Phospholipase A(2) and Lipids as Potential Modulators of c-Raf-1 Kinase

c-Raf-1 is a proximal serine/threonine kinase in the signaling cascade of many mitogens. The cellular mechanisms responsible for regulation of this kinase remain ill-defined. Although c-Raf-1-associated proteins have been identified, including Ras, none of these have been found to activate c-Raf-1 kinase in vitro. To evaluate whether arachidonic acid or one of its products is implicated in c-Raf-1 activation, c-Raf-1 activity was measured in LLC-PK(1) kidney epithelial cells overexpressing the 100 kDa phospholipase A(2) (PLA(2)). As compared to control neomycin plasmid transfected cells, the cells overexpressing PLA(2) had a greater activation of c-Raf-1 in response to A23187 and phorbol ester stimulation. To explore the possibility that c-Raf-1 activity may be modulated directly by lipids, the enzymatic characteristics of c-Raf-1 were determined, and the effects of various possible lipid modulators on c-Raf-1 activity were examined. The K(m) of c-Raf-1 for ATP and mitogen-activiated protein kinase kinase (MAPKK), the only known physiologic substrate of c-Raf-1, were 11.6 &mgr;M and 0.8 &mgr;M, respectively. Of 13 lipids or combinations of lipids tested, including arachidonic acid and several eicosanoids, only phosphatidylserine and diacylglycerol in the presence of CA(2+) (2.5 mM) increased c-Raf-1 kinase activity significantly. The increase (1.5-fold) was approximately two orders of magnitude less than the stimulation of protein kinase C by these lipids. c-Raf-1 kinase activity and immunoreactivity eluted on gel filtration at a predicted molecular mass of greater than 150 kDa, suggesting that active c-Raf-1 is part of a multimeric complex. The absence of immunoreactive Ras in the active fractions confirms that the interaction is not necessary to maintain c-Raf-1 in an active state. In conclusion, a product of PLA(2) may play a role, together with Ras and another unidentified cofactor, in activating c-Raf-1. This lipid mediator(s) may directly or indirectly regulate the activity of c-Raf-1, but the identity of the mediator and its mode of interaction with c-Raf-1 and its associated proteins remain unclear.