Generation of a constitutively active fragment of PKN in microglia/macrophages after middle cerebral artery occlusion in rats.
PKN is a fatty acid- and Rho-activated serine/threonine kinase, which has a catalytic domain highly homologous to that of protein kinase C (PKC). Recent studies have demonstrated that PKN is proteolytically cleaved after apoptotic stimulation and then a constitutively active 55-kDa fragment is generated. However, the role of the 55-kDa fragment are poorly understood. Adult Sprague-Dawley (SD) rats underwent middle cerebral artery occlusion (MCAO), and the temporal and spatial changes in the fragmentation of PKN and of PKC delta were examined by immunoblotting. No proteolytic fragment of PKC delta (about 40 kDa) was detected. The 55-kDa fragment of PKN appeared transiently from 3 days after MCAO at the ipsilateral normal cortex. At the boundary zone of infarction, the 55-kDa fragment was markedly induced from day 5 then peaked on day 21 and persisted until day 28. Analysis of anti-phosphoserine immunoprecipitates with an anti-PKN antibody revealed phosphorylation of the 55-kDa band. Double staining for PKN and Ox42 was used to examine the source of the 55-kDa fragment. PKN immunoreactivity was significantly increased in Ox42-positive cells (microglia/hematogenous macrophages). No DNA laddering and only a few terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells were observed on day 14 in despite of the high level appearance of the 55-kDa band. These results suggest that the constitutively active 55-kDa fragment of PKN does not contribute to apoptosis, but may contribute to a function of microglia/macrophages.
Ueyama
T
T
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Japan.
Ren
Y
Y
Sakai
N
N
Takahashi
M
M
Ono
Y
Y
Kondoh
T
T
Tamaki
N
N
Saito
N
N
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D017209:Apoptosis; D004247:DNA / Q000378:metabolism; D004195:Disease Models, Animal; D004789:Enzyme Activation; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D020244:Infarction, Middle Cerebral Artery / Q000201:enzymology* / Q000473:pathology; D007527:Isoenzymes / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology*; D008297:Male; D017628:Microglia / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051381:Rats; D017207:Rats, Sprague-Dawley
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D010446:Peptide Fragments; D004247:DNA; C497260:Prkcd protein, rat; C061570:protein kinase N; D011493:Protein Kinase C; D051745:Protein Kinase C-delta