In this study, we describe a new family of proteins, the eps8 family, whose founding member, eps8, was previously shown to be a key component of a multimolecular complex endowed with RacGEF activity (Scita et al., 1999
; Innocenti et al., 2003
). Our results show that members of this family link growth factor stimulation to actin organization, generating functional redundancy in the pathways that regulate actin cytoskeletal remodeling.
Members of the eps8 family share a modular organization consisting of a putative PTB domain, a central SH3 domain and a C-terminal effector region. The SH3 domains of eps8Ls display unique binding preferences for peptides containing a proline-X-X-aspartate-tyrosine (pXXDY) consensus and constitute a phylogenetically distinct subfamily within the SH3 domain family (Mongiovi et al., 1999
). In keeping with this, all eps8Ls can associate in vivo, with comparable efficiencies, to Abi1, a known interactor of the SH3 of eps8 that is required for the assembly of the multimeric Rac-GEF complex. However, in the case of eps8L3, this is not sufficient to form an active RacGEF or to substitute for eps8 in eps8 null MEFs, suggesting that additional structural or functional features are required. The C-terminal effector region fulfills this requirement. This region, in eps8, is essential for interaction with Sos-1 and its activation as a RacGEF. In addition, it mediates a direct interaction with F-actin, determining the intracellular localization of the protein at sites where actin is highly dynamic (Scita et al., 2001
). Consistently, eps8L1 and eps8L2, which display the highest homology to the eps8 in the C-terminal effector region, are capable of restoring ruffling response to RTK stimulation in eps8
null fibroblasts. Moreover, they can associate to actin in vivo and accumulate in PDGF-induced ruffles, thus further supporting the notion that relocalization of eps8Ls-based complexes is a prerequisite for their function in vivo. The observation that eps8L3, instead, does not contain a functional effector domain raises the question of its physiological role. We note that eps8L3 displays a punctuate, cytoplasmic staining reminiscent of proteins typically associated with vesicular organelles. Thus, it is tempting to speculate that eps8L3 may be involved in intracellular trafficking, a possibility currently under investigation.
Eps8L1 and eps8L2, similarly to eps8, mediate RTK-dependent actin remodeling, most likely by entering into a RacGEF complex with Abi1 and Sos-1. The existence of a family of proteins capable of forming multisubunit RacGEFs underscores additional complexity in the regulation of Rac. Multiple mechanisms have, indeed, been implicated in the activation of Rac by active plasma membrane receptors (Di Fiore and Scita, 2002
). These rely mainly on the existence of a variety of Rac-specific GEFs (Schmidt and Hall, 2002
), which are all comparably efficient in mediating Rac activation. Physiologically, however, the intracellular and the tissue distribution of the various RacGEFs is thought contribute to their specificity. The tissue distribution of the eps8 family of proteins supports this hypothesis.
Finally, the lack of overt phenotypic alterations in mice lacking eps8
can be rationalized in light of the observation that, at the mRNA level, the pattern of expression of eps8 overlaps with that of eps8L2, which, in turn, exhibits complete functional redundancy with eps8, at the protein level. It is of note that in organisms where eps8
gene duplication has not yet occurred or where only two members have evolved (Caenorhabditis elegans
and in Drosophila
, respectively) the genetic removal of eps8
leads to a number of developmental defects (A. Croce and P.P. Di Fiore, unpublished observations, and K. Moffat, personal communication). In mammalian systems, altered phenotypes (impaired Rac activation and actin remodeling in response to growth factor stimulation), caused by genetic removal of eps8
, could be unmasked only in mouse embryo fibroblasts, which express exclusively eps8
(Scita et al., 2001
, and this study). This suggests that possible alterations in eps8
null mice might be confined to those tissues or organs that do not show redundant expression of other family members, such as the brain. The lack of gross morphological defects in this organ (Scita et al., 2001
, and our unpublished results), however, points to the possibility that more subtle phenotypes, such as behavioral deficits, might be linked to eps8 removal. We are currently investigating this possibility.