Sequence Analysis and Chromosomal Localization of Human Cap Z

Abstract

From a human retinal cDNA library, we have isolated cDNAs that are homologs for the α2 and β subunits of chicken Cap Z. The derived human α subunit shares 95% amino acid identity with the chicken α2 subunit; the β subunit is 99% identical to the chicken subunit residues 1-243. The remaining portion of the human β subunit(244-272) diverges significantly with only 8 out of 29 C-terminal amino acids conserved between the two species. This lack of conservation is of particular interest because the chicken C terminus contains an actin-binding domain. Cosedimentation assays with F-actin show that human Cap Z binds actin with an affinity equal that of chicken Cap Z. These results point to the eight shared amino acids as critical for actin binding, three of which are regularly spaced leucines. These apolar residues and one outside the region of divergence align well with those residues of the actin-binding α-helix proposed for gelsolin segment 1. The apolar residues as well as three polar amino acids are also conserved in other capping, capping and severing, and monomer-binding proteins. Amino acid substitutions in the chicken β subunit of the two most highly conserved leucines result in significant decreases in F-actin binding activity. The human α2 gene (CAPZA2) has been mapped to chromosome 7 position q31.2-q31.3 and the β gene (CAPZB) to chromosome 1 region p36.1. From a human retinal cDNA library, we have isolated cDNAs that are homologs for the α2 and β subunits of chicken Cap Z. The derived human α subunit shares 95% amino acid identity with the chicken α2 subunit; the β subunit is 99% identical to the chicken subunit residues 1-243. The remaining portion of the human β subunit(244-272) diverges significantly with only 8 out of 29 C-terminal amino acids conserved between the two species. This lack of conservation is of particular interest because the chicken C terminus contains an actin-binding domain. Cosedimentation assays with F-actin show that human Cap Z binds actin with an affinity equal that of chicken Cap Z. These results point to the eight shared amino acids as critical for actin binding, three of which are regularly spaced leucines. These apolar residues and one outside the region of divergence align well with those residues of the actin-binding α-helix proposed for gelsolin segment 1. The apolar residues as well as three polar amino acids are also conserved in other capping, capping and severing, and monomer-binding proteins. Amino acid substitutions in the chicken β subunit of the two most highly conserved leucines result in significant decreases in F-actin binding activity. The human α2 gene (CAPZA2) has been mapped to chromosome 7 position q31.2-q31.3 and the β gene (CAPZB) to chromosome 1 region p36.1. INTRODUCTIONCap Z has been identified in chicken as a nonsevering, barbed-end actin-binding protein composed of α and β subunits. In chicken, two cDNAs have been isolated for the α subunit. The α1 and α2 isoforms, which share 85% identity, are the products of two separate genes(1Casella J.F. Casella S.J. Hollands J.A. Caldwell J.E. Cooper J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5800-5804Crossref PubMed Scopus (40) Google Scholar, 2Cooper J.A. Caldwell J.E. Gattermeir D.J. Torres M.A. Amatruda J.F. Casella J.F. Cell Motil. Cytoskeleton. 1991; 18: 204-214Crossref PubMed Scopus (20) Google Scholar); one gene is believed to be responsible for β subunit expression(3Caldwell J.E. Waddle J.A. Cooper J.A. Hollands J.A. Casella S.J. Casella J.F. J. Biol. Chem. 1989; 264: 12648-12652Abstract Full Text PDF PubMed Google Scholar).By capping the barbed end of actin filaments, Cap Z regulates the growth of the actin filament at the barbed end(4Casella J.F. Maack D.J. Lin S. J. Biol. Chem. 1986; 261: 10915-10921Abstract Full Text PDF PubMed Google Scholar). The actin-binding domains of the chicken protein have been mapped by genetic manipulation of the respective subunit genes. Deletion mutations have shown that the C-terminal 55 amino acids of the α1 subunit(5Casella J.F. Torres M.A. J. Biol. Chem. 1994; 269: 6992-6998Abstract Full Text PDF PubMed Google Scholar), and the C-terminal 15 amino acids of the β subunit are necessary for binding to F-actin in