Sprouty and Spred {Sprouty-related EVH1 [Ena/VASP (vasodilator-stimulated phosphoprotein) homology 1] domain} proteins have been identified as antagonists of growth factor signalling pathways. We show here that Spred-1 and Spred-2 appear to have distinct mechanisms whereby they induce their effects, as the Sprouty domain of Spred-1 is not required to block MAPK (mitogen-activated protein kinase) activation, while that of Spred-2 is required. Similarly, deletion of the C-terminal Sprouty domain of Spred-1 does not affect cell-cycle progression of G(0)-synchronized cells through to S-phase following growth factor stimulation, while the Sprouty domain is required for Spred-2 function. We also demonstrate that the inhibitory function of Spred proteins is restricted to the Ras/MAPK pathway, that tyrosine phosphorylation is not required for this function, and that the Sprouty domain mediates heterodimer formation of Spred proteins. Growth-factor-mediated activation of the small GTPases, Ras and Rap1, was able to be regulated by Spred-1 and Spred-2, without affecting receptor activation. Taken together, these results highlight the potential for different functional roles of the Sprouty domain within the Spred family of proteins, suggesting that Spred proteins may use different mechanisms to induce inhibition of the MAPK pathway.
The abundance in vivo of each of three subunits b, OSCP and d, components of the stalk region of the yeast mitochondrial ATP synthase complex, was manipulated by a controlled depletion strategy. Western blots of whole cell lysates were used to study the effect of depletion of each of these subunits on the cellular levels of other subunits of the enzyme complex. A hierarchy of subunit stability was determined and interpreted to indicate the order of assembly of these three subunits of the stalk region. Thus, subunit b is assembled first, followed by OSCP and then by subunit d.
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