Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells

Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-alpha treatment decreased with time in cells grown in control as well as those grown in the presence of alpha-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-alpha treatment in cells grown in the presence of alpha-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-alpha-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-alpha. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.     

Protein kinase and protein phosphatase expression in amyotrophic lateral sclerosis spinal cord

The Kinetworks trade mark multi-immunoblotting technique was used to evaluate the expressions of 78 protein kinases, 24 protein phosphatases and phosphorylation states of 31 phosphoproteins in thoracic spinal cord tissue from control subjects and patients having the sporadic form of amyotrophic lateral sclerosis (ALS). In both the cytosolic (C) and particulate (P) fractions of spinal cord from ALS patients as compared with controls, there were increased levels of calcium/calmodulin-dependent protein kinase kinase (CaMKK; C = 120% increase/P = 580% increase;% change, compared with control), extracellular regulated kinase 2 (ERK2; C = 120% increase/P = 170% increase), G protein-coupled receptor kinase 2 (GRK2; C = 140% increase/P = 140% increase), phospho-Y279/216 glycogen synthase kinase 3 alpha/beta (GSK3alpha/beta; C = 90% increase/P = 220% increase), protein kinase B alpha (PKBalpha; C = 360% increase/P = 200% increase), phospho-T638 PKCalpha/beta (C = 630% increase/P = 170% increase), cGMP-dependent protein kinase (PKG; C = 100% increase/P = 75% increase), phospho-T451 dsRNA-dependent protein kinase (PKR; C = 2600% increase/P = 3330% increase), ribosomal S6 kinase 1 (RSK1; C = 750% increase/P = 630% increase), phospho-T389 p70 S6 kinase (S6K; C = 1000% increase/P = 460% increase), and protein-tyrosine phosphatase 1 delta (PTP1delta; C = 43% increase/P = 70% increase). Cytosolic increases in phospho-alpha-S724/gamma-S662 adducin (C = 15650% increase), PKCalpha (C = 100% increase) and PKCzeta (C = 190% increase) were found in ALS patients as compared with controls, while particulate increases in cAMP-dependent protein kinase (PKA; 43% increase), protein kinase C beta (PKCbeta; 330% increase), and stress-activated protein kinase beta (SAPKbeta; 34% increase) were also observed. Cyclin-dependent kinase-associated phosphatase (KAP) was apparently translocated, as it was reduced (31% decrease) in cytosolic fractions but elevated (100% increase) in particulate fractions of ALS spinal cord tissue. Our observations indicate that ALS is associated with the elevated expression and/or activation of many protein kinases, including PKCalpha, PKCbeta, PKCzeta and GSK3alpha/beta, which may augment neural death in ALS, and CaMKK, PKBalpha, Rsk1, S6K, and SAPK, which may be a response to neuronal injury that potentially can mitigate cell death.     

Protein phosphatase 2A regulates binding of Cdc45 to the prereplication complex

In eukaryotic cells, an ordered sequence of events leads to the initiation of DNA replication. During the G(1) phase of the cell cycle, a prereplication complex (pre-RC) consisting of ORC, Cdc6, Cdt1, and MCM2-7 is established at replication origins on the chromatin. At the G(1)/S transition, MCM10 and the protein kinases Cdc7-Dbf4 and Cdk2-cyclin E cooperate to recruit Cdc45 to the pre-RC, followed by origin unwinding, RPA binding, and recruitment of DNA polymerases. Using the soluble DNA replication system derived from Xenopus eggs, we demonstrate that immunodepletion of protein phosphatase 2A (PP2A) from egg extracts and inhibition of PP2A activity by okadaic acid abolish loading of Cdc45 to the pre-RC. Consistent with a defect in Cdc45 loading, origin unwinding and the loading of RPA and DNA polymerase alpha are also inhibited. Inhibition of PP2A has no effect on MCM10 loading and on Cdc7-Dbf4 or Cdk2 activity. The substrate of PP2A is neither a component of the pre-RC nor Cdc45. Instead, our data suggest that PP2A functions by dephosphorylating and activating a soluble factor that is required to recruit Cdc45 to the pre-RC. Furthermore, PP2A appears to counteract an unknown inhibitory kinase that phosphorylates and inactivates the same factor. Thus, the initiation of eukaryotic DNA replication is regulated at the level of Cdc45 loading by a combination of stimulatory and inhibitory phosphorylation events.     

