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Gamete Biology |
Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, gametogenesis, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The most abundant protein of the fibrous sheath is A-kinase Anchoring Protein-4 (AKAP4). AKAPs anchor cAMP-dependent protein kinases (i.e., protein kinase A [PKA]) in restricted subcellular regions where they phosphorylate nearby proteins in response to cAMP signaling and often form complexes with other components of signal-transduction pathways [3–5]. The Akap4 gene is expressed only in the postmeiotic phase of spermatogenesis [6, 7] and produces two transcripts, one of which encodes a protein that is nine amino acids (aa) longer at the N-terminus than the other [8]. AKAP4 was initially believed to have a single PKA-binding site [9], but subsequently was found to have one site that binds either RI
or RII subunits of PKA, and another site that specifically binds RI subunits [10, 11].
Two other testis-specific AKAPs are present in the fibrous sheath, TAKAP-80 [12] and AKAP3 [13, 14]. Little is known about TAKAP-80, but AKAP3 anchors the testis-specific proteins ropporin and ASP (i.e., AKAP-Associated Sperm Protein) [15]. Ropporin is a fibrous sheath component that binds rhophilin [16], a small GTPase Rho-binding protein abundant in the testis and localized in the principal piece of mouse sperm [17]. The N-terminal region of ropporin and ASP have high sequence similarity with the N-terminal region of the RII subunit of PKA and were hypothesized to regulate motility by competing with RII for binding to AKAPs [15]. It also was hypothesized that AKAPs in sperm serve as scaffold proteins for the Rho signaling pathway and that downstream protein kinases participate in the regulation of sperm motility [15]. Additional fibrous sheath proteins include two glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) [18, 19] and type 1 hexokinase-S (HK1S) [20–23], a unique Mu class glutathione-S-transferase (GSTm5) [24], a calcium-binding tyrosine phosphorylation-regulated protein (CABYR) [25], and an intermediate filament-like protein (FS39) [26]. HK1S is encoded by germ-cell specific Hk1 splice variants [22], but the others are products of genes expressed exclusively in male germ cells, and except for GSTm5, are transcribed only during the postmeiotic phase of spermatogenesis.
Although 13 proteins currently are known to be associated with the fibrous sheath, little is known about how they assemble to form this unique cytoskeletal structure. We report here the results of yeast two-hybrid studies to identify proteins that bind directly to AKAP4, to map the regions on AKAP4 where they bind, and to delimit the regions on these proteins to which AKAP4 binds.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An Akap4 genomic clone was isolated from a P1 library of mouse 129Sv genomic DNA by polymerase chain reaction (PCR) screening (Incyte Genomics, St. Louis, MO). Probes generated from different regions of the Akap4 cDNA sequence by PCR amplification were labeled with [32P]dCTP by random priming (Roche Diagnostics, Indianapolis, IN), and used to determine the locations of Akap4 exons on Southern blots of the cloned mouse genomic DNA. Restriction fragments containing Akap4 DNA were subcloned into Bluescript (Stratagene, La Jolla, CA) and used for detailed restriction enzyme mapping and sequencing. The 
11.8 kilobase (kb) Akap4 gene was sequenced twice, except for 0.5 kb regions within introns 2 and 3 that were highly resistant to sequencing (GenBank accession numbers AF448784, AF448785, and AF448786).
Construction of Akap4 Expression Vectors
Expression vectors for the full-length protein or various deletion mutants were constructed by PCR using the Akap4 cDNA as the template [7] (GenBank accession number U10341). The forward primers introduced EcoRI sites and the reverse primers introduced SalI sites, allowing the PCR products to be ligated into the pAS2-1 yeast expression plasmid (BD Biosciences Clontech, Palo Alto, CA). The PCR reactions, ligation into plasmid pAS2-1, and plasmid propagation were carried out as described previously [10]. These vectors were used to express full-length or mutant forms of AKAP4 in yeast as a fusion protein with the GAL4 DNA-binding domain.
Yeast Cultures
Saccharomyces cerevisiae strain Y190 cells (MATa, ura3-52, his3-
200, ade2-101, trp1-901, leu2-3, 112, gal4gal80, URA::GAL-lacZ, cyhr2, LYS2::GAL-HIS3) were grown in yeast culture media, and synthetic dropout (SD) and dropout supplements (BD Biosciences Clontech) were used to produce media that were deficient in either or both tryptophan or leucine amino acids (SD/Trp- Leu-, SD/Trp-, or SD/Leu-). Transactivation of His3 was assayed using medium deficient in tryptophan, leucine, and histidine (SD/Trp-, Leu-, His-) and supplemented with 25 mM 3-amino-1,2,4-triazole (Sigma, St. Louis, MO).
Yeast Two-Hybrid Library Screening and Analysis
Yeast two-hybrid screens were used to identify proteins (prey) that interact with AKAP4 (bait). A 129Sv-mouse testis cDNA library constructed in the pGAD10 vector (BD Biosciences Clontech) was screened using the yeast two-hybrid method, as described previously [10]. The bait vectors were pAS/Akap4/1–849, encoding the full-length AKAP4 sequence [7], and pAS/Akap4/189–849, encoding the mature form of AKAP4. The full-length and N-terminal 50 aa regions of ropporin were generated by reverse transcriptase-PCR using primers designed from the reported sequence (GenBank accession number AF178531) and cloned into the prey vector. Yeast two-hybrid analysis was used to define the regions of protein-protein interaction between deletion mutants of AKAP4 and other proteins. The specificity of binding was confirmed using a ß-galactosidase expression assay with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside as the substrate on colony lifts of transformants that survived SD/Trp- Leu -His- selection, as described previously [10].
In Vitro Binding Assay
The cDNAs encoding aa 461–579 of AKAP3 and aa 602–849 of AKAP4 were amplified by PCR and cloned into bacterial expression vector pGEX-4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ) or mammalian expression vector pFLAG-CMV-2 (Sigma), respectively. Glutathione S-transferase (GST)-fusion proteins and FLAG-tagged proteins were produced in bacteria and COS-7 cells, respectively, as described previously [27]. GST-AKAP3 fusion protein (1 µg) was immobilized on glutathione-Sepharose resin (Amersham Pharmacia Biotech) and incubated for 3 h at 4°C in 1 ml of binding buffer (140 mM NaCl, 0.1% Triton X-100, 0.1 mM dithiothreitol [DTT], protease inhibitor cocktail [Complete; Roche Applied Science], 20 mM HEPES buffer pH 7.4) with 5 mg of COS-7 cell lysate containing FLAG-AKAP4 protein. The resin was washed four times with binding buffer, proteins were released by treatment with SDS-sample buffer, and the total eluate was subjected to SDS-PAGE. FLAG-AKAP4 was detected by Western blotting using monoclonal antibody M2 to FLAG (Sigma), followed by secondary horseradish peroxidase conjugated goat antiserum to mouse immunoglobulin G (IgG; 1:20 000; Sigma) and enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech), as recommended by the suppliers.
Characterization of Complementary DNAs
Clones were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction kit as recommended by the supplier (PE Applied Biosystems, Foster City, CA). Most clones isolated in the yeast two-hybrid screens contained an incomplete protein-coding region. An additional 5' sequence was determined using mouse testis Marathon-Ready cDNA and the 5' rapid amplification of cDNA ends (RACE) procedure as recommended by the supplier (BD Biosciences Clontech). The resulting PCR fragments were cloned into Bluescript (Stratagene) using NotI and SacI restriction enzyme sites, and transformed into E. coli DH5 competent cells (Life Technologies, Inc., Grand Island, NY). Longer Fsip2 cDNAs also were isolated using the original cDNA as a probe to screen an adult mouse testis 
gtll cDNA library. The GenBank database was searched for homologous sequences using the basic local alignment search tool (BLAST) program (using National Center for Biotechnology Information databases), and sequences were analyzed using GCG programs (Pharmacopeia, Princeton, NJ).
Northern Analysis
Testes were collected from 8- to 28-day-old CD-1 mice, total RNA was isolated using TRIzol (Life Technologies), and the RNA was used to prepare a developmental series Northern blot. Ribonucleic acids (15 µg per lane) were separated on 1.2% agarose gels containing 6.6% formaldehyde, transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH) by capillary action, and attached by UV cross-linking and baking. Blots were probed with cDNAs labeled with [32P]dCTP using a Random Primed DNA Labeling Kit (Roche Diagnostics, Indianapolis, IN) according to the supplier's protocol. The tissue series Northern blot was prepared by the same procedures using 10 µg of total RNA from 10 different somatic tissues, mixed germ cells, and the testis of adult mice. The probes used for Northern analysis corresponded to mouse cDNA base pairs (bp) 2055–2181 of Akap4 [7], bp 1672–1984 of Akap3 [14], bp 722–1123 of Fsip1 (GenBank accession number AF448787), bp 74–422 of the known Fsip2 sequence (GenBank accession number AF448788), and human ß-actin cDNA as a loading control.
Immunohistochemistry
Rabbit antisera were produced against synthetic peptides corresponding to aa 191–204 and 790–803 of AKAP4 [7]. A BLAST search indicated that these sequences are not present in AKAP3 or any other known mouse protein. The rat antiserum rFSP95 raised against recombinant human AKAP3 [13] was the generous gift of Dr. John Herr, University of Virginia. The rFSP95 antiserum was shown to react specifically with the recombinant protein by Western blotting [13]. These antisera were used to determine the tissue distribution of AKAP4 and AKAP3 by immunohistochemistry on paraffin sections of mouse testis fixed in Bouin solution, and to determine the cellular location of AKAP4 and AKAP3 in mouse sperm by indirect immunofluorescence. The AKAP4 antisera were used at 1:1000 dilutions, the rFSP95 antiserum was used at 1:500 dilution on sections, and preimmune antisera were used at the same concentrations for each as negative controls. Resulting antigen-antibody complexes were detected using biotinylated antibodies to rabbit IgG or rat IgG and an Elite ABC avidin-biotin-peroxidase kit (Vector Laboratories, Burlingame, CA) as suggested by the supplier. Comparable results were observed with both AKAP4 antisera. For indirect immunofluorescence, sperm were collected from the cauda epididymis, washed in Dulbecco PBS, and allowed to attach to poly-L-lysine coated slides. They were permeabilized with 100% methanol at -20°C for 1 min and treated for 10 min with 50 mM glycine in PBS and 10% goat serum. Sperm were incubated either in antisera to AKAP4 (1:1000 dilution) or antiserum to AKAP3 (1:100 dilution), or in the respective preimmune sera at the same concentrations for 2 h, and then for 1 h with affinity-purified goat antiserum to rabbit IgG (diluted 1:100) or rat IgG (diluted 1:500) conjugated with fluorescein isothiocyanate (ICN/Cappel, Irvine, CA).
