Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10655-10659, October 1994 The LIM/double zinc-finger motif functions as a dimerization domain RON FEUERSTEIN*, XINKANG WANG*, DECHENG SONG*, NANCY E. COOKE*, AND STEPHEN A. LIEBHABER*t# *Departments of Genetics and Medicine and the tHoward Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6145 Communicated by Y. W. Kan, June 28, 1994

ABSTRACT Protein-protein interactions resulting in MATERIALS AND METHODS dimerization and heterodimerization are ofcentral importance in the control ofgene expression and cell function. that Generation of cDNA Subclones and Fusion Constructs. share the 52-residue LIM/double zinc-finger domain are cDNA fragments corresponding to each of the desired re- involved in a wide range ofdevelopmental and cellular controls. gions of human CRP (hCRP) were generated by the poly- Some of these functions have been hypothesized to involve merase chain reaction (PCR) using primers (sequences avail- protein dimerization. In the present report we demonstrate, able upon request) containing EcoRP restriction sites. For using both in vitro and cell-based studies, that a representative synthesis ofglutathione S-transferase (GST) fusion proteins, LIM protein, human cysteine-rich protein (hCRP), can effi- the pGEX2T(128/129) vector (gft ofM. Blanar, University of ciently homodimerize. The dimerization ability of hCRP is California, San Francisco) was used. mapped to the LIM domains, can be transferred to an unre- Site-directed mutagenesis was carried out by PCR using lated protein by fusion of a single minimal LIM/double oligonucleotide primers (sequences available upon request) zinc-finger segment, occurs in the absence as well as the containing the appropriate point substitutions at amino acids presence of DNA, and appears to depend on coordination of 9 and 62. Each mutation was verified by DNA sequence two zinc atoms in the finger doublet. These observations analysis. support a specific role for protein dimerization in the function Antibody Production. A synthetic peptide representing ofproteins containing the LIM/double zinc-finger domain and residues 83-99 of hCRP was conjugated to keyhole limpet expand the general spec ofpotential interactions mediated hemocyanin by glutaraldehyde crosslinking and used to im- by zinc-finger motifs. munize rabbits (Hazelton Research Products, Lenexa, KS). Clarified antiserum was used without further purification. Proteins capable of dimerization tend to belong to extended Expression and Analysis of Recombinant Proteins. GST families that share particular protein-protein interaction mo- fusion proteins expressed and purifiedfrom clarified bacterial tifs. These motifs, which can mediate both homo- and het- lysate, as described (20), were incubated with a 50% slurry of erodimerization, including the leucine zipper (1, 2), helix- glutathione agarose beads (Sigma) for 15 min at 40C and loop-helix (3, 4), ankyrin (5), and PAS (6) domains. The LIM washed three times with phosphate-buffered saline (PBS), protein family, named for three of the originally identified and the fusion proteins were eluted by incubating beads with members, lin-11 (7), isl-1 (8), and mec-3 (9), is defined by the 2 bead vol of10 mM glutathione/50 mM Tris-HCl, pH 8.0, for presence of one to three repeats of a 52-residue segment 5 min at room temperature. Approximately 5 pg of each containing two adjacent zinc fingers separated by a two- protein was loaded on a 12% polyacrylamide/SDS gel and residue linker, (CX2CX17HX2C)-X2-(CX2CX17CX2C/H/D). electrophoresed for 3 hr at 200 V followed by electrotransfer Of the 12 LIM proteins defined thus far, 7 contain a DNA- to a nitrocellulose membrane and immunoblotting using binding homeodomain adjacent to the LIM domains, and the standard techniques. Antibody binding on Western blots was remaining 5 members lack a homeodomain. The LIM pro- detected by using the ECL system (Amersham). teins are involved in a wide range ofcell functions, including Generation of 32p Labed hCRP Fragments. For radiola- transcription activation [isl-1, lmx-1 (10)], somatic patterning beling, bacterially expressed GST-hCRP fusion proteins [lin-11, mec-3, apterous (11)], focal cell adhesion [zyxin (12)], were incubated with glutathione-agarose beads, and the oncogenic transformation [rhombotin (13, 14)], and immedi- loaded beads were then washed and labeled for60 min at 370C ate-early response to serum stimulation [cysteine-rich pro- in 2 bead vol of lx HMK buffer (21) containing cAMP- tein (CRP) (15)]. There have been reports suggesting the dependent (Sigma) at 1 unit/pl, ['y.32P]ATP involvement of LIM proteins in protein-protein interactions (5000 Ci/mmol, 10 mCi/ml; Amersham; 1 Ci = 37 GBq) at 10 (10, 16, 17). In one case two LIM proteins lacking homeo- puCi/pl, and 1 mM dithiothreitol. After incubation the beads domains, CRP and zyxin, copurified as a complex from focal were washed five times with PBS. For cleavage, the labeling adhesion plaques (12). Although a "docking model" has been putforward which hypothesizes that LIM proteins can dimer- mix was washed once in thrombin cleavage buffer (50 mM ize (18), it has not been demonstrated that this occurs, nor has Tris HCl, pH 8.0/150 mM NaCl/2.5 mM CaCl2/0.1% 2-mer- it been shown that the LIM domain directly mediates such captoethanol) followed by incubation in 2 bead vol of lx interactions. In the present study we demonstrate that a thrombin cleavage buffer containing human thrombin (Sig- representative LIM protein, CRP (19), a highly conserved, ma) at a 1:10 wt ratio of to substrate for 40 min at widely distributed, immediate-early gene containing two re- room temperature. The beads were sedimented and the peated LIM/double zinc-finger domains, can homodimerize labeled CRP fragments were collected from the supernatant. when tested in vitro and in vivo and that the LIM domain is necessary and sufficient for this activity. Abbreviations: CRP, cysteine-rich protein; hCRP, human CRP; GST, glutathione S-transferase; CAT, chloramphenicol acetyltrans- ferase. The publication costs ofthis article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Genetics, University of Pennsylvania, Room 437A Clinical Re- in accordance with 18 U.S.C. §1734 solely to indicate this fact. search Building, Philadelphia, PA 19104. 10655 Downloaded by guest on October 2, 2021 10656 Biochemistry: Feuerstein et al. Proc. Nad. Acad Sci. USA 91 (1994)

A B 'v IN 6 8

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(2-193) ANTI-hCRP - PEPTIDE A (2-110) 1 8 ,71 GST/hCRP WESTERN FUSIONS (62-1 1 8) (80-118) _j C D C, 32P(2-1 93) 32P(1 18-171) 32P(62-1 18) AC' .~ b C lb 0 ,5S~s &4k$'C 40 4 THROMBIN 68 68 W.,- eM -4-- - ilIIE- hCR 43 .1 w ...... W4it 43 t sit-.. THROMBINtT 32P/HMK 29

29

(118-171) 23kD t ._.. a hCRP E-t ;P COMPETITOR

0 (80-118) *e:

FIG. 1. Filter overlay assay demonstrating specificity of CRP dimerization; localization of the interacting region to the LIM domain. (A) Diagram of CRP. CRP contains two LIM/double zinc-finger domains (15, 19, 24). A glycine-rich region of undetermined function is located immediately carboxyl-terminal to each LIM/double finger domain. The position of peptide A, synthesized for epitope-specific antiserum production, is shown. The extent of each CRP segment that was fused to GST is indicated. (B) Expression and analysis of GST/CRP fusion proteins. (Upper) Coomassie blue-stained, 12% polyacrylamide/SDS gel ofpurified GST and five different GST-hCRPfusionproducts. Positions of the molecular mass markers (in kDa) are shown to the left of the gel. (Lower) Western blot analysis of the fusion protein gel shown in Upper with anti-hCRP polyclonal antisera specific to the peptide A epitope. (C) Generation of 32P-labeled hCRP proteins. Bacterially expressed GST-hCRP41-193) fusion protein was labeled by incubation withbovine heart muscle kinase (HMK) and [t32P]ATP, then cleaved with thrombin and analyzed on a 12% polyacrylamide/SDS gel. The diagram of the GST-hCRP fusion protein indicates the approximate positions of the thrombin cleavage site and the HMK phosphorylation site. The 32P-hCRP-(118-171) and 32P-hCRP-(62-118) probes were generated in identical fashion. (D) Overlay binding assay for CRP-CRP interaction. GST-CRP proteins were electrotransferred from 12% polyacrylamide/SDS gels to nitrocellulose membranes, incubated with 32P-labeled hCRP42-193) or the indicated subfiagments in the presence or absence of unlabeled competitors, washed, and autoradiographed. The identity ofeachfusion protein is indicated at the tops ofthe lanes; the positions ofsize markers (kDa) are noted. The identity of the 32P-labeled probe used in each gel is shown at its top. With the 32P-hCRP-(118-171) probe, the middle and bottom membranes were preincubated with 5 pAM unlabeled GST-hCRP-(118-171) or GST-hCRP-(80-118) (estimated 15,000-fold molar excess of competitor).

Filter Overlay Binding Assay. Electrotransfer from SDS/ Two-Hybrid Interaction Assay. VP16 and GAL4 expression polyacrylamide gels to nitrocellulose membranes was carried vectors were gifts of C. Dang (Johns Hopkins University). out at 20 V for 30 min, followed by incubation of the Each ofthe cDNA frMgments used in this assay was generated membranes for 12-15 hr at room temperature in blocking by PCR (see above) and cloned in plasmid pNLVP, which solution (5% bovine serum albumin/50 mM Tris HCl, pH contains the transcriptional activating domain for the Herpes 7.5/150 mM NaCi). When a competition experiment was simplex viral protein VP16. DNA encoding full-length hCRP- performed, the filters were incubated with unlabeled com- (1-193) was inserted into pGAL4 plasmid, which encodes petitors in overlay buffer (OB) (20 mM Hepes, pH 7.5/0.25% amino acids 1-147 of the DNA-binding domain of the yeast gelatin/0.5% bovine serum albumin/0.1% 2-mercaptoetha- protein GAL4. The full-length hCRP-(1-193) construct in- nol/1 mM EGTA/10 mM NaCi) for 2 hr at room temperature cludes an additional 12 amino acids between GAML and hCRP followed by 4 hr in OB containing probe at 600,000 cpm/ml. encoded by the hCRP 5' nontranslated region. NIH 3T3 cells After incubation, membranes were washed twice in universal at 90%o confluency were transfected with combinations of buffer (50 mM Tris HCl, pH 7.5/150 mM NaCl) for 5 min and supercoiled plasmids (total of 30 .g) by Ca3(P4)2 coprecip- once in Sarkosyl buffer (1 M NaC1/0.5% N-lauroylsarcosine/ itation and assayed for chloramphenicol acetyltransferase 0.25% gelatin/50 mM Tris HCI, pH 7.5) for 30 min. Mem- (CAT) activity by the standard technique (23) after 2 days in branes were then dried and autoradiographed. culture. Plasmid pCH110, which contains the /-galacto- Downloaded by guest on October 2, 2021 Biochemistry: Feuerstein et al. Proc. Natl. Acad. Sci. USA 91 (1994) 10657 A C VP16 1 .2 GAL4 TRANSCRIPTIONAL -V r-No ACTIVATION 1.01 4-i ._ .8 GAL4 SITE EIB TATA 6

4-J 4 GAL4 S IT= A + GAL4 SITE EIB TATA 0 .-- - .- --I GAL 4 FUSION (1.193) | (1-193) GAL4 (1-193) V.-193) (1.193) B VP16 FUSION - (2.188)1 VP (2-188) (2-188) (7-79) (62-118)

:. fI "' ;)DIp (7. , 5- xi)JU-COD- -r,' t " I U .13 q,-, I s. (7-79) hCRP FRAGMENTS 4 A62-118) 0. FUSED TO VP16 4- _ (2-188) 1 2 3 4 5 6 7 hCRP FRAGMENT (-t193) FUSED TO GAL4 FIG. 2. Analysis of CRP-CRP interaction by the two-hybrid interaction assay. (A) Two-hybrid protein interaction assay. In the upper part of the diagram, the DNA-binding domain of GAL4 and the transcriptional activation domain of VP16 do not directly interact and the minimal E1B promoter controlling CAT gene expression is not activated. In the lower part ofthe diagram, CAT gene transcription is activated ifGAL4 and VP16 can be brought together by their fusion protein partners. The fusion protein extension on each is a representation of the LIM/double-finger domain. (B) Map of the hCRP cDNA fiagments fused to VP16 and GAL4 fragments. The first two subclones, (7-79) and (62-118), contain the LIM and inter-LIM regions, respectively, while the third, (2-188), encompasses both regions. (C) Two-hybrid interaction results. Each bar on the histogram represents an average taken from at least three separate experiments run in duplicate. A representative CAT analysis is shown below the histogram. Each lane is labeled with the chimeric plasmids transfected alone or in combination into NIH 3T3 cells. Negative controls were included to be sure that hCRP does not bind directly to GALA tGAL4 + VP16-hCRP-(2-188)], to VP16 [GAL4-hCRP- (1-193) + VP16], or act as a transcriptional activator on its own [GAL4-hCRP-(1-193)]. The level of activity of the GAL4-hCRP-(1-193) with VP16-hCRP-(2-188) was 20%6 of that generated by fusing the same GAL4 and VP16 regions to the leucine zipper domains of fos and jun, respectively (data not shown). sidase coding sequence under the control of the simian virus Fig. 1). GST and each of the five GST fusion proteins were 40 early promoter, was cotransfected along with each set of electrotransferred from a polyacrylamide gel to a nitrocellu- test plasmids, and the amount of cell lysate used for individ- lose membrane, allowed to renature in situ, and then incu- ual CAT assays was adjusted to the relative levels of (-ga- bated with a series of32P-labeled protein probes representing lactosidase activity in the extract. CAT assays were quanti- full-length hCRP-(2-193), the isolated second LIM/double- tated by PhosphorImager (Molecular Dynamics) analysis. finger domain hCRP-(118-171), and the inter-LIM segment Means and standard errors for data points were calculated (n hCRP-(62-118) (Fig. 1C). The first two LIM/double-finger- 2 3) by using the paired t test. containing probes [hCRP-(2-193) and hCRP-(118-171)] bound efficiently to each of the GST fusion constructs RESULTS containing one or both LIM domains but bound only at background (GST) levels to the two inter-LIM fiagments The LIM/Double Zinc-Flnger Doman Mediates Protein- (Fig. 1D, Top Left and Center). In contrast, the inter-LIM Protein Interaction in Vitro. Full-length hCRP and four hCRP probe hCRP-(62-118) bound only weakly and equivalently to LIM/double-finger and inter-LIM subsegments were synthe- all fragments at levels comparable to GST binding, indicating sized as GST fusion proteins (Fig. 1A). Sizes of each fusion lack of specificity (Fig. 1D, Top Right). protein expressed in Escherichia coli were in agreement with To document the specificity of the LIM-LIM interaction, the predicted structure: GST, 28 kDa; GST-hCRP-(2-193), 53 binding competitions were carried out (Fig. 1D, Middle and kDa; GST-hCRP-(2-110), 42 kDa; GST-hCRP-(118-171), 36 Bottom). Unlabeled LIM/double-finger domain (118-171) or kDa; GST-hCRP-(62-118), 36 kDa; and GST-hCRP-(80-118), inter-LIM segment (80-118) fusion proteins were incubated 34 kDa (Fig. 1B Upper). As a second check on structure, each with the filter prior to addition of the labeled LIM probe ofthe fusion proteins was probed with a polyclonal antibody (118-171). The unlabeled LIM fragment blocked virtually all raised against 83-99 of hCRP. As expected, four of the five specific binding of the LIM probe, while the inter-LIM fusion proteins contained this inter-LIM epitope (peptide A; segment was ineffective. Downloaded by guest on October 2, 2021 10658 Biochemistry: Feuerstein et al. Proc. Natl. Acad. Sci. USA 91 (1994) A B

2.0

,1 .0 C t C 1.6

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(5Z6 I ~Zn CC b CD 4 -~~1 (C,'¢)t 1S) I/Z .2 CS 0 f' ) ef GAL 4 FUSION (1-193)1-L9341 -1 93)(1-193)(1-193 VP16 FUSION (2-188) ZnCC I ZnCS ZnSC znss I C)0 SC S C)Y Zn

( C)Znt) Ss

1 2 3 4 5 FiG. 3. Fidelity of the zinc fingers is necessary for CRP-CRP interaction. (A) Schematic representation of hCRP, the LIM/double-finger domain subfiagment, and the mutants that were studied. The position(s) of the cysteine-to-serine substitution(s) is shown. Each fragment was ligated into the VP16 vector and then cotransfected along with the GAL4-hCRP-(1-193) plasmid to assess dimerizing potential by the level of CAT activity. (B) The two-hybrid protein interaction assay was carried out and analyzed as described for Fig. 2. Each bar on the histogram represents an average taken from at least three separate experiments run in duplicate. A representative CAT analysis is shown below the histogram. The signal intensity for (3AL4-hCRP-(1-193) cotransfected with VP16hCRP-(ZnCc) was set at 1.0 in each experiment, and all other values were taken as a ratio of this value. The intensity of CAT activity generated with VP16hCRP-(ZnCC) could not be clearly distinguished from VP16-hCRP-(2-188) (P > 0.1) but differed significantly from that obtained with each of the cysteine-to-serine mutants [P < 0.001 (**)]. LIM/Double Zinc-Fine-Mediated Protein Interaction in The Zinc-Finger Doublet Is Necessry and Sufcint for Intact Cells. The studies summarized in Fig. 1 demonstrate Protein-Protein Interaction. The dimerization- efficiency of a that the LIM/double-finger domain can mediate protein- minimal LIM/double-fmnger domain extending from the first protein interaction in vitro. This interaction was further to the last cysteine in the zinc-finger doublet (9-62) was next tested by using a two-hybrid interaction assay (25-27) dia- tested by using the two-hybrid assay (Fig. 3A; ZnCC). Re- grammed in Fig. 2A. In the initial experiment dimerization of markably, this minimal domain was sufficient to mediate the full-length hCRP was tested by fusing hCRP to both the interaction with the GAL4-hCRP-(1-193) fusion protein at GAL4 DNA-binding domain and the VP16 transcriptional 70% of full-length hCRP (Fig. 3B, lanes 1 and 2). The activation domain (Fig. 2B)' and determining if these two importance of the zinc fingers was then directly tested by fusion proteins were brought together by their respective interrupting the coordination of the zinc atoms. Cysteine-to- hCRP extensions. A set of negative controls demonstrated serine substitutions have been shown to result in a 95% that these fusion proteins did not individually activate the reduction in zinc coordination to zinc fingers (28, 29) (Fig. reporter (Fig. 2C, lanes 1-4). When the GAL4-hCRP chimera 3A). When the first, last, or both first and last cysteines ofthe was coexpressed with the VP-hCRP-(2-188) chimera, a sig- minimal LIM domain were replaced by serines, the protein- nificant increase in CAT reporter activity was generated (lane protein interaction was decreased 5-fold (Fig. 3B, lanes 3-5). 5). To localize the interacting segments of hCRP, the first The equivalent impact of all three cysteine-to-serine substi- LIM/double-finger domain, (7-79), and the inter-LIM seg- tutions suggests that both zinc fingers are necessary for ment, (62-118), were separately fused to VP16 and tested for efficient protein-protein interaction. their interactions with GAIA-hCRP (Fig. 2 B and C). In contrast to the minimal interaction by the inter-LIM domain, the isolated LIM/double-finger domain (7-79) mediates DISCUSSION dimerization with hCRP-(1-193) at a level comparable to that The in vitro and cell-based studies presented in this report of the full-length hCRP (Fig. 2C, lanes 5-7). Although the demonstrate that the LIM/double zinc-finger domain of level of (62-118) expression in the. transfected cells is not hCRP is necessary and sufficient for protein dimer formation. directly demonstrated, the parallel absence of significant In vitro dimerization of hCRP was demonstrated in a gel interaction of the. (62-118) segment with full-length hCRP' in overlay binding assay using GST fusion constructs (Fig. 1). the in vitro binding assay confirms the conclusion'that the Protein-protein interaction was specific for the presence of inter-LIM domain is not necessary to mediate CRP-CRP the LIM/double-finger domain and could be reduced by interaction. competition with unlabeled LIM-containing protein seg- Downloaded by guest on October 2, 2021 Biochemistry: Feuerstein et al. Proc. Nati. Acad. Sci. USA 91 (1994) 10659 ments. Furthermore, the fact that the hCRP-(118-171) bound the potential roles for zinc fingers beyond the established with equal intensity to both the first [GST-hCRP-(2-110)] and DNA/RNA-binding activities. second [GST-hCRP-(118-171)] LIM domains suggests that, at least under the conditions of the gel overlay assay, there 1. Landschulz, W. H., Johnson, P. F. & McKnight, S. L. (1988) is no discrimination between the two LIM motifs of hCRP in Science 240, 1759-1764. 2. Turner, R. & Tjian, R. (1989) Science 243, 1689-1694. their protein-binding efficacy. The dihybrid interaction ap- 3. Murre, C., McCaw, P. S. & Baltimore, D. (1989) Cell 56, proach was utilized to determine whether this in vitro assay 777-783. reflects the situation in the intact cell and whether this 4. Benezra, R., Davis, R. L., Lockshon, D., Turner, L. & Wein- interaction is mediated by the LIM domain. The results (Fig. traub, H. (1990) Cell 61, 49-59. 2) demonstrate not only that CRP-CRP protein interaction is 5. Blank, V., Kourilsky, P. & Israel, A. (1992) Trends Biochem. dependent on the LIM region but also that this region is fully Sci. 17, 135-140. sufficient for this interaction, as it is possible to transfer this 6. Huang, Z. J., Edery, I. & Rosbash, M. (1993) Nature (London) 364, 259-262. interactive property to an unrelated protein by the transfer of 7. Freyd, G., Kim, S. K. & Horvitz, H. R. (1990) Nature (Lon- the minimal LIM/double zinc-finger domain [GAL4-hCRP- don) 344, 876-879. (9-62)]. 8. Karlsson, O., Thor, S., Norberg, T., Ohlsson, H. & Edlund, T. The structure of the LIM domain has been recently shown (1990) Nature (London) 344, 879-882. to form a zinc-finger domain, as was originally hypothesized 9. Way, J. C. & Chalfie, M. (1988) Cell 54, 5-16. on the basis of primary structure comparisons (19). The 10. German, M. S., Wang, J., Chadwick, R. B. & Rutter, W. J. evidence supporting this structural conformation consists of (1992) Genes Dev. 6, 2165-2176. 11. Cohen, B., McGuffin, M. E., Pfeifle, C., Segal, D. & Cohen, studies demonstrating an appropriate stoichiometry ofzinc to S. M. (1992) Genes Dev. 6, 715-729. LIM domain (2:1) (12, 30) and the solution ofthe structure of 12. Sadler, I., Crawford, A. W., Michelsen, J. W. & Beckerle, this domain by NMR (31). Whether the protein interactive M. C. (1992) J. Cell Biol. 119, 1573-1587. property of this region reflected intact zinc-finger structure 13. McGuire, E. A., Hockett, R. D., Pollock, K. M., Bartholdi, was studied by selectively destabilizing the zinc finger by M. F., O'Brien, S. J. & Korsmeyer, S. J. (1989) Mol. Cell. site-directed mutation of the coordinating cysteines. The Biol. 9, 2124-2132. results ofthese studies (Fig. 3) demonstrate that dimerization 14. Boehm, T., Foroni, L., Kennedy, M. & Rabbitts, T. H. (1990) is dependent on the intact nature and full coordination ofboth Oncogene 5, 1103-1105. 