vitro(6Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Crossref PubMed Scopus (60) Google Scholar). When expressed as a glutathione S-transferase fusion protein, the C-terminal 25 residues of the β subunit are sufficient to bind monomeric actin(6Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Crossref PubMed Scopus (60) Google Scholar).For comparative studies and as a step toward identifying human mutations, we have isolated and sequenced cDNAs encoding an α and a β subunit for human Cap Z and mapped each subunit gene to its chromosome. We also have expressed the subunits and determined the binding affinity of the human heterodimer to F-actin. Within the divergent β subunit C termini of the Cap Z and other homologous capping proteins, we have identified several conserved residues. We provide the first evidence that many of these conserved amino acids from the Cap Z family of actin-capping proteins align with similar amino acids in actin-capping/severing and monomer binding families of proteins. To confirm the functional importance of these conserved residues, we have made amino acid substitutions for two of the most conserved residues in the β domain of chicken Cap Z and studied the F-actin binding characteristics of the heterodimers (α1/mutated β) in actin cosedimentation and binding assays.MATERIALS AND METHODSScreening of Retinal cDNA LibraryA human retinal cDNA library constructed in λgt10 (kindly provided by Dr. Jeremy Nathans) (7Nathans J. Thomas D. Hogness D.S. Science. 1986; 232: 193-202Crossref PubMed Scopus (1168) Google Scholar) was screened with cDNAs of the chicken α and β subunits. For each of the two screens, approximately 180,000 plaques were transferred onto nitrocellulose, and the filters were prepared for hybridization using standard procedures(8Ausubel F.M. Current Protocols in Molecular Biology. WileyInterscience, New York1987Google Scholar). Hybridizing plaques were isolated and purified by limiting dilution and rescreening.Subcloning into pBS and pGEMPhage DNA was isolated from plate lysates using standard procedures(8Ausubel F.M. Current Protocols in Molecular Biology. WileyInterscience, New York1987Google Scholar). After digesting the purified phage DNA with EcoRI, the cDNA insert was gel purified. The resulting cDNAs, which ranged in size from 1-3 kb, 1The abbreviations used are: kbkilobase(s)UTRuntranslated regionPCRpolymerase chain reactionFISHfluorescence in situ hybridizationDAPI4,6-diamidino-2-phenylindoleYACyeast artificial chromosomebpbase pair5′7MeGppp5′Gp1,5′-(7-methyl)guanosine-P3-5′-guanosine triphosphate. were subcloned into pBluescript KS+ (pBS, Stratagene) and sequenced using the dideoxy chain termination method(9Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52344) Google Scholar). One clone containing the full-length α subunit cDNA (pHα4-1, 1.6 kb) and two clones with the complete β subunit cDNA (pHβ4-2, 1.0 kb and pHβ4-3, 2.2 kb) were sequenced on both strands.α and β cDNAs were transferred into pGEM (Promega) in order to synthesize RNA for in vitro translation. To enhance the level of expression from these constructs, an EcoRI/NcoI fragment containing the 5′-untranslated region (UTR) from the immediate early gene, 475(10Nakabeppu Y. Ryder K. Nathans D. Cell. 1988; 55: 907-915Abstract Full Text PDF PubMed Scopus (531) Google Scholar), was included. Using the forward primer (5′-CCAGAAGGACCATGGCGGAT27-3′) and reverse primer (5′-AAATCTAGATTGACAGTTCAGAATAC940-3′) for the α subunit and the forward primer (5′-GAGACGCCATCATGAGTG7-3′) and the reverse primer (5′-AAATCTAGATGTTATGTGACCTGTCG916-3′) for the β subunit and the templates pHα4-1 and pHβ4-3, cDNAs for each subunit were amplified. In addition, the sequences encoding the initiating methionines for each subunit were modified to include an NcoI or the compatible site, BspHI; an XbaI site was created at the 3′ end of each cDNA (added restriction sites are highlighted in the primers). By digesting the PCR fragment with either NcoI or BspHI and XbaI, the cDNAs were inserted into the pGEM vector behind the 475 5′-UTR. The cDNAs were sequenced again to check for any nucleotide changes created by PCR amplification.Construction of mutations of and were created in the chicken β subunit by PCR using the In the of two mutations, restriction sites were into