Related Experiment Videos
Bacterial lipopolysaccharide regulates the phosphorylation of the 68K protein kinase C substrate in macrophages
A Rosen1, A C Nairn, P Greengard
1Rockefeller University, New York 10021.
Abstract:
Bacterial lipopolysaccharide (LPS) potentiates protein kinase C (PKC)-dependent responses such as the activation of arachidonic acid metabolism in macrophages (Aderem, A. A., Cohen, D. S., Wright, S. D., and Cohn, Z. A. (1986) J. Exp. Med. 164, 165-179). Concomitantly, LPS promotes the myristoylation of a 68K PKC substrate, shown to be equivalent to the 80/87K PKC substrate found in brain and fibroblasts (Aderem, A. A., Albert, K. A., Keum, M. M., Wang, J. K., Greengard, P., and Cohn, Z. A. (1988) Nature 332, 362-364). We have now examined the effect of LPS on the phosphorylation of this 68K PKC substrate. We report here that LPS modifies the kinetics and extent of phosphorylation of the 68K protein. While treatment with LPS alone induces low level phosphorylation of the 68K protein, it markedly increases the rate of subsequent phorbol 12-myristate 13-acetate (PMA)-dependent phosphorylation of this protein. Phosphorylation in LPS-treated macrophages was maximal 1-2 min after administration of PMA, while maximal phosphorylation in macrophages not exposed to LPS was only achieved 6 min after addition of PMA. In addition to increasing the rate of PMA-dependent phosphorylation of the 68K protein in macrophages, LPS also promoted the phosphorylation of a novel peptide on the 68K protein. Thus while PMA stimulated the phosphorylation of two thermolytic phosphopeptides (phosphopeptides 1 and 2), the low level of phosphorylation observed with LPS alone was found to occur on phosphopeptides 1 and 2 as well as on a novel phosphopeptide (phosphopeptide 3). Furthermore, LPS treatment of macrophages potentiated phosphorylation of all three phosphopeptides when the cells were subsequently stimulated with PMA. While phosphorylation stimulated by LPS and PMA was slightly more than additive for phosphopeptides 1 and 2, it was markedly synergistic (increased 14.5-fold) for phosphopeptide 3. Phosphorylation of all three phosphopeptides occurred exclusively on serine. It is possible that LPS-induced myristoylation of the 68K protein directs it to the membrane where its phosphorylation is enhanced by its close association with PKC.
Insights
Bacterial lipopolysaccharide (LPS) enhances the rate and extent of 68K protein phosphorylation by protein kinase C (PKC) in macrophages. LPS also promotes synergistic phosphorylation of a novel peptide, suggesting a role in PKC-dependent signaling pathways.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Bacterial lipopolysaccharide (LPS) is known to potentiate protein kinase C (PKC)-dependent responses in macrophages, including arachidonic acid metabolism.
- LPS also promotes the myristoylation of a 68K PKC substrate, which is also found in brain and fibroblasts.
- The effect of LPS on the phosphorylation of this 68K PKC substrate has not been fully elucidated.
Purpose of the Study:
- To investigate the impact of LPS on the phosphorylation kinetics and extent of the 68K PKC substrate in macrophages.
- To identify novel phosphorylation sites or peptides affected by LPS treatment.
- To understand the synergistic effects of LPS and phorbol 12-myristate 13-acetate (PMA) on 68K protein phosphorylation.
Main Methods:
- Macrophages were treated with LPS alone or in combination with PMA.
- Phosphorylation of the 68K protein and its associated phosphopeptides was analyzed.
- Kinetics of phosphorylation were determined by measuring the time course of PMA-induced phosphorylation in LPS-treated and untreated cells.
- Thermolytic phosphopeptide mapping was used to identify distinct phosphorylation sites.
Main Results:
- LPS treatment alone induced low-level phosphorylation of the 68K protein.
- LPS significantly increased the rate of PMA-dependent phosphorylation, with maximal phosphorylation occurring within 1-2 minutes compared to 6 minutes in untreated cells.
- LPS promoted the phosphorylation of a novel phosphopeptide (phosphopeptide 3) in addition to two known phosphopeptides (1 and 2).
- LPS and PMA treatment resulted in synergistic phosphorylation of phosphopeptide 3 (14.5-fold increase) and additive phosphorylation of phosphopeptides 1 and 2.
- All observed phosphorylation occurred exclusively on serine residues.
Conclusions:
- LPS modifies the phosphorylation kinetics and extent of the 68K PKC substrate in macrophages.
- LPS potentiates PKC-dependent signaling by enhancing the rate of 68K protein phosphorylation and inducing synergistic phosphorylation of a novel serine residue.
- LPS-induced myristoylation may facilitate the 68K protein's localization to the membrane, enhancing its interaction with PKC and subsequent phosphorylation.