ENC-1: A Novel MammalianKelch-Related Gene Specifically Expressed in the Nervous System Encodes an Actin-Binding Protein

Abstract

We have identified and characterized a novel murine gene, , that is an early and highly specific marker of neural induction in vertebrates. ENC-1, which encodes a kelch family related protein, is expressed during early gastrulation in the prospective neuroectodermal region of the epiblast and later in development throughout the nervous system (NS). ENC-1 expression is highly dynamic and, after neurulation, preferentially defines prospective cortical areas. The only apparent expression of ENC-1 outside the NS is restricted to the rostral-most somitomere of the presomitic mesoderm, at the times corresponding to the epithelialization that precedes somite formation. Cellular expression of epitopetagged ENC-1 shows extensive co-localization of ENC-1 with the actin cytoskeleton, and immunoprecipitation studies demonstrate a physical association between ENC-1 and actin. ENC-1 functions as an actin-binding protein that may be important in the organization of the actin cytoskeleton during neural fate specification and development of the NS.

Krp1, a novel kelch related protein that is involved in pseudopod elongation in transformed cells

“…It should also be noted that when Krp1-myc was expressed in 208F cells, and when we examined the low level of endogenous Krp1 that is present in FBR-TAM67 cells, no co-localization with F-actin containing stress ®bres was detected. This result contradicts work presented by Hernandez et al, (1997) and Soltysik-Espanola et al (1999), who reported that two other kelch family members, ENC1 and Mayven, interact with stress ®bres containing F-actin. Together these data suggest that Krp1 only co-localizes with F-actin when it is in the looped membrane ru e-like structure at the tips of pseudopodia.…”

“…Another protein, alpha-scruin of Limulus, which has kelch repeats present at both its amino and carboxyl termini but lacks a BTB/POZ domain, cross-links actin ®laments within the acrosomal processes of sperm (Way et al, 1995a). Further kelch proteins are also thought to interact with actin: murine and human ENC1 (Hernandez et al, 1997(Hernandez et al, , 1998; spe-26 of C. elegans (Varkey et al, 1995); and human Mayven (Soltysik-Espanola et al, 1999). Many other family members are thought not to interact with actin; beta-scruin of Limulus, for instance, is found in sperm acrosomal vesicles, which do not contain actin (Way et al, 1995b).…”

“…Examination of Krp1 expression in pseudopod tips at a higher magni®cation revealed that it was present in membrane ru e-like structures that appeared to be forming a looping conformation (Figure 3d). Because it is known that some kelch family proteins interact with actin (Xue and Cooley, 1993;Hernandez et al, 1997;Soltysik-Espanola et al, 1999), and that the yeast Tea1 kelch family member localizes at the ends of microtubules (Mata and Nurse, 1997), we performed two double immuno¯uorescence experiments on FBR cells: anti-Krp1 antiserum and phalloidin to detect Factin; anti-Krp1 and tubulin antisera (Figure 3b ± d). Both tubulin and Krp1 were present in the cytoplasm, but while Krp1 extended into the loop-like structures at the tips of pseudopodia, tubulin stopped just short of these structures (Figure 3b).…”

“…Cells were washed twice in PBS and then lysed in EBC bu er (50 mM Tris-HCL pH 8, 120 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA) containing 10 mg/ml aprotinin, leupeptin and 1 mM PMSF (Hernandez et al, 1997). Protein concentrations were measured with a commercial reagent (Bio-Rad) and 50 mg of proteins were electrophoresed on either a SDS-7.5% or 10% polyacrylamide gel using standard methods.…”

“…We show that Krp1 is a member of a family of proteins that is de®ned by a carboxyl terminal series of approximately 50 amino acid repeats (the kelch repeats), and a protein ± protein interaction domain, named the BTB/POZ domain, near its amino terminus (Xue and Cooley, 1993;Hernandez et al, 1997;Kim et al, 1998). Members of this family have been shown to be involved in cytoskeleton organization and function (Xue and Cooley, 1993;Hernandez et al, 1997;Kim et al, 1998;Robinson and Cooley, 1997). We report that rat Krp1 is cytoplasmically localized in FBR cells, and accumulates at the tips of pseudopodia, where it co-localizes with F-actin.…”