Immunocytochemical localization of the alpha subspecies of protein kinase C in rat brain.

Abstract

The distribution ofthe a subspecies ofprotein kinase C (PKC) in rat brain was demonstrated immunocytochemically by using polyclonal antibodies raised against a synthetic oligopeptide corresponding to the carboxyl-terminal sequence of a-PKC. The a-PKC-specific immunoreactivity was widely but discretely distributed in both gray and white matter. The immunoreactivity was associated predominantly with neurons, particularly with perikaryon, dendrite, or axon, but little was seen in the nucleus. (2), which correspond to y-, ,1-and 813,-, and a-PKC, respectively (3, 4). These PKC subspecies are subtly different from one another in their kinetic properties, mode of activation, and most likely substrate specificity (1,(5)(6)(7)(8). Early analysis of the mRNA levels has indicated that a-, 1I-, and 81,-PKC are expressed in a variety of tissues, whereas y-PKC is expressed only in the central nervous system (9-12). Enzymatic and immunochemical analysis has also shown that a-PKC (type III) is most commonly distributed in many tissues and cell types (13,14). On the basis of these studies, it has been suggested that a-PKC plays a role of crucial importance in the control of common processes in cell functions (1).Several laboratories have carried out immunocytochemical studies using antibodies specific to each type of . In earlier reports (20-24), with subspeciesspecific antibodies, it was shown that in the rat brain 1,-, 1I-, and y-PKC are differentially distributed in particular cell types, with limited intracellular localization. The present studies were undertaken to identify a-PKC in the rat brain by using immunocytochemistry, and the results show that this PKC subspecies is enriched in particular cell types. MATERIAL AND METHODSPreparation of Antibodies Against a-PKC. The carboxylterminal portion of a-PKC (residues 662-672; Gln-Phe-ValHis-Pro-Ile-Leu-Gln-Ser-Ala-Val) was selected as a sequence specific to a-PKC. The oligopeptide was coupled to keyhole limpet with m-maleimidobenzoic acid N-hydroxysuccinimide ester. Rabbits were immunized with the immunogen by the method described (23,24) and were bled 1 week after the third booster administration. The IgG fraction was obtained from the antisera by affinity chromatography on Sepharose CL-4B coupled to goat anti-rabbit IgG, and the fraction was used as a-PKC-specific antibodies.Immunoblotting Analysis. Specificity of the antibodies was examined by immunoblotting analysis using the three subtypes of rat brain PKC (types I, II, and III) and four subspecies of PKC (a-, 13-, 1I-, and -PKC). These subspecies were purified from COS-7 cells transfected with the respective cDNA-containing plasmids as described (3, 4). The enzyme samples were subjected to NaDodSO4/7.5% polyacrylamide slab gel electrophoresis as described by Laemmli (25) and transferred to nitrocellulose paper. The paper was incubated with the a-PKC-specific antibodies, and immunoreactive bands were visualized by the peroxidaseantiperoxidase method.Immunocytochemical Staining. Frontal sections of the rat brain...

Isozyme specific changes in the expression of protein kinase C isozyme (α-ζ) genes in the hippocampus of rats induced by kindling epileptogenesis

“…The distribution of the PKC mRNA patterns are in good agreement with the patterns reported [4,8,19,31,34,35] and with the distribution of the PKC proteins as visualized by immunohistochemistry [8,19,31]. When the extinction values were corrected for differences in specific activity of the labeled oligonucleotides and differences of exposure duration, it was shown that the expression of the PKC isoforms is subject to a gene-specific and a regionally differential regulation.…”

Induction of Protein Kinase Cδ Subspecies in Neurons and Microglia after Transient Global Brain Ischemia

“…In the present study, most of the PKC mRNAs were eventually de creased in the CAl area, most likely as a consequence of local neuronal degeneration. Because most of the PKC subspecies are expressed in the neurons (Huang et a!., 1988;Ito et a!., 1990), the decline in the expression of PKC isoforms after transient forebrain ischemia may be secondary to neuronal cell death. The remaining expression level of PKCa may therefore occur in either nonneuronal cells (Sieber et a!., 1998) or nonvulnerable CAl neurons.…”

Differential localization of four subspecies of protein kinase C in the rat striatum and substantia nigra

“…The rabbit antisera specific to the PKC subspecies were prepared against synthetic oligopepiides that correspond ;o the carboxvl-terminal nortion of each subsnecies (OFVHPILOSAV for the CX-PKC: SYTNPEI%INV for the PI-PK'C, and%VNSEFLKPEVKS for the BII-PKC, Hosoda et al, 1989;Saito et al, 1989;Ito et al, 1990). The monoclonal antibody to r-PKC, obtained by immunizing mice with the purified PKC from the soluble fraction of rat brain, was characterized (Hashimoto et al, 1988).…”

“…Among various tissues, the enzyme is most concentrated in the CNS (Minakuchi et al,198 1). Immunocytochemical and biochemical studies in this and other laboratories have revealed a differential regional and cellular localization of each PKC subspecies in the brain (Kitano et al, 1987;Hidaka et al, 1988;Huang et al, 1988;Kose et al, 1988;Saito et al, 1988Saito et al, , 1989Hosoda et al, 1989;Ito et al, 1990). Particularly, the r-PKC subspecies is found only in the CNS (Ohno et al, 1987).…”