Effects of magnesium sulphate following spinal cord injury in rats

Neutrophil infiltration has been implicated in the secondary destructive pathomechanisms after initial mechanical injury to the spinal cord. Tissue myeloperoxidase (MPO) activity has been shown to be an exclusive indicator of the extent of post-traumatic neutrophil infiltration. We have studied the effect of magnesium sulphate on MPO activity after spinal cord injury in rats. Rats were randomly allocated into 5 groups. Group 1 was control and normal spinal cord samples were obtained after clinical examination. Forty g-cm contusion injury was introduced to Group 2. Group 3 was vehicle, 1 ml of physiological saline was injected post-trauma. Group 4 was given 30 mg/kg methylprednisolone sodium succinate (MPSS) immediately after trauma. Group 5 was given 600 mg/kg magnesium sulphate immediately after trauma. Animals were examined by inclined plane technique of Rivlin and Tator 24 h after trauma. Spinal cord samples obtained following clinical evaluations. Magnesium sulphate treatment improved early functional scores and decreased MPO activity. These findings revealed that magnesium sulphate treatment possesses neuroprotection on early clinical results and on neutrophil infiltration after acute contusion injury to the rat spinal cord.     

Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast

Background Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28–cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis.Results The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55Δ cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids.Conclusions We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.     

Axonal transport in rats rendered paraplegic following a single subarachnoid injection of either batrachotoxin or 6-aminonicotinamide into the spinal cord

Batrachotoxin (BTX) or 6-aminonicotinamide (6-AN) when injected into the subarachnoidal space of the lumbar spinal cord block fast axonal transport of 3H-protein in motor nerves. Axonal transport recovers partially within one day after administering BTX while the effect of 6-AN lasts for more than 21 days. These observations are discussed in relation to the onset and recovery of membrane depolarization observed in the extensor muscle.     

SEMA3A regulates developing sensory projections in the chicken spinal cord

The present study explores the role of SEMA3A (collapsin-1) in the temporal and spatial regulation of developing sensory projections in the chick spinal cord. During development, SEMA3A mRNA (SEMA3A) is first expressed throughout the spinal gray matter, but disappears from the dorsal region when small caliber (trkA(+)) sensory axon collaterals first grow into the dorsal horn. In explant cultures of spinal cord segments with attached sensory ganglia, the spatial extent of SEMA3A expression varied in different explants, but in each case the growth of trkA(+) sensory collaterals was largely excluded from areas of SEMA3A expression. To test if SEMA3A had a direct effect on sensory axon growth, we injected recombinant protein into the explants before placing them in culture. Increased levels of SEMA3A substantially reduced the ingrowth of trkA(+) axons, whereas trkC(+) axon collaterals were not affected. Consistent with the insensitivity of trkC(+) collaterals to SEMA3A, these collaterals did not express neuropilin-1, a receptor for SEMA3A. The inhibitory effects of SEMA3A on trkA(+) axons within the spinal cord suggests that the fall in SEMA3A expression in the dorsal horn may contribute to the initiation of growth of these axons into gray matter. In addition, the observation that trkA(+) axons frequently grew close to but rarely over areas of SEMA3A expression suggests that semaphorin may act principally as a short-range guidance cue within the spinal cord.     

Permanent alterations of spinal cord reflexes following nerve lesion in newborn rats

Sciatic nerve lesion in newborn rats is known to cause degeneration of a large number of axotomized motoneurones and spinal ganglion cells. Some of the surviving motoneurones exhibit abnormal firing properties and the projection pattern of central terminals of sensory neurones is altered. We report here on long-term changes in spinal cord reflexes in adult rats following neonatal nerve crush. In acutely spinalized and anaesthetized adult rats 4-6 months old in which the sciatic nerve had been crushed on one side at birth, the tibial nerve, common peroneal nerve or sural nerve were stimulated on the reinnervated and control side and reflex responses were recorded from the L5 ventral spinal roots. Ventral root responses (VRRs) to tibial and peroneal nerve stimulation on the side of the nerve lesion were significantly smaller in amplitude representing only about 15% of the mean amplitude of VRRs on the control side. The calculated central delay of the first, presumably monosynaptic component of the VRR potential was 1.6 ms on the control side while the earliest VRR wave on the side of the nerve lesion appeared after a mean central latency of 4.0 ms that seems too long to be of monosynaptic origin. These results suggest that neonatal sciatic nerve injury markedly alters the physiological properties and synaptic connectivity in spinal cord neurones and causes a marked depression of spinal cord responses to peripheral nerve stimulation.     

A mapping study of caspase-3 activation following acute spinal cord contusion in rats.

