Src kinase and PI 3-kinase as a transduction pathway in ceramide-induced contraction of colonic smooth muscle

Abstract

Ceramide mediates sustained contraction of smooth muscle cells. C2 ceramide induced a rapid increase in Src kinase activity within 15 s, peaked at 1 min, and was sustained up to 8 min. Contraction and Src kinase activity were inhibited in cells incubated in Ca2+-free medium containing 2 mM EGTA and in cells preincubated with herbimycin A, a Src kinase inhibitor. Immunoblotting using a phosphospecific anti-Src (416Y) antibody showed a ceramide-induced increase in pp60 src tyrosine phosphorylation. Immunoprecipitation using an anti-phosphotyrosine antibody followed by Western immunoblotting using a monoclonal IgG anti-phosphoinositide 3-kinase NH2 terminal-SH2 domain antibody showed a ceramide-induced increase in phosphoinositide 3-kinase (PI 3-kinase) tyrosine phosphorylation at a protein mass corresponding to 85 kDa, the regulatory subunit of PI 3-kinase, which contains the Src kinase binding site. PI 3-kinase phosphorylation was inhibited by herbimycin A and by the PI 3-kinase inhibitors wortmannin and LY-294002. Preincubation of cells with herbimycin A or PI 3-kinase inhibitors also resulted in an inhibition of mitogen-activated protein (MAP) kinase p42 and p44 activities as seen on Western blots. In summary, we found that 1) the maintenance of sustained contraction is dependent on extracellular Ca2+; 2) ceramide activates a nonreceptor tyrosine kinase pathway through activation of pp60 src and PI 3-kinase; and 3) the converging signals are probably through activation of MAP kinase.

Ceramide Regulates Lipopolysaccharide-Induced Phosphatidylinositol 3-Kinase and Akt Activity in Human Alveolar Macrophages

“…This is not the first study to show that ceramide can activate PI 3-kinase and Akt. Human colonic smooth muscle cells have been shown to have PI 3-kinase activation downstream of ceramide (72,82). The other significant study demonstrating ceramide activation of PI 3-kinase is that by Hanna and Brindley (54), demonstrating that TNF-induced ceramide in fibroblasts activated PI 3-kinase via tyrosine kinase activity and p21…”

Functional Dichotomy of Protein Kinase C (PKC) in Tumor Necrosis Factor-α (TNF-α) Signal Transduction in L929 Cells

“…Given the facts that agents that activate the PI 3-kinase/Akt1 pathway trigger an anti-apoptotic signal (59) and that ceramide regulates phospholipase D and PKC (12,60,61) and leads to activation of apoptotic signaling, it is conceivable that TNF-␣ and/or ceramide could inhibit PKC␣ through regulation of phosphoinositide-dependent kinase-1. In fact, several recent studies have reported that TNF-␣ and/or ceramide activates PI 3-kinase (62,63), whereas Zundel and Giaccia (64) have shown the inhibition of PI 3-kinase/Akt kinase by ceramide. A few other studies have demonstrated the inhibition of Akt kinase activity by ceramide, but the effect of ceramide on PI 3-kinase activity was not significant (65,66).…”

Cell-permeable ceramides increase basal glucose incorporation into triacylglycerols but decrease the stimulation by insulin in 3T3-L1 adipocytes

“…23 Therefore, the effects of short-chain ceramides are mainly direct rather than involving metabolism to sphingosine or sphingosine 1-phosphate. 23,24 TNFa and ceramides stimulate PI 3-K in 3T3 L1 adipocytes, 22,25 fibroblasts, 23,24 smooth muscle cells, 26 osteoclasts 45 and macrophages. 46 These increases in PI 3-K are associated with protection from apoptosis and increased division rather than cell death.…”

“…22 Our later studies with Rat2 fibroblasts demonstrate that TNFa, sphingomyelinase and C 2 -ceramide rapidly stimulate tyrosine kinases, production of Ras-GTP and its association with activated PI 3-K. 23,24 Furthermore, incubation of 3T3-L1 adipocytes with TNFa increases the tyrosine phosphorylation of IRS-1 and its binding to PI 3-K. 25 Ceramides also activate Src and PI 3-K in smooth muscle cells. 26 In L6 myocytes, ceramides inhibit insulin-stimulated glucose uptake, but this did not depend upon decreased IRS-1 phosphorylation, or PI 3-K activity. 27 Ceramides appear to inhibit insulin-stimulated glucose uptake downstream of PI 3-K at the level of protein kinase B (PKB).…”