Abstract
Treatment of Swiss 3T3 cells with sphingosine, a potential breakdown product of all sphingolipids, induced tyrosine phosphorylation of multiple substrates including bands of M(r) 110,000-130,000 and M(r) 70,000-80,000. Tyrosine phosphorylation in response to sphingosine occurred in a concentration dependent manner (EC50 = 10 microM) and developed gradually reaching half maximum and maximum effects at 20 and 60 min, respectively. The dihydroenantiomere of sphingosine, DL-threo-dihydrosphingosine, neither induced tyrosine phosphorylation nor interfered with sphingosine-stimulated tyrosine phosphorylation. Focal adhesion kinase (p125FAK) and paxillin were identified as prominent substrates for sphingosine-stimulated tyrosine phosphorylation. Cell permeable ceramides also stimulated tyrosine phosphorylation of the M(r) 110,000-130,000 band as well as p125FAK, but the effect was less pronounced than that of sphingosine. Tyrosine phosphorylation by sphingosine could be dissociated from both protein kinase C activation and Ca2+ mobilization from intracellular stores. Sphingosine stimulated striking actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells. The kinetics of actin stress fiber formation and tyrosine phosphorylation in response to sphingosine closely paralleled. Cytochalasin D, which disrupts the network of actin microfilaments, completely inhibited sphingosine induced tyrosine phosphorylation. In addition, tyrosine phosphorylation of p125FAK and paxillin in response to sphingosine was completely prevented when cells were stimulated in the presence of platelet-derived growth factor at a concentration (30 ng/ml) that caused disruption of the actin cytoskeleton. Our results demonstrate, for the first time, that sphingosine induces p125FAK and paxillin tyrosine phosphorylation, actin stress fiber formation and focal adhesion assembly in Swiss 3T3 cells.
Highlights
Tential breakdown product of all sphingolipids, induced synthesis, sphingosine elicits rapid mobilization of Ca2+from tyrosine phosphorylation of multiple substrates includ- intracellular stores [8, 9] and formation of phosphatidic acid ing bands of M, 110,000-130,000 and M, 7 0, 0 0 0 - 8 0, 0 0 0 . ~ - throughactivation of phospholipaseD [10]
Tyrosine Here we report that sphingosine stimulates tyrosine phosphosphorylation of ~ 1 2 an5d p~ax~illin in response to phorylation of multiple proteins in Swiss 3T3 cells including sphingosine was completelyprevented when cells were ~ 1 2 an5d p~axi~llin and induces striking actin stress fiber stimulated in the presence of platelet-derived growth factor at a concentration (30 ng/ml)that caused disrupformation andfocal adhesion assembly in thesecells
The kinetics of sphingosine induced tyrosine phosphorylation in Swiss 3T3 cells is shown in Fig. 1(lower panel)
Summary
The kinetics of sphingosine induced tyrosine phosphorylation in Swiss 3T3 cells is shown in Fig. 1(lower panel). Phosphorylation-Sphingosine induces activation of phospholipase D and mobilization of Ca2+ in intactcells [8,9,10] which or received a n equivalent amountof solvent (-) and subsequently lysed, could stimulate PKC, a potential pathway leading to tyrosine and lysates were further analyzed by immunoprecipitation with anti- phosphorylation of ~ 1 2 an5d ~pax~illin~(17, 19) Known thata thigh concentrations (>20 p ~ s)phingosine is an inhibitor of PKC in Swiss 3T3 cells [6].At the concentrations used in this study(12.5 PM), sphingosine induced a veryslight
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