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
B Amess1, H A Manjarrez-Hernandez, S A Howell
1Laboratory of Protein Structure, National Institute for Medical Research, Mill Hill, London, UK.
Abstract:
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 Ser7, Ser11 and Ser18, and, with two less potent phorbol esters, at Ser7 and Ser18. Okadaic acid effected a net phosphorylation of Ser7 and/or Ser11. Solid-phase sequencing showed that, in vitro, the order of initial rate of phosphorylation was Ser11 greater than Ser7 greater than Ser18, while Ser18 was preferentially phosphorylated when either Ser7 or Ser11 was occupied. No significant phosphorylation of Ser15 was detected.
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