Western Analysis
Testes were collected from 20- to 36-day-old C57/BL6 mice and crude protein extracts were isolated by homogenizing in lysis buffer (140 mM NaCl, 0.1% Triton X-100, and 20 mM HEPES pH 7.4, containing Complete proteinase inhibitor cocktail [Roche Diagnostics]), at a concentration of 50 mg (wet weight)/ml. Lysates corresponding to 0.5 mg (wet weight) for each age were separated by SDS-PAGE using 10% (w/v) acrylamide gel and transferred onto Imobilon nylon membrane (Milllipore Corp., Bedford, MA). The blots were incubated overnight at 4°C in 5% (w/v) skim milk in TBST buffer (150 mM NaCl, 0.1% Tween-20, 50 mM Tris-HCl pH 7.4). Blots were then probed with antisera to AKAP4 (1:5000 dilution in TBST), with antiserum to AKAP3 (1:1000 dilution in TBST), or with the respective preimmune sera at the same concentrations. Secondary antibodies were horseradish peroxidase-conjugated goat antiserum to rabbit IgG (1:20 000; Santa Cruz) or to rat IgG (1:20000; ICN-Cappel), respectively, and visualized using ECL reagents (Amersham Pharmacia Biotech) according to the manufacturer's protocols.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparison of the Akap4 cDNA and genomic sequences of the mouse established that the gene contains seven exons and six introns spanning approximately 11.8 kb (Fig. 1). We refer to the two transcripts produced by alternative splicing as Akap4a [7], derived from exons 1 and 3–7, and Akap4b [6], derived from exons 2–7. Exon 2 is noncoding, whereas exon 6 encodes more than 80% of the protein. Exon 1 encodes 9 aa at the N-terminus of the AKAP4a precursor protein that are not present in AKAP4b. Proteolytic processing of the precursors yields identical mature proteins [28]. The partial gene structure reported previously [28] was confirmed, and an additional sequence containing exons 1 and 2 was determined. The deduced protein sequence was 99% identical to that reported by Carrera et al. [6] and 94% identical to that reported by Turner et al. [28].
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Identification of Proteins That Interact With AKAP4
A bait vector encoding full-length AKAP4a was used in yeast two-hybrid screens of a mouse testis cDNA library. Eight cDNA clones were isolated and characterized, five of which encoded the RI subunit of PKA [10], whereas the others encoded two novel proteins, FSIP1 and FSIP2. Additional screens were carried out using the mature form of AKAP4 (aa 189–849) as bait. Seven clones were isolated and characterized, four of which encoded RI and three encoded AKAP3. The longest Akap3 cDNA was identical to 386 bp of the mouse Akap3 cDNA sequence reported previously [14] (GenBank accession number AF093406). Neither screen retrieved AKAP4 clones and homologous interactions were not observed in direct two-hybrid assays (data not shown). However, two-hybrid assays found that AKAP4 aa 125–311 in the bait vector interacted strongly with the N-terminal 50 aa of ropporin, whereas the binding with full-length ropporin was negligible (data not shown).
The Fsip1 cDNA encoded an open reading frame between bp 169 and bp 1476, a polyadenylation signal at bp 1648–1653 and a polyA tract beginning at bp 1670. An in-frame stop codon 159 bp upstream of the putative translation initiation codon was located using the 5' RACE procedure. The deduced FSIP1 protein is 435 aa in length and is rich in glutamine and asparagine, resulting in a calculated molecular weight of 49 950 and a pI of 4.65. Chou-Fassman analysis predicted the presence of alpha-helices in the C-terminal portion of the protein. The Fsip1 cDNA was 99% identical to a mouse testis cDNA reported previously (GenBank accession number AK017026).
The 357 bp Fsip2 cDNA was used as a probe to screen a mouse testis cDNA library and sequencing of overlapping clones added 1638 bp to the Fsip2 sequence. An open reading frame was present throughout the entire 1995 bp sequence. This sequence is 99% identical to nucleotides 3120–5115 of a 20 kb mRNA predicted from genomic sequencing (GenBank accession number XM_141020) that encodes a 6781 aa protein with a molecular weight of 760 512.
Expression of Transcripts for Fibrous Sheath Proteins
Northern analysis with total RNA from 11 mouse tissues and from mixed germ cells was used to determine the tissue distribution and size of the Fsip1 and Fsip2 transcripts. Transcripts 1.6 and 2 kb in length for Fsip1 and approximately 20 kb in length for Fsip2 were detected only in testis and mixed germ cells (Fig. 2A). In addition, Northern analysis also was used to determine when Akap4, Akap3, Fsip1, and Fsip2 are transcribed during spermatogenesis. The first wave of spermatogenesis in the postnatal period is relatively synchronous and the age when a transcript is first observed correlates with the appearance of specific types of spermatogenic cells [29]. A Northern blot with RNA from testes of mice 8 to 28 days of age was hybridized with each of the cDNAs. The 2.9 kb Akap4 mRNA, 3.1 kb Akap3 mRNA, and 1.6 and 2 kb Fsip1 mRNAs were detected first on Day 18, corresponding to the beginning of the postmeiotic phase of spermatogenesis (Fig. 2B). However, Fsip2 transcripts were first observed on Day 16 (Fig. 2B), corresponding to the latter part of the meiotic phase of spermatogenesis.
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Localization of Proteins
Sections of mouse testis were immunostained to determine when AKAP3 and AKAP4 are synthesized. Spermiogenesis is divided into 16 steps, on the basis of the size and shape of the nucleus, acrosome, and cell body [30–32]. This process takes more than 2 wk in mice, and as the sperm forms, the spermatid nucleus changes from round to elongate and the chromatin condenses. AKAP4 was first detected in both the cytoplasm and flagellum of step 14 (stage II–III) condensing spermatids and was not observed in the cytoplasm of condensing spermatids beyond step 15 (Fig. 3A). AKAP3 was first detected in the cytoplasm of step 4 (stage IV) round spermatids, but was not observed in the flagellum until 4 days later, in step 9 (stage IX) elongating spermatids. AKAP3 was no longer observed in the cytoplasm of step 14 condensing spermatids (Fig. 3B). Sections treated with preimmune serum using the same conditions exhibited no staining for either protein (data not shown).
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Indirect immunofluorescence microscopy was used to determine the distribution of AKAP3 and AKAP4 in epididymal sperm. AKAP4 (Fig. 3C) and AKAP3 (Fig. 3D) were detected only in the principal piece of the flagellum.
Western blotting with crude extracts of testes from 20- to 36-day-old mice was used to verify when AKAP4 and AKAP3 are synthesized. Both the precursor and mature forms of AKAP4, approximately 110 kDa and 82 kDa, respectively, were observed at Day 30 and thereafter (Fig. 3E), corresponding to the appearance of elongated spermatids. The approximately 110 kDa AKAP3 protein was first observed at Day 26, corresponding to the appearance of round spermatids, and increased in amount through Day 36 (Fig. 3F). These proteins were not detected on Western blots with preimmune serum (data not shown). Although both AKAP3 and AKAP4 have approximately 110 kDa forms, only the 82 kDa protein was recognized by the antiserum to AKAP4, indicating that the antisera do not cross-react.
Mapping Protein-Protein Binding Regions
Deletion mutagenesis and yeast two-hybrid assays were used to map the regions on AKAP4 to which AKAP3, FSIP1, and FSIP2 bind. A series of Akap4 cDNA constructs were prepared that expressed AKAP4 with successive truncations at the N-terminal end, the C-terminal end, or both. The original clones of Akap3, Fsip1, and Fsip2 in the prey vector and Akap4 cDNAs in bait vectors were cotransformed into yeast grown under selection conditions and colonies containing interacting peptide sequences were identified. AKAP3 bound to construct aa 750–849 (Fig. 4A) and to mature AKAP4 (construct aa 189–849), but not to the full-length precursor (construct aa 1–849) (data not shown). FSIP1 bound to construct aa 351–721, which includes most of the region between PKA binding domain B and the AKAP3-binding region, but did not bind to constructs aa 189–692 or aa 359–849 (Fig. 4A). FSIP2 bound to construct aa 202–247, containing a region near the N-terminal end of the processed protein and encompassing PKA-binding domain A (Fig. 4A, Fig. 5), but did not bind to constructs aa 237–849 or aa 125–218 of AKAP4.
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Yeast two-hybrid assays and deletion mutagenesis also were used to map the AKAP4 binding regions on AKAP3 and FSIP1. AKAP4 bound to construct aa 489–561 of the 864 aa AKAP3 protein and to construct aa 378–435 of the 435 aa FSIP1 protein (data not shown). The AKAP4-binding region on FSIP2 was not mapped further, but the cDNA originally isolated encoded only 122 aa of the predicted 6781 aa FSIP2 protein (GenBank accession number XP_141020), indicating that a very small region of the protein is required for binding to AKAP4.
