Abstract
The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors.
Highlights
From the $Department of Biochemistry, University of Massachusetts Medical School, Worcester,Massachusetts 01655 and the Departments of ~ M e and W~Bioc~kmi~stsy,Duke Uniuers ~Medical Center, ~ ~ r No~rthmCaro,lina27710
Theresults obtained from the studies described in this report demonstrate that sphingosine is a potent inhibitor of protein kinase C in intact A431 cells
Treatment ofA431 cellmonolayers with sphingosine caused an inhibition of the binding of [3H]PDBUto cellular receptors (Table 11).Second, sphingosineinhibited the phosphorylation of two protein kinase C substrates (EGF receptor threonine 654 and transferrin receptor serine 24) in A431 cells treated with phorbol ester (Figs.10 and 11)
Summary
Regulation of EGF Receptor Phosphorylation by Sphingosine"A431 cells wereincubated for 24 h with [32P]phosphate in order to achieve steady-state labeling of the intracellular phosphate pools. Treatment of the cells with higher concentrations of sphingosine (>25 PM) resulted in a marked decrease in the phosphorylation state of many cellular proteins, including the EGF receptor (datanot shown). This general decrease in protein phosphorylation observed with high concentrations of sphingosine may reflect cytotoxic effects including cell permeabilization and thesubsequent release of nucleotides into the medium [19]. This action of sphingosine was not observed at lower concentrations which cause an increase in the phosphorylation state of the EGFreceptor (Fig. 1). It wasobserved that the effect of sphingosine to increase the phosphorylation state of the EGF
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