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.

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