Distinct requirements for the Sprouty domain for functional activity of Spred proteins

Abstract

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.

Sprouty proteins: modified modulators, matchmakers or missing links?

“…SPRED1 and 2 are tyrosine phosphorylated by stem cell factor, PDGF, and EGF and, like SPRY, SPRED can inhibit ERK phosphorylation stimulated by NGF, and FGF , Bundschu et al 2007. It is presently unclear how SPRED proteins differ from SPRY proteins in exercising their respective inhibitory effects on the Ras/ERK pathway, however, studies have shown that, under certain conditions, both the EVH1 and the CRD are indispensable for this function (King et al 2005). SPRED3 shows less inhibitory activity than SPRED1 and 2, suggesting that a functional KBD may also be required.…”

“…In addition, replacing the CRD of the relatively weak inhibitor SPRED3 with that of SPRED1 results in the chimera inhibiting ERK phosphorylation as strongly as SPRED1 itself . Furthermore, there is an apparently functional variation among the CRDs of the SPRED family; King et al (2005) provided evidence that while the CRD mediates heterodimer formation, the CRD of SPRED2 is required for ERK inhibition, whereas the equivalent domain of SPRED1 is not, indicating that the two isoforms use different mechanisms for the inhibition of the ERK pathway. A recent report implicated the CRD of SPRY in mediating its binding to Caveolin1 (Cabrita et al 2006), another protein that also binds to SPRED (Nonami et al 2005) and, although the region for mediating this binding was not demonstrated, they revealed that various SPRY2 mutants (including the CRD mutation at R242D) failed to bind to caveolin and concomitantly showed a compromised ability to downregulate ERK phosphorylation.…”

“…As yet, no bona fide interacting partner for the EVH1 domain of SPRED has been identified, while a number of proteins bind to the common CRD of both SPRED and SPRY family members. Complicating matters further, both SPRED proteins can homodimerize and heterodimerize via the CRD (King et al 2005), and the possibility remains that SPRED and SPRY proteins can heterodimerize via the same mechanism.…”

A shared molecular mechanism underlies the human rasopathies Legius syndrome and Neurofibromatosis-1

“…Mammals contain three homologs, Spred 1, Spred 2, and Spred 3, each of which can negatively regulate the Ras/ Raf/mitogen-activated protein kinase (MAPK) pathway (Kato et al 2003;King et al 2006). However, the precise mechanism by which the Spreds act remains unclear, and it has been reported to function both upstream of and downstream from Ras (Wakioka et al 2001;King et al 2005). Overexpression of Spred1 can increase Raf's recruitment to the plasma membrane, where it associates with Ras without stimulating Raf activation (Wakioka et al 2001).…”

“…Yet, this mechanism has never been fully refined and fails to explain how Spred1 prevents Raf activation. A separate report suggests that Spred1 may prevent Ras activation, as evidenced by decreased Ras-GTP levels when Spred1 is overexpressed (King et al 2005).…”