Activation of protein kinase C in vitro and in intact cells or synaptosomes determined by acetic acid extraction of MARCKS.
MARCKS is a widespread cellular phosphoprotein that migrates at 80-87 kDa on polyacrylamide gels. It is phosphorylated apparently exclusively by protein kinase C (PKC) and its phosphorylation in intact cells can be used as an index of intracellular PKC activation. Most methods for the determination of its phosphorylation state in vitro or in intact cells rely on two-dimensional gel electrophoresis to detect the protein with a pI of 4.6; however, this does not readily allow for multiple samples to be simultaneously and rapidly processed. Here we utilize the acid solubility of MARCKS to develop a novel extraction procedure. MARCKS was found to be soluble in 40% acetic acid and can be extracted quantitatively and rapidly from phosphorylation mixtures in vitro or from labeled intact cells. Acetic acid has the advantage that it is volatile and readily removed and precipitates protein in the presence of SDS, and the extracted protein is more readily resuspended in sodium dodecyl sulfate (SDS) sample buffer. Two extraction methods were developed, one for extraction of MARCKS from [gamma-32P]ATP-labeled subcellular fractions or intact synaptosomes labeled with 32Pi and one for extraction from 32Pi-labeled cultured nucleated cells, the latter requiring an additional protein recovery and DNA removal step. With this procedure MARCKS phosphorylation in intact synaptosomes was shown to be reversibly stimulated upon depolarization and MARCKS phosphorylation was found to be an early event in the activation of cultured smooth muscle cells by angiotensin II.
Robinson
P J
PJ
Endocrine Unit, John Hunter Hospital, Hunter Region Mail Centre, N.S.W., Australia.
Liu
J P
JP
Chen
W
W
Wenzel
T
T
D000085:Acetates; D019342:Acetic Acid; D000818:Animals; D002540:Cerebral Cortex / Q000201:enzymology; D004789:Enzyme Activation; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D012995:Solubility; D013574:Synaptosomes / Q000201:enzymology
D000085:Acetates; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D019342:Acetic Acid