15. Wang, X., Lee, G., Liebhaber, S. A. & Cooke, N. E. (1992)J. of the adjacent zinc fingers. The sum of these results allows Biol Chem. 267, 9176-9184. us to conclude that the double zinc-finger domain functions 16. Leonard, J., Serup, P., Gonzalez, G., Edlund, T. & Montminy, in protein-protein interactions. M. (1992) Proc. Natl. Acad. Sci. USA 89, 6247-6251. The in vitro evidence and the in vivo evidence for the 17. Xue, D., Tu, Y. & Chalfie, M. (1993) Science 261, 1324-1328. protein interactive property of the LIM domain presented in 18. Rabbitts, T. H. & Boehm, T. (1990) Nature (London) 346, 418. this report are mutually supportive. On the basis of the 19. Liebhaber, S. A., Emery, J. G., Urbanek, M., Wang, X. & N. E. Nucleic Res. structure ofthe proteins in the LIM family and precedents in Cooke, (1990) Acidi 18, 3871-3879. 20. Smith, D. B. & Johnson, K. S. (1988) Gene 67, 31-40. other systems, it has been suggested that such interactions 21. Blanar, M. A. & Rutter, W. J. (1992) Science 256, 1014-1018. are central to the functioning of the LIM proteins in their 22. Cooke, N. E. & Liebhaber, S. A. (1993) Human Protein Data, native cellular roles (18). For example, there are striking ed. Haeberli, A. (VCH, Weinheim, F.R.G.). parallels between the proteins in the helix-loop-helix (HLH) 23. Gorman, C. M., Merlino, G. T., Wiiling, M. C., Pastan, I. family and the LIM family. Both the LIM and HLH proteins & Howard, B. H. (1982) Mol. Cell Biol. 2, 1044-1051. can be divided into two classes: those that contain and those 24. Michelsen, J. W., Schmeichel, K. L., Beckerle, M. C. & that lack definable DNA-binding domains. These DNA- Winge, D. R. (1993) Proc. Natl. Acad. Sci. USA 9, 4404-4408. 25. Fields, S. & Song, 0. (1989) Nature (London) 340, 245-246. binding domains are homeodomains in the LIM family and 26. Chien, C. T., Bartel, P. L., Sterlanz, R. & Fields, S. (1991) basic domains in the HLH family (22). MyoD, from the HLH Proc. Natl. Acad. Sci. USA 88, 9578-9582. family, contains a basic DNA-binding domain and activates 27. Fearon, E. R., Finkel, T., Gillison, M. L., Kennedy, S. P., transcription when dimerized with a second HLH protein Cosella, J. F., Tomoselli, G. F., Morrow, J. S. & Van Dang, C. also containing a DNA-binding domain. This interaction is (1992) Proc. Natl. Acad. Sci. USA 89, 7958-7962. blocked when MyoD dimerizes with Id, an HLH protein 28. Severne, Y., Wieland, S. T., Schaffner, W. & Rusconi, S. lacking a DNA-binding domain (4). LIM proteins might (1988) EMBO J. 7, 2503-2508. interact similarly: LIM/homeodomain proteins might trans- 29. Webster, L. C., Zhang, K., Chance, B., Ayene, I., Culp, J. S., activate by forming dimers and might be silenced by com- Huang, W. J., Wu, F. Y. H. & Ricciardi, R. P. (1991) Proc. Natl. Acad. Sci. USA 88, 9989-9993. bining with a LIM protein lacking a homeodomain (18). Such 30. Kosa, J. L., Michelsen, J. W., Louis, H. A., Olsen, J. I., combinatorial interactions of proteins in the LIM family Davis, D. R., Beckerle, M. C. & Winge, D. R. (1994)Biochem- might modulate and gene expression. The istry 33, 468-477. present data establish a possible mechanism by providing 31. Perez-Alvarado, G. C., Miles, C., Michelsen, J. W., Louis, H., evidence that the LIM domain can mediate protein-protein Winge, D. R., Beckerle, M. C. & Summers, M. F. (1994) association. In this way these findings also serve to extend Nature Struct. Biol. 1, 388-398. Downloaded by guest on October 2, 2021