The Kinetworksä 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/calmodulindependent protein kinase kinase (CaMKK; C ¼ 120% increase/P ¼ 580% increase;% change, compared with control), extracellular regulated kinase 2 (ERK2; C ¼ 120% increase/P ¼ 170% increase), G protein-coupled receptor kinase 2 (GRK2; C ¼ 140% increase/P ¼ 140% increase), phospho-Y279/216 glycogen synthase kinase 3 a/b (GSK3a/b; C ¼ 90% increase/P ¼ 220% increase), protein kinase B a (PKBa; C ¼ 360% increase/P ¼ 200% increase), phospho-T638 PKCa/b (C ¼ 630% increase/P ¼ 170% increase), cGMP-dependent protein kinase (PKG; C ¼ 100% increase/ P ¼ 75% increase), phospho-T451 dsRNA-dependent protein kinase (PKR; C ¼ 2600% increase/P ¼ 3330% increase), ribosomal S6 kinase 1 (RSK1; C ¼ 750% increase/P ¼ 630% increase), phospho-T389 p70 S6 kinase (S6K; C ¼ 1000% increase/P ¼ 460% increase), and protein-tyrosine phosphatase 1 d (PTP1d; C ¼ 43% increase/P ¼ 70% increase). Cytosolic increases in phospho-a-S724/c-S662 adducin (C ¼ 15650% increase), PKCa (C ¼ 100% increase) and PKCf (C ¼ 190% increase) were found in ALS patients as compared with controls, while particulate increases in cAMPdependent protein kinase (PKA; 43% increase), protein kinase C b (PKCb; 330% increase), and stress-activated protein kinase b (SAPKb; 34% increase) were also observed. Cyclindependent 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 PKCa, PKCb, PKCf and GSK3a/b, which may augment neural death in ALS, and CaMKK, PKBa, Rsk1, S6K, and SAPK, which may be a response to neuronal injury that potentially can mitigate cell death.

Phosphatidylinositol 3-kinase (PI3-K) is a growth factoractivated 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.

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-speci®c PKC inhibitors showed that Ca 21 -and lipid-dependent PKC isoforms (cPKCs), speci®cally PKCb1, 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.

Enzyme activities and protein levels of several protein and lipid kinases were measured in postmortem

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.