R. Wagey

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

The Kinetworks™ 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 α/β (GSK3α/β; C = 90% increase/P = 220% increase), protein kinase B α (PKBα; C = 360% increase/P = 200% increase), phospho‐T638 PKCα/β (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 δ (PTP1δ; C = 43% increase/P = 70% increase). Cytosolic increases in phospho‐α‐S724/γ‐S662 adducin (C = 15650% increase), PKCα (C = 100% increase) and PKCζ (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 β (PKCβ; 330% increase), and stress‐activated protein kinase β (SAPKβ; 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 PKCα, PKCβ, PKCζ and GSK3α/β, which may augment neural death in ALS, and CaMKK, PKBα, Rsk1, S6K, and SAPK, which may be a response to neuronal injury that potentially can mitigate cell death.

Dysregulation of phosphatidylinositol 3-kinase and downstream effectors in human breast cancer †

Phosphatidylinositol 3‐kinase (PI3‐K) is a growth factor‐activated transforming lipid (and protein) kinase, involved in cell motility and invasion, that has multiple effectors. Relatively little is known about its expression and enzymatic activity in human breast cancer. Since growth factor receptors are amplified in breast cancer, and the tumor suppressor PTEN may be mutated in human breast cancer, it was hypothesized that PI3‐K and its downstream effectors would be activated in this disease. In 11 resected tumors analyzed for expression of this kinase, a mean 3‐fold increase in protein expression was observed over the corresponding adjacent control tissue. Using an in vitro lipid kinase assay of the immunoprecipitated PI3‐K protein, a greater than 2‐fold increase in activation was observed. These changes were observed in the absence of an activation of either protein kinase B (PKB, akt1) or p70 S6 kinase (p70 S6K). However, p21‐activated kinase (Pak), p38 mitogen‐activated protein kinase (p38 MAPK) and mitogen‐activated protein kinase‐activated protein kinase 2 (MAPKAPK 2) were all overexpressed and demonstrated increased enzyme activity. It may be concluded that aberrant mitogenic signaling in human breast cancer in vivo involves Pak, p38 MAPK and MAPKAPK2 downstream of PI3‐K, but neither of PKB or p70 S6K. It is proposed that this pathway may serve as a useful targeting nexus for investigation of small molecule inhibitors in human breast cancer.

Increased [35S]glutathione binding sites in spinal cords from patients with sporadic amyotrophic lateral sclerosis

Recent observations have suggested abnormalities in the gene for superoxide dismutase (SOD1) in patients with the familial form of amyotrophic lateral sclerosis (ALS). As SOD activity has secondary effects on glutathione (GSH), we have evaluated [35S]GSH binding in spinal cord sections from patients who died with sporadic ALS and control subjects. [35S]GSH binding sites were present in the grey matter of spinal cords in both the dorsal and ventral horns. ALS patients showed significantly increased [35S]GSH binding (+16%) in the dorsal and ventral grey horns compared to controls. Scatchard analysis of saturation binding data revealed that increased [35S]GSH binding was due to changes in the number rather than the affinity of GSH binding sites. These findings add support to a role for GSH in the mechanism loading to the pathogenesis of sporadic ALS.

Phosphatidylinositol 3-kinase: increased activity and protein level in amyotrophic lateral sclerosis.

Abstract: Enzyme activities and protein levels of several protein and lipid kinases were measured in postmortem tissue from patients who died with amyotrophic lateral sclerosis (ALS) as well as from control subjects. Patients who died with ALS had increased activities and protein levels of phosphatidylinositol 3‐kinase (PI 3‐K) in particulate fractions of spinal cord tissue compared with control subjects. The PI 3‐K activity increased with PI 3‐K protein level, indicating no change in specific PI 3‐K activity in ALS. No differences in PI 3‐K activities were found in cytosolic fractions of spinal cord, or in motor and visual cortices, from ALS patients compared with those from controls. PI 3‐K activities and protein levels were unchanged in brain tissue from patients who died with Alzheimers disease compared with those from controls. PI 3‐K is a lipid kinase that is important for cell survival and is activated in response to many growth factors. Increased PI 3‐K activities in particulate fractions of spinal cord from ALS patients may be related to the increase of PI 3‐K protein levels found in this tissue. The protein kinases Erk2, protein kinase B (PKB), and p70 ribosomal S6 kinase (S6K) showed no differences in activities in spinal cord tissue between ALS patients and controls. However, the amounts of PKB and S6K protein were significantly higher in ALS patients, whereas Erk2 protein amount was unchanged compared with controls. Protein kinase C activity was increased in spinal cord tissue from ALS patients, which is consistent with our previous report. The increased activity of PI 3‐K in spinal cord tissue from patients with ALS implicates the involvement or activation of PI 3‐K in ALS, as either a cause or a consequence of the neuron loss. The lack of up‐regulation in the activities of PKB and S6K in ALS tissue supports an impairment in signal transduction cascades mediated by PI 3‐K in this neurodegenerative disease.

Modulation of NMDA-mediated excitotoxicity by protein kinase C

Excessive activation of N‐methyl‐d‐aspartate (NMDA) receptors leads to cell death in human embryonic kidney‐293 (HEK) cells which have been transfected with recombinant NMDA receptors. To evaluate the role of protein kinase C (PKC) activation in NMDA‐mediated toxicity, we have analyzed the survival of transfected HEK cells using trypan blue exclusion. We found that NMDA‐mediated death of HEK cells transfected with NR1/NR2A subunits was increased by exposure to phorbol esters and reduced by inhibitors of PKC activation, or PKC down‐regulation. The region of NR2A that provides the PKC‐induced enhancement of cell death was localized to a discrete segment of the C‐terminus. Use of isoform‐specific PKC inhibitors showed that Ca2+‐ and lipid‐dependent PKC isoforms (cPKCs), specifically PKCβ1, was responsible for the increase in cell death when phorbol esters were applied prior to NMDA in these cells. PKC activity measured by an in vitro kinase assay was also increased in NR1A/NR2A‐transfected HEK cells following NMDA stimulation. These results suggest that PKC acts on the C‐terminus of NR2A to accentuate cell death in NR1/NR2A‐transfected cells and demonstrate that this effect is mediated by cPKC isoforms. These data indicate that elevation of cellular PKC activity can increase neurotoxicity mediated by NMDA receptor activation.

A role for amplified protein kinase C activity in the pathogenesis of amyotrophic lateral sclerosis

Abstract: Amyotrophic lateral sclerosis (ALS) is a human neurodegenerative disorder of unknown origin that is characterized by progressive degeneration of corticospinal tracts and anterior horn cells in the brainstem and spinal cord. Previous studies have indicated that motoneuron degeneration associated with ALS may be triggered by mechanisms leading to increased intracellular Ca2+. In the present report, Ca2+‐activated phospholipid‐dependent protein kinase C (PKC) was evaluated in cervical spinal cords from ALS patients and control subjects. In patients who died with ALS, PKC histone H1 phosphotransferase activity was significantly increased by 330% in cytosolic‐ and 118% in particulate‐derived extracts compared with controls. This increase in PKC phosphotransferase activity appeared to be partially due to an increase in the amount of PKC protein present in ALS spinal cord tissue. PKC histone H1 phosphotransferase activities of cytosolic‐ and particulate‐derived extracts from motor and visual cortex of ALS patients and controls were not statistically different, nor were there differences in PKC histone H1 phosphotransferase activity in platelets and leukocytes. The specific nature of PKC alterations in affected regions of the CNS supports a role for PKC in the events leading to motoneuron death in sporadic ALS.

Characterization, distribution, and protein kinase C-mediated regulation of [35S]glutathione binding sites in mouse and human spinal cord.

Abstract: We have characterized a high‐affinity [35S]‐glutathione ([35S]GSH) binding site in mouse and human spinal cord. [35S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High‐affinity [35S]GSH binding was saturable, showing a Bmax of 72 fmol/mg of protein and a KD of 3.0 nM for mouse spinal cord and a Bmax of 52 fmol/mg of protein and a KD of 1.6 nM for human spinal cord. [35S]GSH binding was displaceable by GSH, l‐cysteine, and S‐hexyl‐GSH, but not by glutamate, glycine, or NMDA. These [35S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [35S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13‐diacetate for 1 h before ligand binding. Phorbol ester treatment increased [35S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine, a general protein kinase inhibitor. [35S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS.

Abnormalities of protein kinases in neurodegenerative diseases

In neurodegenerative diseases such as ALS and AD there is evidence for abnormal regulation of protein kinases. In these diseases, altered activities and protein levels of several specific kinases suggest that abnormal phosphorylation is present and this aberrant phosphorylation may be involved in the pathogenesis of these diseases. The observation that regulation of the NMDA receptor ion channel is altered in tissue from ALS patients may arise from the abnormal phosphorylation state of the protein kinase regulating NMDA receptor function. Whether the abnormalities of these protein kinases is a primary event leading to altered receptor regulation or vice versa is still poorly understood. The seemingly multiple pathogenic mechanisms of ALS and AD create complexity in assessing a primary cause that may lead to cell death. The mechanisms causing cell death (apoptosis or necrosis) may be overlapping with integrated events among the components interacting and contributing to a final pathway for neuron death. Thus, evidence of impairment in protein kinase signalling in these diseases may be a primary cause, a secondary event, or a compensatory mechanism. To further study this issue, different model systems could be beneficial to obtain a better understanding of these diseases.

Amyotrophic lateral sclerosis: quantitative autoradiography of [3H]MK-801/NMDA binding sites in spinal cord

We have characterized a high-affinity binding site for [3H]MK-801, an NMDA receptor ion channel antagonist, in cervical spinal cords from patients who have died with amyotrophic lateral sclerosis (ALS) and from control subjects. In cervical spinal cord [3H]MK-801 labelled at least two binding sites, the highest affinity site having a Kd of between 9-16 nM. No significant differences in affinity were observed between spinal cords from ALS patients or controls. In spinal cords from ALS patients, large reductions in [3H]MK-801 receptor binding (between 30-40% reductions) were detected in both the dorsal and ventral horns. These data may reflect the death of receptor-bearing cells, or a form of receptor regulation.

Protein kinase C activity and protein levels in Alzheimer's disease

Patients with Alzheimers disease (AD) have been reported to have abnormalities in the levels and activities of protein kinase C (PKC) in brain and other tissues. We have measured Ca2+-activated, phospholipid-dependent PKC activities and levels in cerebral cortex from frontal, motor, temporal and parietal regions, as well as in leukocytes and platelets from AD patients and controls. No significant differences in PKC histone H1 phosphotransferase activity were seen in frontal, motor, temporal or parietal cortex, or in leukocytes and platelets from AD patients and controls. Elevated PKC protein was present in cytosolic fractions from frontal cortex, but not in other brain regions, or in leukocytes and platelets. These data suggest that abnormalities of PKC phosphorylating activity are absent in AD.