Characterization of protein kinase C isoforms in primary cultured cerebellar granule cells.
Protein kinase C (PKC) is a family of serine/threonine kinases comprised of 10 isoforms. Although commercial antibodies are available for all 10 isoforms, the specificity of these antibodies has been questioned. We have identified immunoblot conditions in which commercially purchased PKC antibodies are specific for their respective isoform. We then used these conditions to determine that PKC isoforms alpha, betaI, betaII, delta, epsilon, gamma, lambda, theta, and zeta are present in rat primary cultured cerebellar granule cells (CGCs) 6-14 days in vitro (DIV). This PKC profile is identical to that observed in cerebellar homogenates taken from 6-, 14- and 21-day-old rats. Western blot analysis indicated that the classical and the atypical PKC isoforms were more prevalent in the cytosolic subcellular fraction compared to the particulate fraction under basal conditions. Immunoreactivity for the novel isoforms tended to be higher in the particulate fraction under basal conditions. Phorbol 12-myristate 13-acetate (PMA) treatment resulted in translocated immunoreactivity from the cytosolic to the particulate fraction for all of the classical and novel PKC isoforms, but not for the atypical isoforms. However, the degree of translocation as well as the speed of translocation varied among the isoforms. The stability of the individual isoforms after PMA-induced activation also varied among the isoforms. Differences in these parameters were dependent upon culture batches and PKC isoform groups. We have identified experimental conditions in which reproducible results can be obtained with primary cultured CGCs in the study of PKC. We discuss possible solutions for problems encountered when utilizing primary cultured neurons to study PKC-mediated signal transduction.
Popp
R Lisa
RL
Department of Pharmacology and Neuroscience, School of Medicine, Texas Tech University Health Sciences Center, 3601 4th Street STOP 6592, Lubbock, TX 79430-0002, USA. lisa.popp@ttuhsc.edu
Velasquez
Oscar
O
Bland
Jason
J
Hughes
Peter
P
D000818:Animals; D000831:Animals, Newborn; D000918:Antibody Specificity; D002478:Cells, Cultured; D002525:Cerebellar Cortex / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D019169:Jurkat Cells; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D020033:Protein Isoforms / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D011493:Protein Kinase C / Q000032:analysis / Q000276:immunology* / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020033:Protein Isoforms; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate