Protein kinase C isoforms in human neutrophil cytoplasts.

Granule-poor human neutrophil cytoplasts, prepared without heat or cytochalasin B treatment so as to preserve both motile function and activatable respira- tory burst oxidase, were investigated for their content of several isoforms of protein kinase C (P1(C). Immunoblot- ting with isoform-specific rabbit antibodies (Abs) to PKC revealed that both the a-specifIc and the i3(I and II)- specific Abs recognized a protein band of 78 kd comigrat- ing with PKC from rat brain cytosol. The 'y-specific anti- serum did not detect any protein of this molecular mass. The cytoplast 13-PKC band was more readily detected than the cytoplast a-PKC band. Antibodies to 131- or 13-Il- specific PKC sequences showed the /311 subtype to be the predominant form of f3-PKC, although some /31 was also found. The identity of the 78-kd cytoplast bands as PKC was established by the fact that phorbol ester treatment of intact cytoplasts induced translocation of the bands from cytosol to membrane fractions. However, whereas PKC specific activity was similar in cytoplast lysates and brain cytosol, immunoreactivity of cytoplast a- and /3- PKC bands was considerably less than that of rat brain. Hydroxylapatite chromatography of partially purified cytoplast PKC revealed two major peaks of PKC activity precisely coeluting with brain a- and 13-PKC and display- ing comparable enzymatic activities despite the relatively weak immunoreactivity of cytoplast a- and 13-PKC. To our knowledge, this is the first demonstration that human neutrophil-derived cytoplasts contain a, /31, and /311 forms of PKC and that each isoform translocates from cytosol to membrane upon exposure to phorbol ester at concentrations that induce superoxide production. In ad- dition, our evidence raises the possibility that cytoplasts may also possess other isoforms of PKC that we are un- able to detect with our a, /3, and y antibodies. Finally, the granule-poor cytoplasts seem a particularly useful prepa- ration in which to examine the role of individual PKC isoforms in neutrophil activation. J. Leukoc. Biol. 51: 84-92; 1992.