Multisite phosphorylation of the 80 kDa (MARCKS) protein kinase C substrate in C3H/10T1/2 fibroblasts Quantitative analysis of individual sites by solid-phase microsequencing

• Published in: FEBS letters Vol. 297; no. 3; pp. 285 - 291
• Main Authors: Arness, Bob, Manjarrez-Hernandez, H.Angel, Howell, Steve A., Learmonth, Michele, Aitken, Alastair
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 10.02.1992; Elsevier
• Subjects: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 12-deoxyphorbol-13-phenylacetate-20-acetate; 12-O-tetradecanoylphorbol-13-acetate; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cell Line; Chromatography, High Pressure Liquid; DOPPA; EDC; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; fibroblasts; Fibroblasts - enzymology; Fundamental and applied biological sciences. Psychology; Kinase C; MARCKS protein; Mice; Mice, Inbred C3H; Molecular Sequence Data; Phosphopeptides - metabolism; Phosphorylation; PKC; protein kinase C; Protein Kinase C - metabolism; SAP A; sapintoxin A; SDS; sites; sodium dodecylsulphate; Spectrometry, Mass, Fast Atom Bombardment; Substrate Specificity; substrates; TFA; TPA; Transferases; trifluoroacetic acid; Phosphorylation; SAP A; SDS; TPA; DOPPA; EDC; PKC; MARCKS protein; Kinase C; TFA; Protein kinase C; Enzyme; Tumor promotor; Rodentia; 3T3-Cell line; Activation; Substrate analog; Vertebrata; Mammalia; Mouse; Fibroblast; Quantitative analysis
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(92)80557-W

A synthetic peptide, KKKKRFSFKKSFKLSGFSFKK, containing the phosphorylation sites of the acidic 80–87 kDa protein kinase C substrate was used to identify phosphopeptides in enzyme digests of this protein from mouse fibroblast C3H/10T1/2 cells. Stimulation of phosphorylation occurred, in vivo, with TPA at Ser 7, Ser 11 and Ser 18, and, will two less potent phorbol esters, at Ser 7 and Ser 18. Okadaic acid effected a net phosphorylation of Ser 7 and/or Ser 11. Solid-phase sequencing showed that, in vitro, the order of initial rate of phosphorylation was Ser 11 > Ser 7 > Ser 18, while Ser 18 was preferentially phosphorylated when either Ser 7 or Ser 11 was occupied. No significant phosphorylation of Ser 15 was detected.