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 language | English |
|---|---|
| Pages (from-to) | 23-29 |
| Number of pages | 7 |
| Journal | Archives of Biochemistry and Biophysics |
| Volume | 250 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 1986 |
Fingerprint
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Phosphorylation of proteins in human neutrophils activated with phorbol myristate acetate or with chemotactic factor. / Pontremoli, Sandro; Melloni, Edon; Salamino, Franca; Sparatore, Bianca; Michetti, Mauro; Sacco, Oliviero; Horecker, B. L.
In: Archives of Biochemistry and Biophysics, Vol. 250, No. 1, 1986, p. 23-29.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Phosphorylation of proteins in human neutrophils activated with phorbol myristate acetate or with chemotactic factor
AU - Pontremoli, Sandro
AU - Melloni, Edon
AU - Salamino, Franca
AU - Sparatore, Bianca
AU - Michetti, Mauro
AU - Sacco, Oliviero
AU - Horecker, B. L.
PY - 1986
Y1 - 1986
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0022886504&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0022886504&partnerID=8YFLogxK
U2 - 10.1016/0003-9861(86)90697-1
DO - 10.1016/0003-9861(86)90697-1
M3 - Article
C2 - 3767374
AN - SCOPUS:0022886504
VL - 250
SP - 23
EP - 29
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 1
ER -