Characterization of a selective protein kinase C substrate derived from the MARCKS phosphorylation site domain for use in brain tissue homogenates.
Protein kinase C (PKC) isozymes play crucial roles in neuronal signal transduction and can regulate transmitter release, ion channels, neural development, and plasticity. In vitro assays of PKC are frequently used to associate PKC activity with cellular function, and the availability of selective PKC substrates can facilitate such studies. We have characterized a commercially available 12 amino acid peptide derived from the myristoylated alanine-rich C kinase substrate (MARCKS-PSD, Calbiochem) for use in crude rat brain homogenates. Assays were performed at 25 degrees C for 10 min (linear up to 12 min) using optimal concentrations of calcium and lipid cofactors. Kinetic analysis of MARCKS-PSD phosphorylation by PKC purified from rat brain gave a K(m) of 2.3 microM, which was similar to the K(m) of 2.8 microM obtained using rat brain cortical homogenates. The selective PKC inhibitor bisindolylmaleimide reduced phosphorylation of MARCKS-PSD in a concentration-dependent manner, with greater than 95% inhibition at 1.0 microM. MARCKS-PSD was more potent than another widely used selective PKC substrate (neurogranin((28-43)) and was a good substrate for human recombinant PKC alpha, delta, and epsilon but not zeta. The ontogeny of PKC activity was examined in the cortex and cerebellum. PKC activity was low at birth and reached adult levels by 21 days of age in both regions. Calcium-independent PKC activity in brain homogenates could be measured with MARCKS-PSD and accounted for approximately 25 and 10% of total activity in 1-day-old and adult rat cortex, respectively. These results suggest that the MARCKS-PSD peptide can be used as a selective PKC substrate in rat brain homogenates.
Mundy
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WR
Neurotoxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
Sutton
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LD
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D013379:Substrate Specificity
D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C