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.
Bonventre
Joseph V.
JV
Medical Services, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Charlestown and Boston, USA.
Kyriakis
John M.
JM
Spech
Richard
R
Witzgall
Ralph
R
Force
Thomas
T