Differential expression of MARCKS and other calmodulin-binding protein kinase C substrates in cultured neuroblastoma and glioma cells.
Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [3H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional 3H-myristoylated proteins of 50 and 40-45 kDa were observed in cell extracts heated to > 80 degrees C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca2+, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [3H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced rechange of the protein on nitrocellulose. Addition of beta-12-O-tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.
Rosé
S D
SD
Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
Cook
H W
HW
Palmer
F B
FB
Ridgway
N D
ND
Byers
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D019922:GAP-43 Protein; D015870:Gene Expression; D005910:Glioma / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D019922:GAP-43 Protein; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C000619711:Marcks protein, mouse; C000619712:Marcks protein, rat; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009227:Myristic Acids; D009419:Nerve Tissue Proteins; D011506:Proteins; D012333:RNA, Messenger; D019814:Myristic Acid; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate