Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ.
The LIM motif defines a double zinc finger structure found in proteins involved in cell fate determination and growth control. LIM proteins, which include LIM homeo domain, LIM kinase, focal adhesion, and LIM-only proteins, usually contain two or more LIM motifs clustered at their amino- or carboxy-terminal end. At present, the mode of action of the LIM domain is not clear. In this study we have analyzed the binding properties of LIM motifs in the cellular environment. We show that MLP, CRP, and betaCRP define a subclass of LIM-only proteins with unique dual subcellular localization in the nucleus and along actin-based filaments in the cytosol. A double MLP construct that accumulated nearly exclusively along actin filaments promoted myogenic differentiation efficiently, arguing for a functional role of cytoskeleton-associated MLP. Binding of MLP to the actin cytoskeleton is specifically attributable to its second LIM motif. An additional LIM motif potentiates binding. Potentiating LIM motifs can be interchanged, resulting in differential targeting of interacting proteins. To analyze LIM-LIM interactions in situ, this property was exploited to develop a hybrid interaction approach based on the relocalization of LIM-containing constructs to the actin cytoskeleton. These experiments revealed the existence of marked selectivity in the interactions of single LIM motifs, and among LIM domains from different LIM-homeo domain and LIM-only proteins. Furthermore, the analysis suggested that the LIM motif has two interacting interfaces. On the basis of these findings, we propose that LIM motifs function as specific adapter elements to promote the assembly and targeting of multiprotein complexes.
Arber
S
S
Friedrich Miescher Institute, Basel, Switzerland.
Caroni
P
P
D016475:3T3 Cells; D008841:Actin Cytoskeleton / Q000737:chemistry; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; D060850:LIM-Homeodomain Proteins; D008667:Metalloproteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D011485:Protein Binding; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D016335:Zinc Fingers
D000199:Actins; D030161:Avian Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; D060850:LIM-Homeodomain Proteins; C513044:Lmo1 protein, mouse; D008667:Metalloproteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C074459:ap protein, Drosophila; C090114:cysteine and glycine-rich protein 3; C081447:cysteine-rich protein, avian; C074370:cysteine-rich protein, mammalian