% of the human kinome and regulate diverse cellular behaviors. PKC catalytic activity requires priming phosphorylations at three conserved sites within the kinase domain. Here we demonstrate that priming of PKC is dependent on the conformation of the nucleotide binding pocket but not on its intrinsic kinase activity. Inactive ATP binding site mutants are unprimed, but they become phosphorylated upon occupancy of the ATP binding pocket with inhibitors of PKC. We have exploited this property to screen for PKC inhibitors in vivo. Further, we generated a distinct class of kinase-inactive mutants that maintain the integrity of the ATP binding pocket; such mutants are constitutively primed and functionally distinct from ATP binding site mutants. These data demonstrate that autophosphorylation is not required for PKC priming and show how ATP pocket occupation can enable a kinase to mature as well as function.

The regulated assembly of a PKC|[epsiv]| complex controls the completion of cytokinesis

The cell cycle is exquisitely controlled by multiple sequential regulatory inputs to ensure fidelity. Here we demonstrate that the final step in division, the physical separation of daughter cells, is controlled by a member of the PKC gene superfamily. Specifically, we have identified three phosphorylation sites within PKCɛ that control its association with 14-3-3. These phosphorylations are executed by p38 MAP kinase (Ser 350), GSK3 (Ser 346) and PKC itself (Ser 368). Integration of these signals is essential during mitosis because mutations that prevent phosphorylation of PKCɛ and/or PKCɛ binding to 14-3-3 also cause defects in the completion of cytokinesis. Using chemical genetic and dominant-negative approaches it is shown that selective inhibition of PKCɛ halts cells at the final stages of separation. This arrest is associated with persistent RhoA activation at the midbody and a delay in actomyosin ring dissociation. This study therefore identifies a new regulatory mechanism that controls exit from cytokinesis, which has implications for carcinogenesis.

Negative feedback at kinetochores underlies a responsive spindle checkpoint signal.

Kinetochores are specialized multi-protein complexes that play a crucial role in maintaining genome stability. They bridge attachments between chromosomes and microtubules during mitosis and they activate the spindle assembly checkpoint (SAC) to arrest division until all chromosomes are attached. Kinetochores are able to efficiently integrate these two processes because they can rapidly respond to changes in microtubule occupancy by switching localized SAC signalling ON or OFF. We show that this responsiveness arises because the SAC primes kinetochore phosphatases to induce negative feedback and silence its own signal. Active SAC signalling recruits PP2A-B56 to kinetochores where it antagonizes Aurora B to promote PP1 recruitment. PP1 in turn silences the SAC and delocalizes PP2A-B56. Preventing or bypassing key regulatory steps demonstrates that this spatiotemporal control of phosphatase feedback underlies rapid signal switching at the kinetochore by: allowing the SAC to quickly transition to the ON state in the absence of antagonizing phosphatase activity; and ensuring phosphatases are then primed to rapidly switch the SAC signal OFF when kinetochore kinase activities are diminished by force-producing microtubule attachments.

Recognition of an intra-chain tandem 14-3-3 binding site within PKCepsilon

The phosphoserine/threonine binding protein 14‐3‐3 stimulates the catalytic activity of protein kinase C‐ε (PKCε) by engaging two tandem phosphoserine‐containing motifs located between the PKCε regulatory and catalytic domains (V3 region). Interaction between 14‐3‐3 and this region of PKCε is essential for the completion of cytokinesis. Here, we report the crystal structure of 14‐3‐3ζ bound to a synthetic diphosphorylated PKCε V3 region revealing how a consensus 14‐3‐3 site and a divergent 14‐3‐3 site cooperate to bind to 14‐3‐3 and so activate PKCε. Thermodynamic data show a markedly enhanced binding affinity for two‐site phosphopeptides over single‐site 14‐3‐3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14‐3‐3 ligand. This dual‐site intra‐chain recognition has implications for other 14‐3‐3 targets, which seem to have only a single 14‐3‐3 motif, as other lower affinity and cryptic 14‐3‐3 gatekeeper sites might exist.