Phosphorylation of proteins in human neutrophils activated with phorbol myristate acetate or with chemotactic factor

Sandro Pontremoli, Edon Melloni, Franca Salamino, Bianca Sparatore, Mauro Michetti, Oliviero Sacco, B. L. Horecker

Research output: Contribution to journalArticlepeer-review

Abstract

In human neutrophils stimulated with phorbol myristate acetate (PMA) or with the chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLF) a number of proteins are phosphorylated, including proteins recovered in the membrane fraction corresponding to molecular masses of 130, 78, 46, 40, and 34 kDa and proteins recovered in the cytosol fraction corresponding to molecular masses of 65, 55, 48, 38, 36, 30, and 22 kDa. Phosphorylation of the membrane proteins was fourfold greater in cells stimulated with PMA, as compared to cells stimulated with fMLF, whereas both activators induced similar phosphorylation of proteins recovered in the cytosol fraction. Phosphorylation of membrane proteins appeared to be mediated by native protein kinase C (PKC) translocated from the cytosol to the plasma membrane. Thus phosphate incorporation was inhibited by retinal and a similar pattern of incorporation was reproduced in a reconstituted system composed of isolated cell membranes and purified PKC. Phosphorylation of cytosol proteins, on the other hand, appeared to be mediated by the proteolytically modified form of PKC. In this case, phosphate incorporation was inhibited by leupeptin, which prevents the conversion of native PKC to the proteolytically modified form. The phosphorylation pattern was reproduced when isolated cytosol fractions were incubated with the proteolytically modified form of the enzyme but not with the native PKC. These results demonstrate that responses to stimuli such as PMA or fMLF are mediated by different forms of PKC and that the proteolytically modified form is responsible for the major responses elicited by fMLF.

Original languageEnglish
Pages (from-to)23-29
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume250
Issue number1
DOIs
Publication statusPublished - 1986

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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