the nucleotide for To the two C-terminal PCR were for each restriction The PCR were using a forward primer containing a site and a reverse primer with either the restriction site or The forward used for the 3′ PCR were the of the reverse used in of the PCR the reverse primer was the primer in pGEM 3′ to the After the PCR products were with and either or and the 3′ PCR were with either or and The and 3′ were at the restriction site and for the fragment containing the β C-terminal domain in When the encoding was to include an site, was to to a site, was of was using a similar forward primer and reverse primer were made which the β subunit and a restriction These substitutions To the amino acid at position were at and in the forward to the constructs, a PCR fragment was using the forward primer and the reverse a 3′ PCR fragment was made using the forward primer and The were with the restriction and at the The resulting fragment was used to the in The nucleotide changes at and of these in several amino acid substitutions at of nucleotide were for sequences homologous to human Cap Z using the from the for using the from the of the cDNAs, the human α2 and β subunit were to two by The for the α2 were and those for the β were and Using the for of DNA from each of the PubMed Scopus Google Scholar) were of were the at at the for α and for β and at of to chromosome 7 was by and PCR of a of S. A. J. PubMed Scopus Google Scholar, J. D. S. K. M.A. K. F. J. J. 1994; 55: Google Scholar) and a chromosome artificial chromosome 1992; PubMed Scopus Google Scholar). The human α2 cDNA was used as a in the hybridization PCR products were using the to human α2 gene was on chromosome 1 by in situ hybridization to PubMed Scopus Google Scholar, J. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, J. 1994; PubMed Scopus Google Scholar). cDNA the was with the 1 PubMed Scopus Google Scholar). After hybridization and the were and using J. 1994; PubMed Scopus Google Scholar). The and the were of the to a was determined by the on the PubMed Scopus Google and protein was in vitro using and (Promega) as J.F. Torres M.A. J. Biol. Chem. 1994; 269: 6992-6998Abstract Full Text PDF PubMed Google Scholar). For 1 of each was used of and was was from the For each approximately 8 of RNA were in a containing of of of RNA for each was used in the of the of were in these to the resulting of the of protein was on the of and as J.F. Torres M.A. J. Biol. Chem. 1994; 269: 6992-6998Abstract Full Text PDF PubMed Google Scholar).For binding assays and to heterodimer were on a 1 The was and at a of in were in from each were in to the products were on and by of the were using the and by to the of each and cosedimentation of the subunits and heterodimers with chicken F-actin was by of the in vitro with F-actin for 15 in the or of Cap Z as J.F. Torres M.A. J. Biol. Chem. 1994; 269: 6992-6998Abstract Full Text PDF PubMed Google Scholar). After a for in a the or were on a the binding of the and proteins to the was that heterodimer was used and the of actin was After the three in the the the of in the was determined by of the proteins were using the of and D. PubMed Scopus Google Scholar) as modified by Full Text PDF PubMed Scopus Google and Amino human cDNAs were isolated from a human retinal library for both the Cap Z α and β subunits. In the α plaques were 15 plaques with the β For each the cDNA from plaques were and of the cDNAs of the α or β subunit for the human α2 and β cDNAs are and the of to the identity was with three and of which complete subunit cDNA been identified as Cap of the human α nucleotide that shared identity with the chicken α2 and identity with The amino acid only between the human and chicken α2 subunits out of human β from shared nucleotide identity with the chicken β nucleotide resulting in 99% amino acid identity between chicken and The remaining of the human β subunit from the chicken β that a nucleotide segment that to the C-terminal domain of the chicken β subunit is in the human a that are similar nucleotide to a region region in chicken have region for the C terminus of the human β subunit. The human and chicken C termini in and share only Cosedimentation and with Cap divergence of the human β in a actin-binding domain of the chicken subunit the of the human protein actin-binding activity. To human α and β subunits by in vitro were for to a protein and bind to In vitro human α and β subunits on a a at When both α and β were two were One from the at a position similar to that of the the other at in a position identical to chicken Cap Z. of on a gel and approximately of each subunit by and for and of the subunits and heterodimer with only the heterodimer with actin be by the of chicken Cap Z. studies with the heterodimers a of approximately 7 of human Cap Z α and β subunit were in vitro or in the of The products were on a and were of α and β products show one two are α2 and β subunits are In the one at a position similar to the subunits and the other at a position identical to chicken Cap Z of Cap Z products with and are shown cosedimentation of in vitro human Cap Z subunits and heterodimer with was with and chicken Cap Z. The were on a gel and by between the C-terminal of and Cap Z β and of the human protein to bind actin its to the actin-binding domain in the chicken protein of the C-terminal amino acid C-terminal domains of human amino and chicken amino 8 amino acids that were identical Within the Cap Z family of actin-binding proteins, these 8 residues are conserved with the of the at position of the amino acids also to be or as shown in the for Cap Z and The most of the was an of regularly spaced leucines that with and of human and between the β subunit C-terminal domain of Cap Z and homologs and other capping, capping and severing, and monomer-binding proteins. portion of the of the C-terminal domains of human and chicken Cap Z β J.E. Waddle J.A. Cooper J.A. Hollands J.A. Casella S.J. Casella J.F. J. Biol. Chem. 1989; 264: 12648-12652Abstract Full Text PDF PubMed Google Scholar). the amino acid in the human and chicken sequences and the the site of divergence between the two C In the C-terminal those amino acids that are conserved in human and chicken are with an C-terminal from β subunits of other Cap Z homologs J.A. Cooper J.A. Biol. Cell. PubMed Scopus Google Scholar, C. S. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, J.F. Cooper J.A. J. Cell Biol. 1992; PubMed Scopus Google Scholar) are and a of those amino acids that are in at three out of the sequences is In the an of amino acids containing three regularly spaced is In the portion of the sequences from actin-binding proteins D. D. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar, C. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) are with the Cap Z β identical or amino acids the families of proteins. The of the first and residues of the are apolar amino acids that are believed to be in actin binding of gelsolin are The amino acids to be conserved in several actin-binding proteins are in the by to other actin-binding proteins, as the D. D. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar), J. A. J. D. J. Cell Biol. 1988; PubMed Scopus Google Scholar, F. A. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar, Science. PubMed Scopus Google Scholar, J. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, J. 1991; PubMed Scopus Google Scholar), and monomer-binding C. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) proteins, we a similar of apolar residues that with the of Cap Z and In addition, an apolar amino acid to the region of divergence in the human and chicken C termini with human position was amino acids that with Cap Z and were also residues that with and were those with position were of Cap the two most highly conserved leucines in the actin-binding domain were critical for binding, amino acid substitutions at and of the chicken β subunit were These proteins were first studied by actin cosedimentation assays The cosedimentation with α1 and the β subunits the order of actin-binding of the proteins, these cosedimentation results be by heterodimer the products were also purified by gel of the β subunits heterodimers that a of protein was responsible for the in These in and similar to Cap Z. To the of α and β the purified were on and of the of α and β subunits which were to the of the subunits determined by the of into each cosedimentation with α1 and β subunits. are shown cosedimentation of in vitro chicken Cap Z α1 and β subunits. was with and chicken Cap Z. The were on a gel and by containing the β subunit