Characterization of Two Isoforms of the Skeletal Muscle LIM Protein 1, SLIM1

Abstract

We have cloned and characterized a novel isoform of the skeletal muscle LIM protein 1 (SLIM1), designated SLIMMER. SLIM1 contains an N-terminal single zinc finger followed by four LIM domains. SLIMMER is identical to SLIM1 over the first three LIM domains but contains a novel C-terminal 96 amino acids with three potential bipartite nuclear localization signals, a putative nuclear export sequence, and 27 amino acids identical to the RBP-J binding region of KyoT2, a murine isoform of SLIM1. SLIM1 localized to the cytosol of Sol8 myoblasts and myotubes. SLIMMER was detected in the nucleus of myoblasts and, following differentiation into myotubes, was exclusively cytosolic. Recombinant green fluorescent protein-SLIM1 localized to the cytoplasm and associated with focal adhesions and actin filaments in COS-7 cells, while green fluorescent protein-SLIMMER was predominantly nuclear. SLIMMER truncation mutants revealed that the first nuclear localization signal mediates nuclear localization. The addition of the proposed nuclear export sequence decreased the level of exclusively nuclear expression and increased cytosolic SLIMMER expression in COS-7 cells. The leucine-rich nuclear export signal was required for the export of SLIMMER from the nucleus of myoblasts to the cytoplasm of myotubes. Collectively, these results suggest distinct roles for SLIM1 and SLIMMER in focal adhesions and nuclear-cytoplasmic communication.The LIM domain is a double zinc finger motif that mediates the protein-protein interactions of transcription factors, signaling, and cytoskeleton-associated proteins (1-4). LIM is an acronym of the three transcription factors, Lin-11, Isl-1, and Mec-3, in which the motif was first identified (5). The LIM domain contains 50 -60 amino acids, with the consensus sequence (CX 2 CX 17-19 HX 2 C)X 2 (CX 2 CX 16 -20 CX 2 (H/D/C)). The conserved cysteine and histidine residues form two zinc-binding pockets stabilizing the tertiary structure of the protein (6, 7). Despite their structural resemblance to GATA-1 zinc fingers, there is no evidence that LIM domains bind DNA directly. Instead, an increasing number of studies implicate LIM domains in protein-protein interactions that regulate development, cellular differentiation, and the cytoskeleton (8, 9).It has been proposed that LIM proteins form a scaffold upon which the coordinated assembly of proteins occurs (8,10). No single LIM domain-binding motif has been identified. LIM domains can associate with other LIM domains to form homoor heterodimers (8, 11). In addition, LIM domains also bind tyrosine-containing motifs, PDZ domains, ankyrin repeats, and helix-loop-helix domains in proteins lacking LIM domains including tyrosine and serine/threonine kinases, cytoskeletal proteins, and transcription factors (12)(13)(14)(15)(16).LIM proteins have been demonstrated in both the nucleus and cytoplasm. LIM homeodomain and LMO proteins are nuclear proteins, which regulate transcription by forming complexes with other transcription factors (2, 10). Members of the cysteine-rich...

SLIMMER (FHL1B/KyoT3) Interacts with the Proapoptotic Protein Siva-1 (CD27BP) and Delays Skeletal Myoblast Apoptosis

“…4C, a and d-f) and HA-SLIM-MER (Fig. 4F, a and c-e) localized to the nucleus in myoblasts as we reported previously (15). Following the differentiation of myoblasts to myotubes, SLIMMER, like Siva-1, exhibited a prominent cytoplasmic distribution (Fig.…”

“…We previously identified SLIMMER as a FHL1 splice isoform of unknown function (15). In this study, we have identified the proapoptotic protein Siva-1 (24) as a SLIMMER-specific binding partner.…”

“…4C-c). The ability of SLIMMER to shuttle in and out of the nucleus is conferred by its bipartite nuclear localization signal and leucine-rich nuclear export sequence (15). Endogenous Siva-1 and SLIMMER co-localized in the nucleus of myoblasts (Fig.…”

“…SLIMMER is generated by inclusion of an alternative exon 4b, leading to a 200-bp insert and frameshift after LIM domain 3. SLIMMER contains the FHL1 N-terminal three-anda-half LIM domains, followed by a unique C terminus with bipartite nuclear localization signals, a leucine-rich nuclear export sequence, and an RBP-J-binding site (15,16).…”

“…Structure-The fhl1 gene contains five exons (1-5) and encodes three different isoforms (FHL1, SLIMMER, and KyoT2) by alternate mRNA splicing (15) (Fig. 1).…”