The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

Abstract

PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein ␣-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal ␤-TM). The interaction between Enigma and skeletal ␤-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal ␤-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal ␤-TM in transfected cells. The association of Enigma with skeletal ␤-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells. INTRODUCTIONConserved protein interaction domains found in many proteins with diverse functions provide molecular recognition essential for assembling multiprotein complexes (Pawson and Scott, 1997). LIM and PDZ domains are two protein interaction motifs that are widely distributed in cells of both plants and animals (Gill, 1995;Fanning and Anderson, 1996). LIM domains, named for the three homeodomain proteins in which they were first recognized (lin-11, isl-1, and mec-3) (Jurata and Gill, 1998) are cysteine-rich modules that contain two coordinated Zn 2ϩ atoms (PerezAlverado et al., 1994). Nuclear LIM domains interact with nuclear LIM interactor (Agulnick et al., 1996, Jurata et al., 1996 and transcription factors (Wadman et al., 1994), whereas cytoplasmic LIM domains bind to protein kinases (Wu and Gill, 1994;Kuroda et al., 1996;Wu et al., 1996), other LIM domains (Schmeichel and Beckerle, 1994), and cytoskeletal targets (Crawford and Bekerle, 1992;Arber and Caroni, 1996;Pomies et al., 1997). PDZ domains, named for the three proteins in which they were first recognized (postsynaptic density-95, discs large, and zo1 tight junction protein) (Fanning and Anderson, 1996), are ϳ85 amino acid ␤-barrel structures (Doyle et al., 1996) that bind to the consensus sequence (Ser/Thr)-X-(Val/Leu/Ile) contained in targets, most commonly at the carboxyl terminus (Kim et al., 1995;Kornau et al., 1995;Songyang et al., 1997). PDZ domains are also reported to recognize internal consensus sites (Shieh and Zhu, 1996), other PDZ domains , spectrinlike repeats (Xia et al., 1997), LIM domains (Cuppen et al., 1998), and unspecified sites (Tsunoda et al., 1997). Several PDZ ...

Human balanced translocation and mouse gene inactivation implicate Basonuclin 2 in distal urethral development

“…Targeted mutations of two other LIM/PDZ genes encoding actin-associated LIM protein or ZASP/Cypher/Oracle in mice have been found to cause congenital heart and muscle pathology. 25,26 Expression of both BNC2 and LMO7 was enriched in human fetal heart and penis compared with other tissues. However, alteration of LMO7 expression is unlikely to cause the observed phenotypes: the breakpoint was 3¢ of this gene, whereas long-range position effects are generally associated with breakpoints in 5¢ regulatory regions.…”

Unusual binding interactions in PDZ domain crystal structures help explain binding mechanisms

“…[13][14][15][16][17][18] PDLIM7 is known to bind b-tropomyosin via its PDZ domain and, therefore, also acts to direct LIM-binding proteins to the cytoskeleton. 19 A similar association has not yet been observed for PDLIM5, which is, however, of interest due to its potential as a susceptibility gene for schizophrenia. 20 The NMR structures of the apo PDZ domains from human PDLIM4 [RIKEN structural genomics initiative (RSGI)] and PDLIM6 21 have been solved previously, as well as the NMR structures of mouse PDLIM3 (94% sequence identity to human PDLIM3 over the PDZ domain) and PDLIM6 (100% identical) also from RSGI (PDB IDs 1EEG, 1RGW, 1V5L, 1WJL, respectively).…”

“…In the case of PDLIM7, the construct contained the same C-terminus (ITSL) as b-tropomysin, its known binding partner. 19 All of the structures were determined by molecular replacement starting with models of previously determined PDZ domains, followed by alteration to the correct sequence and addition of coordinates for missing atoms including the C-terminal extensions and solvent molecules where appropriate.…”

Identification and characterization of genes upregulated in cells transformed by v-Jun

“…Both domains are protein-protein interaction motifs. A relative of RIL, the protein termed Enigma, functions as adaptor molecule, mediating the assembly of multiprotein complexes and coupling cell surface receptors to downstream components of cell signaling Guy et al, 1999;Wu et al, 1996). The PDZ motif of RIL has been shown to bind the protein phosphatase PTP-BL as well as the LIM domain of RIL itself (Cuppen et al, 1998).…”