Spinal cord injury (SCI) initiates a cascade of biochemical changes that results in necrotic and apoptotic cell death. There is evidence that caspase-3 activation and apoptotic cell death occur within hours after SCI. However, the time course and cellular localization of activated caspase-3 has not been examined. Such information is essential because caspase-3-independent apoptotic pathways do exist. In this experiment, we describe the distribution of and cell types containing activated caspase-3 at 4 hr, 1 day, 2 days, 4 days, and 8 days following SCI in rats. Numerous caspase-3-positive cells were observed at 4 hr and 1 day postinjury and colocalized most often with CC1, a marker for oligodendroglia. Both markers disappeared near the injury epicenter over the next several days. Activated caspase-3 was again present in the injured spinal cord on postoperative day 8, which coincided with a reemergence of CC1-positive cells. Many of these CC1-positive cells again colocalized activated caspase-3. NeuN-positive neurons of the dorsal horn were occasionally immunopositive for activated caspase-3 at early time points. OX42-positive microglia/macrophages rarely contained activated caspase-3. The results indicate a biphasic pattern of caspase-3 activation during the first 8 days postinjury, suggesting that at least two mechanisms activate caspase-3 following SCI. This time-course study provides a framework for investigating and understanding the different signaling events contributing to this biphasic pattern of caspase-3 activation.     

Protein phosphatase 2A regulates deoxycytidine kinase activity via Ser-74 dephosphorylation

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn²⁺-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.     

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABalphaC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.     

Enkephalin-like immunofluorescence in nerves of the rat iris following systemic capsaicin injection

A network of nerve fibers with an enkephalin-like immunoreactivity was demonstrated in rat iris whole mounts. Systemic administration of capsaicin in doses which caused partial (5 mg/kg) or complete (50 mg/kg) disappearance of substance P-containing fibers in the iris did not cause degeneration of enkephalin-positive nerve fibers. The enkephalin-immunoreactive network seemed intact also after a capsaicin dose of 250 mg/kg. In fact, the fluorescence intensity of the nerve fibers showing enkephalin-immunoreactivity was often increased three days after a capsaicin injection in a dose of 50 mg/kg. The mechanism behind this effect of capsaicin remains to be elucidated, but could be due either to a direct effect on the enkephalin-positive nerves or involve the disappearance of substance P nerves and/or a simultaneous inflammatory response. However, an increased fluorescence intensity of the enkephalin-immunoreactive fibers was sometimes seen also without capsaicin treatment.     

Protein phosphatase 2Cγ regulates the level of p21 by Akt signaling

PP2Cγ is a splicing factor that dephosphorylates specific substrates required for the formation of the spliceosome. In a previous study, we reported that the degradation of p21^Cip1/WAF1was affected by PP2Cγ, causing an accumulation of cells in S phase. Here, we demonstrate that the PP2Cγ-induced degradation of p21^Cip1/WAF1 is mediated by Akt signaling. In cells expressing PP2Cγ, Akt1 protein was phosphorylated. When PP2Cγ expression was knocked down, the phosphorylation of Akt1 was reduced and the level of p21^Cip1/WAF1 protein was increased. Interestingly, the stability of p21^Cip1/WAF1 was highly maintained in Akt1-depleted cells despite the ectopic expression of PP2Cγ. Taken together, these results suggest that PP2Cγ is a novel regulator of p21^Cip1/WAF1 protein stability via the Akt signaling pathway.     

Cathepsin B mRNA and protein expression following contusion spinal cord injury in rats

We provide the first data that cathepsin B (Cath B), a lysosomal cysteine protease, is up-regulated following contusion-spinal cord injury (SCI). Following T12 laminectomy and moderate contusion, Cath B mRNA and protein expression profiles were examined from 2 to 168 h post-injury in rats using real-time PCR and immunoblots, respectively. Contusion injury significantly increased [mRNA]Cath B in the injury site and adjacent segments over sham injury levels. While the largest [mRNA]Cath B induction (20-fold over naive) was seen in the injury site, the caudal segment routinely yielded [mRNA]Cath B levels greater than 10-fold over naive. Interestingly, sham injury animals also experienced mRNA induction at several time points at the injury site and in segments rostral and caudal to the injury site. Contusion injury also significantly elevated levels of Cath B proenzyme protein (37 kDa) over sham injury in the injury site (48, 72 and 168 h post-injury). Furthermore, significant protein increases of single and double chain Cath B (both active forms) occurred at the injury site at 72 and 168 h post-injury. Similar significant increases in Cath B protein levels were seen in areas adjacent to the injury site. The induction of Cath B mRNA and protein expression following contusion injury is previously undescribed and suggests that Cath B may potentially be involved in the secondary injury cascade, perhaps for as long as 1 week post-injury.