Synergistic activation of protein kinase C alpha, -beta I, and -gamma isoforms induced by diacylglycerol and phorbol ester: Roles of membrane association and activating conformational changes
Sj. Slater et al., Synergistic activation of protein kinase C alpha, -beta I, and -gamma isoforms induced by diacylglycerol and phorbol ester: Roles of membrane association and activating conformational changes, BIOCHEM, 38(12), 1999, pp. 3804-3815
Protein kinase C alpha (PKC alpha) has been shown to contain two discrete a
ctivator sites with differing binding affinities for phorbol esters and dia
cylglycerols. The interaction of diacylglycerol with a low-affinity phorbol
ester binding site leads to enhanced high-affinity phorbol ester binding a
nd to a potentiated level of activity [Slater, S. J., Ho, C., Kelly, M. B.,
Larkin, J, D., Taddeo, F. J., Yeager, M, D., and Stubbs, C. D. (1996) J, B
iol. Chem. 271, 4627-4631]. In this study, the mechanism of this enhancemen
t of activity was examined with respect to the Ca2+ dependences of membrane
association and accompanying conformational changes that lead to activatio
n. The association of PKC alpha with membranes containing 12-O-tetradecanoy
lphorbol 13-acetate (TPA) or 1,2-dioleoylglycerol (DAG), determined fi om t
ryptophan to dansyl-PE resonance energy transfer (RET) measurements, was fo
und to occur at relatively low Ca2+ levels (less than or equal to 1 mu M).
However, PKC alpha was found to be inactive even though membrane associatio
n was complete at these Ca2+ levels and further titration of Ca2+ to a conc
entration of similar to 100 mu M was required for activation, This increase
in Ca2+ concentration also led to a further increase in RET, which was due
to a Ca2+-induced activating conformational change, as verified by an acco
mpanying increase in the PKC alpha tryptophan fluorescence anisotropy. Coad
dition of DAG and TPA resulted in a reduction in the Ca2+ levels required f
or both the conformational change and enzyme activation. Also, it was found
that incubation of the enzyme with TPA alone resulted in a time-dependent
increase in the Ca2+-independent PKC alpha activity, the rate and extent of
which was further enhanced upon coaddition with DAG, The results suggest t
hat the enhanced level of activity induced by coaddition of DAG and TPA inv
olves both Ca2+-dependent and Ca2+-independent activating conformational ch
anges which result in active conformers of PKC alpha distinct from those fo
rmed by interaction with either activator separately.