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Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family.

Reczek D, Berryman M, Bretscher A - J. Cell Biol. (1997)

Bottom Line: Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast.These findings show that EBP50 is a physiologically relevant ezrin binding protein.Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

View Article: PubMed Central - PubMed

Affiliation: Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

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Isolation and characterization of the human  placental N-ERMAD binding candidates. (A) An affinity binding assay similar to  that shown in Fig. 2, was  scaled up 100-fold, and  bound proteins were eluted  with urea. A silver-stained  12% gel of the peak fractions  is shown; the region in which  the binding proteins migrate  is bracketed. (B) Enlarged  view to show resolution of  the placental candidates into  three species: α, β, and γ. (C)  Binding protein heterogeneity is due to phosphorylation.  The proteins were recovered  from ezrin–N-ERMAD agarose beads by elution with NaI,  treated with alkaline phosphatase, and then analyzed on a 10% gel. Lane 1, untreated sample;  lane 2, phosphatase-treated sample; lane 3, phosphatase-treated  sample in the presence of phosphatase inhibitors. The arrow indicates the migration position of alkaline phosphatase. DF, dye  front.
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Figure 3: Isolation and characterization of the human placental N-ERMAD binding candidates. (A) An affinity binding assay similar to that shown in Fig. 2, was scaled up 100-fold, and bound proteins were eluted with urea. A silver-stained 12% gel of the peak fractions is shown; the region in which the binding proteins migrate is bracketed. (B) Enlarged view to show resolution of the placental candidates into three species: α, β, and γ. (C) Binding protein heterogeneity is due to phosphorylation. The proteins were recovered from ezrin–N-ERMAD agarose beads by elution with NaI, treated with alkaline phosphatase, and then analyzed on a 10% gel. Lane 1, untreated sample; lane 2, phosphatase-treated sample; lane 3, phosphatase-treated sample in the presence of phosphatase inhibitors. The arrow indicates the migration position of alkaline phosphatase. DF, dye front.

Mentions: A scaled-up version of the affinity binding assay was used to acquire sufficient amounts of each candidate for sequence analysis. The placental 50–53-kD bands were significantly enriched in the peak fractions (Fig. 3 A). Under these conditions, three major placental polypeptide bands, which we designate α, β, and γ, were resolved (Fig. 3 B). Amino acid analysis of each of these bands indicated essentially identical compositions, suggesting that they might be posttranslationally modified species of the same polypeptide. Although antiphosphotyrosine immunoblots indicated that the heterogeneity was apparently not the result of tyrosine phosphorylation (data not shown), treatment of the placental candidates with calf intestinal alkaline phosphatase resulted in a collapse of most or all of these species into a major polypeptide band migrating at 50 kD (Fig. 3 C, lane 2). Control experiments in which the enzyme was omitted, or phosphatase inhibitors were included, showed no detectable change in the migration of p50 α, β, or γ (Fig. 3 C, lanes 1 and 3). Therefore, most or all of the heterogeneity of the placental species is due to varying degrees of serine and/or threonine phosphorylation of a 50-kD polypeptide. We refer to this collection of polypeptides as EBP50.


Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family.

Reczek D, Berryman M, Bretscher A - J. Cell Biol. (1997)

Isolation and characterization of the human  placental N-ERMAD binding candidates. (A) An affinity binding assay similar to  that shown in Fig. 2, was  scaled up 100-fold, and  bound proteins were eluted  with urea. A silver-stained  12% gel of the peak fractions  is shown; the region in which  the binding proteins migrate  is bracketed. (B) Enlarged  view to show resolution of  the placental candidates into  three species: α, β, and γ. (C)  Binding protein heterogeneity is due to phosphorylation.  The proteins were recovered  from ezrin–N-ERMAD agarose beads by elution with NaI,  treated with alkaline phosphatase, and then analyzed on a 10% gel. Lane 1, untreated sample;  lane 2, phosphatase-treated sample; lane 3, phosphatase-treated  sample in the presence of phosphatase inhibitors. The arrow indicates the migration position of alkaline phosphatase. DF, dye  front.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139813&req=5

Figure 3: Isolation and characterization of the human placental N-ERMAD binding candidates. (A) An affinity binding assay similar to that shown in Fig. 2, was scaled up 100-fold, and bound proteins were eluted with urea. A silver-stained 12% gel of the peak fractions is shown; the region in which the binding proteins migrate is bracketed. (B) Enlarged view to show resolution of the placental candidates into three species: α, β, and γ. (C) Binding protein heterogeneity is due to phosphorylation. The proteins were recovered from ezrin–N-ERMAD agarose beads by elution with NaI, treated with alkaline phosphatase, and then analyzed on a 10% gel. Lane 1, untreated sample; lane 2, phosphatase-treated sample; lane 3, phosphatase-treated sample in the presence of phosphatase inhibitors. The arrow indicates the migration position of alkaline phosphatase. DF, dye front.
Mentions: A scaled-up version of the affinity binding assay was used to acquire sufficient amounts of each candidate for sequence analysis. The placental 50–53-kD bands were significantly enriched in the peak fractions (Fig. 3 A). Under these conditions, three major placental polypeptide bands, which we designate α, β, and γ, were resolved (Fig. 3 B). Amino acid analysis of each of these bands indicated essentially identical compositions, suggesting that they might be posttranslationally modified species of the same polypeptide. Although antiphosphotyrosine immunoblots indicated that the heterogeneity was apparently not the result of tyrosine phosphorylation (data not shown), treatment of the placental candidates with calf intestinal alkaline phosphatase resulted in a collapse of most or all of these species into a major polypeptide band migrating at 50 kD (Fig. 3 C, lane 2). Control experiments in which the enzyme was omitted, or phosphatase inhibitors were included, showed no detectable change in the migration of p50 α, β, or γ (Fig. 3 C, lanes 1 and 3). Therefore, most or all of the heterogeneity of the placental species is due to varying degrees of serine and/or threonine phosphorylation of a 50-kD polypeptide. We refer to this collection of polypeptides as EBP50.

Bottom Line: Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast.These findings show that EBP50 is a physiologically relevant ezrin binding protein.Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

View Article: PubMed Central - PubMed

Affiliation: Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

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