BiochemistryCloning and characterization of hdlg: The human homologue of the Drosophila discs large tumor suppressor binds to protein 4.1 (PSD-

hDlg, the human homologue of the Drosophila Discs-large (Dlg) tumor suppressor protein, is known to interact with the tumor suppressor protein APC and the human papillomavirus E6 transforming protein. In a two-hybrid screen, we identified a 322-aa serine͞threonine kinase that binds to the PDZ2 domain of hDlg. The mRNA for this PDZ-binding kinase, or PBK, is most abundant in placenta and absent from adult brain tissue. The protein sequence of PBK has all the characteristic protein kinase subdomains and a C-terminal PDZ-binding T͞SXV motif. In vitro, PBK binds specifically to PDZ2 of hDlg through its C-terminal T͞SXV motif. PBK and hDlg are phosphorylated at mitosis in HeLa cells, and the mitotic phosphorylation of PBK is required for its kinase activity. In vitro, cdc2͞cyclin B phosphorylates PBK. This evidence shows how PBK could link hDlg or other PDZ-containing proteins to signal transduction pathways regulating the cell cycle or cellular proliferation.

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. In this investigation, synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, glycophorin C, p55, and their non-erythroid homologues, interact with the cytoskeleton to exert their physiological effects.

hDlg is the human homolog of the Drosophila Discslarge tumor suppressor. As a member of the MAGUK (membrane-associated guanylate kinase) family of scaffolding proteins, hDlg is composed of three PDZ (PSD-95, Dlg, and ZO-1) repeats, an SH3 (Src homology 3) motif, and a GUK (guanylate kinase-like) domain. Additionally, hDlg contains two regions of alternative splicing. Here we identify a novel insertion, I1B, located Nterminal to the PDZ repeats. We further analyze the tissue-specific combinations of insertions and correlate those results with the distribution of protein isoforms. We also identify the functions of the two alternatively spliced regions. The N-terminal alternatively spliced region is capable of binding several SH3 domains and also moderates the level of protein oligomerization. Insertions in the second region are responsible for determining the localization of hDlg, with insertion I3 targeting the protein to the membrane regions of cell-cell contact and insertion I2 targeting the protein to the nucleus.First identified from human B lymphocyte cDNA, hDlg encodes a 100-kDa protein sharing ϳ60% similarity with Drosophila Dlg and 70% similarity with rat SAP90/PSD-95 (1). Both hDlg mRNA and protein are abundant in human and murine tissues (1). Consistent with the cellular localization of Dlg to septate junctions, hDlg localizes to regions of cell-cell contact in human epithelial cells (1). Dlg and hDlg are members of the MAGUK 1 family of proteins typically involved in protein scaffolding and cell signaling. MAGUK family members contain three core domains: either one or three PDZ repeats, an SH3 motif, and a GUK domain homologous to guanylate kinase enzymes.hDlg has been shown to interact with a number of cytosolic structural proteins. Protein 4.1 was the first protein shown to bind to hDlg (1). Two members of the ERM (ezrin, radixin, moesin) subfamily of cytoskeletal proteins with homology to protein 4.1 also bind to hDlg and, in turn, form a complex with the membrane glycoprotein CD44 (2). The GUK domain recruits a number of proteins including the GKAP/SAPAP/DAP family of proteins, BEGAIN and MAP1A (3,4).Additionally, hDlg binds to several proteins involved in cell cycle regulation and tumorigenesis. It has been recently demonstrated that PBK, a mitotic serine/threonine kinase, interacts with hDlg (5). The APC tumor suppressor forms a complex with hDlg that blocks progression to the S phase of the cell cycle (6). The human papilloma virus E6 and adenomavirus E4-ORF1 oncoproteins also interact with hDlg. Furthermore, mutant E6 and E4 lacking sequences required for hDlg binding loose their oncogenic capacity (7,8).Several alternatively spliced isoforms of hDlg have been described (Figs. 1 and 6). An alternatively spliced insertion called I1 (1) is located between the unique N-terminal domain of hDlg (NAG) and the first PDZ repeat. The region between the SH3 and GUK domains of hDlg was first characterized as containing two alternatively spliced insertions, I2 and I3 (1). In the same region, a third altern...

Abstract. hDlg, a human homologue of the Drosophila Dig tumor suppressor, contains two binding sites for protein 4.1, one within a domain containing three PSD-95/DIg/ZO-1 (PDZ) repeats and another within the alternatively spliced I3 domain. Here, we further define the PDZ-protein 4.1 interaction in vitro and show the functional role of both 4.1 binding sites in situ. A single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2 (PDZ1-2) contains the protein 4.1-binding site. Both this PDZ1-2 site and the 13 domain associate with a 30-kD NH2-terminal domain of protein 4.1 that is conserved in ezrin/radixin/moesin (ERM) proteins. We show that both protein 4.1 and the ezrin ERM protein interact with the murine form of hDlg in a coprecipitating immune complex. In permeabilized cells and tissues, either the PDZ1-2 domain or the 13 domain alone are sufficient for proper subcellular targeting of exogenous hDlg. In situ, PDZ1-2-mediated targeting involves interactions with both 4.1/ERM proteins and proteins containing the COOH-terminal T/SXV motif. I3-mediated targeting depends exclusively on interactions with 4.1/ERM proteins. Our data elucidates the multivalent nature of membrane-associated guanylate kinase homologue (MAGUK) targeting, thus beginning to define those protein interactions that are critical in MAGUK function. , 1994), is a member of a superfamily known as membrane-associated guanylate kinase homologues (MAGUKs) . The amino acid sequence of hDlg includes a core arrangement of several domains conserved in other MAGUK proteins: a domain that is homologous to the known guanylate kinases (GUK), a Src Homology domain 3 (SH3) motif, and three repetitive sequence motifs known as PSD-95/Dlg/ZO-1 (PDZ) repeats. PDZ sequences (Kennedy, 1995) have previously been described as GLGF repeats (Cho et al., 1992) and Dig Homology region domains . The MAGUK superfamily currently consists of three subgroups distinguished by size and variations in their domain organization (Fig. 1). While the PDZ-SH3-GUK core arrangement is well conserved among family members, several proteins contain i. Abbreviations used in this paper: dig, Drosophila discs-large; ERM, ezrin/radixin/moesin; GST, glutathione-S-transferase; GUK, guanylate kinase; MAGUK, membrane-associated guanylate kinase homologue; PDZ, PSD-95/DIg/ZO-1; PDZ1-2, a single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2; SH3, Src Homology domain 3; TB, targeting buffer; T/SXV, threonine/serine, X amino acid, valine.additional sequences that are probably essential to their differing cellular functions. For example, hDlg contains an NH2-terminal domain that is not found in other MAGUKs , and ZO-1 Willot et al., 1993) and ZO-2 (Jesaitis and Goodenough, 1994) both contain unique COOH-terminal domains. More recently, a rat neuronal homologue of hDlg (SAP97; Muller et al., 1995) has also been shown to contain the additional sequence domains characteristic of the human protein. The observation that recessive mutations ...