(Received for publication, March 8, 1995; and in revised form, August 16, 1995)
From the
Here we describe a family of closely related LIM domain proteins in avian cells. The LIM motif defines a zinc-binding domain that is found in a variety of transcriptional regulators, proto-oncogene products, and proteins associated with sites of cell-substratum contact. One type of LIM-domain protein, called the cysteine-rich protein (CRP), is characterized by the presence of two LIM domains linked to short glycine-rich repeats and a potential nuclear localization signal. We have identified and characterized two evolutionarily conserved members of the CRP family, CRP1 and CRP2, in chicken and quail. Expression of the genes encoding both CRP1 and CRP2 is differentially regulated in normal versus transformed cells, raising the possibility that members of the CRP family may function in control of cell growth and differentiation.
A number of genes are specifically and rapidly up-regulated in
response to growth factor stimulation(1) . The expression of
these primary response, or immediate early, genes is independent of new
protein synthesis and requires only the activation of pre-existing
transcriptional regulators(2) . The primary response genes
encode proteins including transcription factors, proto-oncogene
products, and other regulatory proteins that facilitate the transition
of the cell from an arrested to a proliferative growth state and
stimulate differentiation(3, 4, 5) . The
first cysteine-rich protein family member to be described (referred to
here as CRP1) ()was shown to be encoded by the primary
response gene, CSRP1, that exhibits serum induction coordinate
with c-myc expression(6, 7) .
From
analyses of both human and chicken cDNA, genomic DNA, and protein
sequences(6, 7, 8, 9) , it has been
determined that CRP1 contains two copies of a specific amino acid
sequence motif termed LIM. The LIM motif displays the consensus amino
acid sequence
CXCX
HX
CX
CX
CX
CX
(C/H/D) (8) . Spectroscopic studies of LIM domains derived from a
number of different proteins have revealed that the LIM domain
specifically coordinates two zinc
ions(10, 11, 12, 13) . The solution
structure of a LIM domain derived from chicken CRP1 has been solved by
two-dimensional NMR and illustrates that the LIM domain is itself a
bipartite structure with spatially distinct modules focused around each
metal binding site (14) . Interestingly, although the LIM
domain has been clearly demonstrated to function in specific
protein-protein interactions (15, 16, 17) ,
the tertiary fold of one zinc-binding module within the LIM domain is
essentially identical to that found in well characterized DNA-binding
zinc fingers(14) . It remains to be determined whether the
structural features of the LIM domain reflect a biologically
significant ability to associate with nucleic acids as well as
proteins.
The LIM motif was first identified in three developmentally regulated transcription factors, C. elegans Lin-11, rat Isl-1, and C. elegans Mec-3, from which the term LIM is derived(18, 19) . The LIM domain is often found in association with obvious functional domains, such as a DNA-binding homeodomain (18, 19) or a kinase domain(20) . However, the LIM domain may also represent the primary sequence element in a protein. Examples of such ``LIM only'' proteins include CRP1(6, 7, 8, 9) , the cysteine-rich intestinal protein(21) , and rhombotin(22, 23, 24, 25) . Interestingly, although the LIM-only proteins lack DNA-binding homeodomains, they may also function in the regulation of cell growth and differentiation. For example, rhombotin-2 is a proto-oncogene product that is required for erythroid differentiation during mouse development (26) and overexpression of rhombotin genes in the thymus of transgenic mice results in T-cell acute lymphoblastic leukemia(27, 28) .
CRP1 has been purified to homogeneity from chicken smooth muscle, and many of its biochemical and biophysical properties have been characterized(9) . Binding studies have revealed that CRP1 interacts directly with another LIM protein called zyxin(8, 9) . Both zyxin and CRP1 are localized at sites of membrane-substratum contact in association with the actin cytoskeleton(9) . Together these proteins are postulated to perform a regulatory or signaling function at the adhesive membrane(8, 9, 15) .
Recently, the level of a quail transcript that encodes a LIM domain protein with a high degree of structural similarity to CRP1 was shown to be dramatically reduced in avian fibroblasts transformed by retroviral oncogenes or chemical carcinogens(29) . The amino acid sequence of the predicted quail protein was 79.8% identical to that of human CRP1(29) , and the protein was therefore postulated to represent the quail homologue of human CRP1. Subsequently, the complete amino acid sequence of chicken CRP1 was deduced from a cDNA clone and shown to be 90.6% identical to human CRP1(9) . Because the quail protein, although obviously closely related to CRP1, was significantly less similar to human CRP1 than was chicken CRP1, we postulated that the quail protein represented a new member of the CRP family, a CRP2. In this report, we demonstrate that both chicken and quail display multiple genes encoding CRP proteins. We have characterized two members of this gene family, CSRP1 and CSRP2, in both species.
Chicken genomic DNA containing a CSRP2 fragment was isolated by the polymerase chain reaction using primers corresponding to nucleotides 354-374 and 558-577 of CSRP2-TM1 cDNA. Sequencing of the genomic DNA fragment was conducted using methods described above.
As described above, previous work had suggested that multiple CSRP family members might be represented in the vertebrate genome. Here we have explored the possibility that two avian species, the chicken and the quail, express multiple forms of CRP. In this paper, the cysteine-rich protein that was originally described in human (6, 7) and chicken (8, 9) is referred to as CRP1. The closely related gene product that was originally described in quail (29) is here referred to as CRP2 to indicate that we consider it to be a member of the same family. The CSRP gene symbols refer to genes that encode members of the CRP family of proteins.
Figure 1: Nucleotide sequence of quail CSRP1 cDNA and deduced amino acid sequence of the quail CRP1 protein. The nucleotide and amino acid sequence positions are numbered in the right and left margin, respectively. Translational start and stop codons and the polyadenylation signal are underlined. The conserved cysteine and histidine residues that define the two LIM domains are boxed. Glycine residues that occur in the glycine-rich repeat following each LIM domain are circled. The sequence is deposited in the EMBL data base under the accession number Z28333.
Figure 2: Nucleotide sequence of chicken CSRP2 cDNA and deduced amino acid sequence of the chicken CRP2 protein. A, a schematic representation of the isolated CSRP2 cDNA clones used to generate the composite CSRP2 cDNA. B, the nucleotide (numbered on the right) and deduced amino acid (numbered on the left) sequences of the composite cDNA clone (CSRP2) are shown. The translational initiation codon, stop codon, and the polyadenylation signal are underlined. Cysteine and histidine residues that contribute to the LIM consensus motif are boxed. Glycine residues that are found in the repeat adjacent to each LIM domain are circled. The sequence is deposited in the EMBL data base under the accession number X84264.
In multiple CSRP2 cDNAs that were characterized, none extended the 3` end beyond what was observed for CSRP2-TM1. Likewise, none of the clones isolated in the original screen contained any useful 5` sequence beyond what was found in CSRP2-TM1. Therefore, in order to identify cDNA clones that contained the 5` end of the coding sequence and the 5`-untranslated region corresponding to the CSRP2 mRNA, we used two strategies. First, we employed a modified primer extension-5`-RACE technique (36) to identify the 5` end of the CSRP2 cDNA; a representative cDNA clone derived from this screen is referred to as CSRP2-5`-RACE (Fig. 2A). In addition, we screened an independently generated chicken embryo fibroblast cDNA library (37) with the quail CSRP2 probe. Four clones that were identified in this screen displayed 5` extensions of the cDNA insert that contained the remainder of the coding sequence as well as some of the 5`-untranslated region as observed in the CSRP2-5`-RACE cDNA.
The nucleotide and deduced amino acid sequences of the composite CSRP2 cDNA derived from the fusion of CSRP2-TM1 with the product of the 5`-RACE is shown in Fig. 2B. The initiation codon, termination codon, and polyadenylation signal are underlined. The predicted chicken CRP2 protein is 194 amino acids in length with a calculated molecular weight of 20,925 and a predicted unmodified pI of 8.68. As in the case of other CRP family members, chicken CRP2 displays two LIM domains with associated glycine-rich motifs and a potential nuclear localization signal. The metal-coordinating cysteine and histidine residues that contribute to the LIM consensus are boxed in Fig. 2B. The chicken CRP2 amino acid sequence is 76.6% identical to chicken CRP1 and 99.5% identical to quail CRP2, with only a single conservative amino acid substitution at residue 95 distinguishing the chicken and quail CRP2 homologues.
We have extended these studies here to evaluate whether CSRP1 and CSRP2 behave in a similar manner when cells are challenged with a transforming factor. The levels of CSRP1 and CSRP2 mRNAs in normal chicken embryo fibroblasts, normal quail embryo fibroblasts, and transformed quail embryo fibroblasts were evaluated by Northern analysis (Fig. 3). In chicken embryo fibroblasts, the CSRP1 probe recognizes two transcripts, a minor species of 1.4 kb and a major species of 1.0 kb, but only a single transcript of 1.0 kb is detected in quail embryo fibroblasts (Fig. 3, A and C). The CSRP2 probe hybridizes to a single transcript of 0.9 kb that is present in both chicken and quail embryo fibroblasts (Fig. 3, B and D). CSRP1 expression is strongly decreased in the v-myc-transformed quail embryo fibroblast line Q8 (Fig. 3, A and C). However, longer exposures of the autoradiographs (not shown) reveal the presence of residual CSRP1 mRNA in transformed cells. In contrast, there is a nearly complete loss of CSRP2 transcripts in such cells (Fig. 3, B and D), in confirmation of our previous results. The level of mRNA encoding the enzyme glyceraldehyde-3-phosphate dehydrogenase was used as an internal control for the quality and amount of mRNA present in each lane (39) .
Figure 3:
Expression of CSRP1 and CSRP2 mRNAs in normal and transformed avian fibroblasts. Northern blot
analyses of poly(A) RNA (2 µg/lane) from chicken
embryo fibroblasts (CEF), quail embryo fibroblasts (QEF), and the v-myc-transformed quail fibroblast
line Q8 (Q8) are shown. RNAs were hybridized with a
P-labeled chicken CSRP1 probe (4.1
10
cpm) (A), a chicken CSRP2 probe (4.6
10
cpm) (B), a quail CSRP1 probe
(6.0
10
cpm) (C), or a quail CSRP2 probe (5.4
10
cpm) (D). The
autoradiographs were exposed for 4.5 h (A and B) or
2.5 h (C and D) using intensifying screens. The
positions of ribosomal RNAs are indicated in the margin.
Hybridization with a
P-labeled quail
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (4.9
10
cpm; 4-h exposure) was used as an internal
control.
In order to rule out the possibility that the suppression of CSRP1 and CSRP2 expression occurs only in long term transformed cell lines such as Q8, we analyzed their expression in a conditional transformation system and during the process of the initial establishment of oncogene-induced transformation. In quail embryo fibroblasts transfected with proviral DNA from a temperature-sensitive mutant of Rous sarcoma virus, expression of CSRP2 is strongly induced upon shift of the cells from the permissive to the nonpermissive temperature and strongly reduced again upon shift from the nonpermissive to the permissive temperature (Fig. 4A). Thus, suppression of CSRP2 expression directly correlates with the morphologically transformed state of the cells at the permissive temperature. Likewise, in quail embryo fibroblasts freshly infected with the avian MH2 retrovirus and passaged three times until complete morphological transformation of the culture was observed, complete suppression of CSRP2 expression and full spread of virus-induced transformation coincide (Fig. 4B). Collectively, these data are a strong indication of a direct correlation between cell transformation and the suppression of CSRP2 expression. The level of transcripts encoding CRP1 is also negatively correlated with the degree of cell transformation; however, the changes in CSRP1 mRNA levels are not as great as we observe for CSRP2 transcripts (Fig. 4, A and B). The distinct changes in the levels of CSRP1 and CSRP2 transcripts in response to transformation indicate that the abundance of CSRP1 and CSRP2 transcripts is independently regulated; our results do not distinguish whether transcript level is controlled at the level of transcription, mRNA degradation, or both.
Figure 4:
Correlation between CSRP gene
expression and cell transformation. A, a Northern blot
analysis of total RNAs (30 µg/lane) from the following cellular
sources is shown: quail embryo fibroblasts (lane 1); quail
embryo fibroblasts transformed by the temperature-sensitive protein
product of the v-src oncogene of tsLA29 and kept at
37 °C (lane 2) or shifted to 40.5 °C for 1 day (lane 3) or 2 days (lane 4); quail embryo fibroblasts
transformed by tsLA29 at 37 °C and then shifted and kept
at 40.5 °C (lane 5) or shifted back to 37 °C for 1 day (lane 6) or 2 days (lane 7). B, a Northern
blot analysis of total RNAs (30 µg/lane) from the following
cellular sources is shown: quail embryo fibroblasts (lane 1);
quail embryo fibroblasts at day 7 (lane 2), day 16 (lane
3) or day 29 (lane 4) postinfection with the avian
retrovirus MH2 carrying the two oncogenes v-myc and
v-mil; the MH2-transformed quail fibroblast line MH2-A10 (lane 5). RNAs from both filters (A and B)
were first hybridized with a P-labeled quail CSRP2 probe (3.4
10
cpm), and the autoradiograph was
exposed for 7.5 h using an intensifying screen. The filters were
stripped and then hybridized with a
P-labeled quail CSRP1 probe (6.7
10
cpm), and the
autoradiograph was exposed for 15 h. The positions of ribosomal RNAs
are indicated in the margin. Hybridization with a
P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (4.9
10
cpm; 8-h exposure)
was used as an internal control.
We have investigated whether the two transcripts in chicken cells that hybridized with the CSRP1 probe are derived from a single gene or represent unique transcripts from closely related genes. Inspection of the previously reported sequence of chicken CSRP1 cDNA (9) revealed that two potential polyadenylation recognition motifs were present: an AATAAA sequence at nucleotides 942-947 and a CACTG recognition element (44) at nucleotides 1312-1316. Utilization of both of these signals would be predicted to give rise to mRNA transcripts of approximately 1.4 and 1.0 kb, consistent with what is observed by Northern analysis (Fig. 3). Characterization of a number of CSRP1 cDNA clones isolated from a chicken embryo fibroblast cDNA library revealed that two classes of cDNAs were present. The two classes were indistinguishable in their coding sequences and varied only in their 3`-untranslated regions, in particular with respect to the position of the poly(A) tail (Fig. 5). In the large sized minor transcript, referred to as CSRP1.1 mRNA, the CACTG site appears adjacent to the poly(A) tail in a 3`-untranslated region of 668 nucleotides, whereas the small sized major transcript, referred to as CSRP1.2 mRNA, appears to result from the alternative use of the AATAAA signal to generate a 3`-untranslated region of only 310 nucleotides excluding the poly(A) tail.
Figure 5: Differential polyadenylation of chicken CSRP1 transcripts. The two chicken CSRP1 transcripts observed by Northern analysis (cf. Fig. 3, A and C) result from alternative polyadenylation. The 3`-untranslated regions of the longer transcript (CSRP1.1) and the shorter transcript (CSRP1.2) are shown. The sequences corresponding to polyadenylation recognition signals (AATAAA and CACTG) are underlined.
Figure 6:
Chicken and quail CSRP1 and CSRP2 genes represent distinct genetic loci. Chicken (A and B) and quail (C and D) genomic DNA
(10 µg/lane) were digested with BamHI, EcoRI, or PstI, and the digests were analyzed by Southern blotting using P-labeled chicken CSRP1 (A), chicken CSRP2 (B), quail CSRP1 (C), or
quail CSRP2 (D) cDNA clones as hybridization probes.
The positions of DNA size markers are indicated in the margin.
Interestingly, low stringency Southern blots also revealed the presence of some minor cross-hybridizing DNA fragments that were not detected in the high stringency screens with either the CSRP1 or CSRP2 probes (not shown). These bands may represent sequences derived from more distantly related CSRP family members. Indeed, a recent report describes another avian gene, MLP, that encodes a protein with characteristics similar to CRP1 and CRP2(45) . The muscle LIM protein (MLP) exhibits two LIM domains with the spacing found in CRP1 and CRP2 proteins, adjacent glycine-rich repeats, and a potential nuclear localization signal, the three structural hallmarks of CRP family members (see below). Searches of the EMBL/GenBank data bases revealed that CRP1 and CRP2 are the most closely related sequences to MLP that have been reported to date. Because of the significant sequence relationships and similarities in global protein organization between MLP and the two CRP family members described here, it is likely that MLP and CRPs are evolutionarily related. We suggest that it would be appropriate to refer to MLP as CRP3 to indicate its relationship to members of the CRP family of proteins.
