Our previous study suggested that
N,N-dimethylsphingosine, but not unsubstituted
sphingosine, could be a modulator of
protein kinase C in
epidermoid carcinoma A431 cells, since
N,N-dimethyl-D-erythrosphingenine showed a stronger stereospecific effect on
protein kinase C activity in comparison with N,N-dimethyl-L-erythrosphingenine, unsubstituted D- or L-erythrosphingenine, and
gangliosides (Igarashi, Y., Hakomori, S., Toyokuni, T., Dean, B., Fujita, S., Sugimoto, M., Ogawa, T., El-Ghendy, K., and Racker, E. (1989) Biochemistry 28, 6796-6800). Other studies also indicated that commercial
sphingosine preparation has an enhancing effect on
epidermal growth factor (
EGF) receptor kinase activity in A431 cells (Davis, R. J., Girones, N., and Faucher, M. F. (1988) J. Biol. Chem. 263, 5373-5379; Faucher, M. F., Girones, N., Hannun, Y. A., Bell, R. M., and Davis, R. J. (1988) J. Biol. Chem. 263, 5319-5327). In the present paper, we report (i) the effect of
N,N-dimethylsphingosine as compared with lyso-
glycosphingolipids and other
sphingolipid breakdown products on
EGF receptor autophosphorylation and (ii) demonstration of endogenous
N,N-dimethylsphingosine synthesis and the virtual absence of unsubstituted
sphingosine in A431 cells. The autophosphorylation of
EGF receptor in the absence of
detergent was strongly enhanced by
N,N-dimethyl-D-erythrosphingenine; this effect was even obvious in the absence of
EGF and synergistic in the presence of
EGF. Similar enhancing activity was not produced by N,N-dimethyl-L-erythrosphingenine, D- and L-erythrosphingenine, N-monomethyl-D-erythrosphingenine, N-acetyl-D-erythrosphingenine, or the five lyso-
glycosphingolipids tested. Labeling of
sphingosine in A431 cells by culturing in medium containing [3H]Ser for various durations, followed by extraction and isolation of
sphingolipids by standard procedures, resulted in clear bands corresponding to
N,N-dimethylsphingosine and
ceramide, whereas the band corresponding to
sphingosine was virtually absent. The bands corresponding to
N,N-dimethylsphingosine and
ceramide intensified when cells were treated with metabolic inhibitor for
UDP-Glc:Cer beta-Glc
transferase (which causes accumulation of
ceramide). These results indicate that
N,N-dimethylsphingosine acts as a stereospecific enhancer for
EGF receptor kinase and is able to produce
EGF-like activity in vitro even in the absence of
EGF and
detergent. Under physiological conditions,
N,N-dimethylsphingosine is the major catabolite resulting from
ceramide breakdown.