by gel for chicken β and several β subunits containing mutations for the amino acid substitutions at or were in The α and β subunit were and the products gel on a The is an of the Cap Z products containing the mutations at β position the products which have mutations at β position in two are for both of mutations in the β one at a position similar to chicken Cap Z and the other at the position of the subunits of either or from each of heterodimers are shown in of the subunit of heterodimers and binding of the heterodimers to of the of subunits and gel the of the The and are for each the the of the the of from the α and β subunits in the purified is the for a is the actin-binding by the binding containing the β subunits were for to bind to The results of the cosedimentation with determined from the binding assays show that actin binding of Cap Z is the at position is the at position each amino acid substitutions show in actin binding at either with a significantly with apolar amino as and also of the Cap Z α and β of and were determined by PCR of PubMed Scopus Google Scholar). PCR products of the size for the α2 and β subunit were only from the containing human chromosome 7 and human chromosome the of a of containing of human chromosome 7 S. A. J. PubMed Scopus Google Scholar, J. D. S. K. M.A. K. F. J. J. 1994; 55: Google Scholar) was by the PCR The results that the gene mapped to the region The PCR were used to a human chromosome library 1992; PubMed Scopus Google Scholar) and and were identified These clones were to and shown to be of a of clones the to the gene S. A. J. PubMed Scopus Google Scholar). and kb) were to both and the two are by a of The results of these studies a order of the DNA for the the gene at clones the and are The of the DNA used to the are shown the the region to which the DNA be The and are from the The other DNA have been S. A. J. PubMed Scopus Google gene was to chromosome 1 region by The of hybridization of the cDNA clone to chromosome was approximately was used to the to chromosome which was in with the PCR of were and the results that mapped to to of chromosome 1 the using the β subunit the of on chromosome between and Cap results show that the of human Cap Z is 95% identical to the heterodimer of chicken Cap Z. One of the in was the in conservation to in the C-terminal domain of the β subunit. The nucleotide that a segment in the chicken β cDNA is in the human This results in a region in chicken the for the human β subunit C chicken β subunit cDNA with a C terminus similar to human clone has been identified and the Cooper J.A. J. Cell Biol. 1994; PubMed Scopus Google Scholar). the is for either the chicken or human β the most is that the portion of has been from the β subunit human α1 and were isolated in lack of be to the of the cDNA be or in in the these be in the of in an between capping proteins of and chicken Cap Z have a conservation of in the C-terminal region of the β C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Crossref PubMed Scopus (60) Google Scholar). was that an binding domain was mapped to region of the chicken β subunit. When expressed as a fusion protein, the C-terminal region actin monomeric actin with a of between and monomer binding or barbed-end capping proteins significant C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Crossref PubMed Scopus (60) Google the human heterodimer with its β C-terminal binds actin with an affinity equal to or the two of chicken Cap J.F. Torres M.A. J. Biol. Chem. 1994; 269: 6992-6998Abstract Full Text PDF PubMed Google Scholar). with the chicken protein, the human subunits one for affinity The in binding lack of identity with the chicken actin-binding domain to the eight conserved residues as critical for actin Amino in the C-terminal in those conserved amino acids is a that is in in other Cap Z This is the C-terminal 25 amino acids of the chicken to an actin-binding show that amino acid substitutions at either polar or apolar residues the capping to bind actin the of the subunit to with the subunit. The leucines at and of Cap Z align with leucines a highly conserved amino acid in the gelsolin and protein families and in a of actin-binding D.J. F.M. Sci. 1989; Full Text PDF PubMed Scopus Google Scholar, J. Cell Biol. 1989; PubMed Scopus (20) Google