Figure 7: Comparison of the amino acid sequences of CRP family members. An alignment of the amino acid sequences of chicken (c) CRP1(9) , quail (q) CRP1 (Fig. 1), human (h) CRP1(6) , chicken CRP2 (Fig. 2), quail CRP2(29) , and chicken MLP/CRP3 (45) is shown. Residues conserved in all sequences are marked by asterisks below the bottom line. The conserved Cys and His residues of the LIM domains are indicated by crosses, and the conserved glycine residues of the glycine-rich repeats are marked by dashes above the top line.
Figure 8: Domain structure of selected LIM-proteins. A, a schematic diagram of the structures of representative chicken (c) or human (h) LIM proteins including members of the CRP family is shown. LIM motifs and glycine-rich repeats are shown as open and black boxes, respectively. The amino- and carboxyl-terminal amino acid residues and the first and last residues of the LIM domains are numbered. Sources for the amino acid sequences are as follows: chicken CRP1(9) , chicken CRP2 (Fig. 2), chicken MLP/CRP3(45) , human TTG1(22) , chicken zyxin(8) . B, the spacing of the eight metal-coordinating amino acid residues (shown in boldface letters) within all LIM domains of the proteins shown in panel A is compared. Among the members of the CRP family (CRP1, CRP2, MLP/CRP3), the spacing is absolutely conserved. Numbering of LIM domains refers to their order of appearance in these proteins relative to the amino terminus and does not reflect a structural classification.
On the other hand, there is growing evidence that LIM domains mediate specific protein-protein interactions. Direct evidence for a functional role of LIM domains in protein-protein interactions was recently provided for zyxin and CRP1, two interacting chicken LIM-proteins(8, 9, 15) . In addition, a LIM protein has been shown to interact with tyrosine-containing tight turn motifs present in the cytoplasmic domain of the insulin receptor(17) . LIM domains have also been implicated in homotypic, intermolecular interactions(16) . Moreover, interactions of LIM domain proteins with proteins containing basic helix-loop-helix motifs known to be involved in protein dimerization have been demonstrated(47, 48) . Thus, the LIM domain can clearly support specific associations with partner proteins. Proteins that display multiple LIM domains may serve as adaptor molecules or as scaffolds for the coordinated, localized assembly of multimeric protein complexes (15) .
The detailed biological role of the CRP family of proteins is not well understood. In general, LIM domain proteins, in particular those that contain additional homeodomains, have been implicated in regulatory processes important for development and cellular differentiation(18, 19, 49) . Many of the LIM-only proteins also appear to function in these broad processes. For example, the rhombotins were originally identified at chromosomal translocations and shown to be involved in tumorigenesis (22, 23, 24, 27, 28) , and recent studies on their tissue-specific expression and in vivo function have revealed that rhombotins have essential roles in normal development(25, 26) . For the CRP protein family, a proposed role in regulatory processes was most clearly confirmed for the MLP protein that was isolated from a subtracted cDNA library enriched in genes induced in skeletal muscle by denervation and then shown to be a positive regulator of myogenesis(45) . For chicken CRP1, direct interaction with zyxin, an adhesion plaque protein, has been demonstrated, and it was postulated that both proteins may function as components of a signal transduction pathway that mediates adhesion-stimulated changes in gene expression (8, 9) . The expression of the human CSRP1 gene was shown to be induced as a primary response to serum in quiescent cells, with a serum induction profile similar to that of c-myc, and expression that continues, like that of c-myc, in logarithmically growing cells(7) . This is in agreement with our results reported here and previously (29) on CSRP1 and CSRP2 expression in continuously growing normal avian fibroblasts. The strong suppression of CSRP genes, in particular of CSRP2, in all transformed cells tested may be connected with a regulatory function of CRP proteins in ordered cell growth. Although there is strong circumstantial evidence for the involvement of CRP proteins in regulatory processes important for cell growth and differentiation, definitive characterization of their biological functions and distinction between the functions of the individual members of the CRP family of closely related LIM only proteins will depend on the elucidation of their biochemical functions and on the identification of their cellular targets.