The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein-protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.

Grating and plaid masks indicate linear summation in a contrast gain pool

In human vision, the response to luminance contrast at each small region in the image is controlled by a more global process where suppressive signals are pooled over spatial frequency and orientation bands. But what rules govern summation among stimulus components within the suppressive pool? We addressed this question by extending a pedestal plus pattern mask paradigm to use a stimulus with up to three mask components: a vertical 1 c/deg pedestal, plus pattern masks made from either a grating (orientation = -45°) or a plaid (orientation = ±45°), with component spatial frequency of 3 c/deg. The overall contrast of both types of pattern mask was fixed at 20% (i.e., plaid component contrasts were 10%). We found that both of these masks transformed conventional dipper functions (threshold vs. pedestal contrast with no pattern mask) in exactly the same way: The dipper region was raised and shifted to the right, but the dipper handles superimposed. This equivalence of the two pattern masks indicates that contrast summation between the plaid components was perfectly linear prior to the masking stage. Furthermore, the pattern masks did not drive the detecting mechanism above its detection threshold because they did not abolish facilitation by the pedestal (Foley, 1994). Therefore, the pattern masking could not be attributed to within-channel masking, suggesting that linear summation of contrast signals takes place within a suppressive contrast gain pool. We present a quantitative model of the effects and discuss the implications for neurophysiological models of the process. © 2004 ARVO.

Adaptation and gain pool summation:Alternative models and masking data

Foley [J. Opt. Soc. Am. A 11 (1994) 1710] has proposed an influential psychophysical model of masking in which mask components in a contrast gain pool are raised to an exponent before summation and divisive inhibition. We tested this summation rule in experiments in which contrast detection thresholds were measured for a vertical 1 c/deg (or 2 c/deg) sine-wave component in the presence of a 3 c/deg (or 6 c/deg) mask that had either a single component oriented at -45° or a pair of components oriented at ±45°. Contrary to the predictions of Foley's model 3, we found that for masks of moderate contrast and above, threshold elevation was predicted by linear summation of the mask components in the inhibitory stage of the contrast gain pool. We built this feature into two new models, referred to as the early adaptation model and the hybrid model. In the early adaptation model, contrast adaptation controls a threshold-like nonlinearity on the output of otherwise linear pathways that provide the excitatory and inhibitory inputs to a gain control stage. The hybrid model involves nonlinear and nonadaptable routes to excitatory and inhibitory stages as well as an adaptable linear route. With only six free parameters, both models provide excellent fits to the masking and adaptation data of Foley and Chen [Vision Res. 37 (1997) 2779] but unlike Foley and Chen's model, are able to do so with only one adaptation parameter. However, only the hybrid model is able to capture the features of Foley's (1994) pedestal plus orthogonal fixed mask data. We conclude that (1) linear summation of inhibitory components is a feature of contrast masking, and (2) that the main aftereffect of spatial adaptation on contrast increment thresholds can be assigned to a single site. © 2002 Elsevier Science Ltd. All rights reserved.