Scholar) of gelsolin fragment PubMed Scopus Google Scholar) with actin that a of apolar residues between 1 and of actin and is with residues. From several of the residues of the highly conserved amino acid in gelsolin as well as several amino acids are in PubMed Scopus Google Scholar). The position identified in the Cap Z family of proteins well with the apolar residues of gelsolin that are proposed to with the region of in Cap Z is to be α studies have to show between Cap Z and other actin-binding proteins. between chicken Cap Z β subunit residues and residues to a gelsolin residues Science. PubMed Scopus Google Scholar). Within segment of a of amino acids is which is to be in binding J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The of the provide the first evidence of a functional between the actin-binding domains of Cap Z and of the and The that a has been three families of actin-binding proteins is and that binding sites in three families have from a of is any genetic result from mutations in the human Cap Z genes. the region of chromosome 7 are chromosome J. 1988; PubMed Scopus Google Scholar). The of these a the of and an are J. 1988; PubMed Scopus Google Scholar). are on the of chromosome be to the with the of with The and have been used in several J. A. Cell 1989; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1988; PubMed Scopus Google Scholar), the of to in these and other the of to chromosome also provide a DNA for similar studies of chromosome 1 INTRODUCTIONCap Z has been identified in chicken as a nonsevering, barbed-end actin-binding protein composed of α and β subunits. In chicken, two cDNAs have been isolated for the α subunit. The α1 and α2 isoforms, which share 85% identity, are the products of two separate genes(1Casella J.F. Casella S.J. Hollands J.A. Caldwell J.E. Cooper J.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5800-5804Crossref PubMed Scopus (40) Google Scholar, 2Cooper J.A. Caldwell J.E. Gattermeir D.J. Torres M.A. Amatruda J.F. Casella J.F. Cell Motil. Cytoskeleton. 1991; 18: 204-214Crossref PubMed Scopus (20) Google Scholar); one gene is believed to be responsible for β subunit expression(3Caldwell J.E. Waddle J.A. Cooper J.A. Hollands J.A. Casella S.J. Casella J.F. J. Biol. Chem. 1989; 264: 12648-12652Abstract Full Text PDF PubMed Google Scholar).By capping the barbed end of actin filaments, Cap Z regulates the growth of the actin filament at the barbed end(4Casella J.F. Maack D.J. Lin S. J. Biol. Chem. 1986; 261: 10915-10921Abstract Full Text PDF PubMed Google Scholar). The actin-binding domains of the chicken protein have been mapped by genetic manipulation of the respective subunit genes. Deletion mutations have shown that the C-terminal 55 amino acids of the α1 subunit(5Casella J.F. Torres M.A. J. Biol. Chem. 1994; 269: 6992-6998Abstract Full Text PDF PubMed Google Scholar), and the C-terminal 15 amino acids of the β subunit are necessary for binding to F-actin in vitro(6Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Crossref PubMed Scopus (60) Google Scholar). When expressed as a glutathione S-transferase fusion protein, the C-terminal 25 residues of the β subunit are sufficient to bind monomeric actin(6Hug C. Miller T.M. Torres M.A. Casella J.F. Cooper J.A. J. Cell Biol. 1992; 116: 923-931Crossref PubMed Scopus (60) Google Scholar).For comparative studies and as a step toward identifying human mutations, we have isolated and sequenced cDNAs encoding an α and a β subunit for human Cap Z and mapped each subunit gene to its chromosome. We also have expressed the subunits and determined the binding affinity of the human heterodimer to F-actin. Within the divergent β subunit C termini of the Cap Z and other homologous capping proteins, we have identified several conserved residues. We provide the first evidence that many of these conserved amino acids from the Cap Z family of actin-capping proteins align with similar amino acids in actin-capping/severing and monomer binding families of proteins. To confirm the functional importance of these conserved residues, we have made amino acid substitutions for two of the most conserved residues in the β domain of chicken Cap Z and studied the F-actin binding characteristics of the heterodimers (α1/mutated β) in actin cosedimentation and binding