The interaction between AKAP3 and AKAP4 also was examined using an in vitro binding assay. The recombinant GST-AKAP3 (aa 461–579) fusion protein produced in E. coli was bound to glutathione-Sepharose resin, COS-7 lysates containing the recombinant FLAG-AKAP4 (aa 602–849) fusion protein was added, and nonspecifically associated proteins were removed by washing. Specifically bound proteins were released with SDS sample buffer, subjected to SDS-PAGE, and Western blotting was carried out using an antibody to the FLAG peptide. FLAG-AKAP4 was found to bind to GST-AKAP3, but not to GST alone (Fig. 4B), consistent with the yeast two-hybrid results. Initial attempts to demonstrate AKAP4-FSIP1 binding have not been successful, apparently due to low solubility of the recombinant proteins. AKAP4-FSIP2 binding has been examined so far only using yeast two-hybrid assays. The AKAP4-FSIP1 and AKAP4-FSIP2 interactions will be examined in detail in future studies using other in vivo and in vitro systems.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found that in addition to the RI and RII subunits of PKA, AKAP4 anchors AKAP3 and two novel proteins, FSIP1 and FSIP2. We also found that Akap4, Akap3, and Fsip1 transcripts were first detected in the testis on postnatal Day 18, corresponding to the appearance of round spermatids during the first wave of spermatogenesis. AKAP3 immunostaining was first observed in the cytoplasm of round spermatids (step 4), suggesting that translation begins soon after transcription. However, a delay of more than 4 days occurred between the appearance of AKAP3 protein in the cytoplasm and its integration into the fibrous sheath. In previous studies, precursors of the longitudinal columns were first seen in the rat in round spermatids, whereas precursors of the ribs were first seen in late elongating spermatids [33–35]. This suggests that AKAP3 is incorporated into the fibrous sheath concurrently with formation of the ribs, but after formation of the longitudinal columns has begun. Furthermore, immunogold labeling of AKAP3 in human sperm occurred over the ribs, but not over the longitudinal columns [13]. These observations strongly suggest that AKAP3 is involved in formation of the rib precursors.
AKAP4 immunostaining was not seen until late in spermiogenesis, during steps 14 and 15, and the protein was promptly integrated into the longitudinal columns and ribs of the fibrous sheath. Previous studies found that formation of the definitive fibrous sheath occurs from distal to proximal during steps 14 and 15 in mice [36] and during a comparable period in rats [33–35]. AKAP4 constitutes about 40% of the 4 M urea-insoluble components of the fibrous sheath [37], and is present in both the longitudinal columns and ribs of the fibrous sheath [8]. In addition, targeted mutagenesis of the Akap4 gene results in sperm that contain precursors of the longitudinal columns and ribs, but lack a fully formed fibrous sheath [38]. These observations strongly suggest that incorporation of AKAP4 into the longitudinal columns and ribs is a major step in completion of assembly of the definitive fibrous sheath.
Yeast two-hybrid screens with either the precursor or mature forms of AKAP4 did not retrieve clones encoding AKAP4, and significant interactions were not seen between mature forms of AKAP4 in two-hybrid assays. However, the mature form and not the precursor form of AKAP4 binds AKAP3, indicating that removal of the 188 aa N-terminus from AKAP4 is necessary to make available the C-terminal binding site for AKAP3. To our knowledge, this is the first report of direct binding between two AKAPs. Furthermore, AKAP3 is present in the flagellum before AKAP4 and the processed form of AKAP4 is first detected in the flagellum [8]. This suggests that completion of rib formation involves processing of AKAP4 in the flagellum and the subsequent binding of mature AKAP4 to AKAP3 present in the rib precursors. However, AKAP4 is appreciably more abundant than AKAP3, but has only one AKAP3 binding site. In addition, AKAP4 is present in both the longitudinal columns and ribs, and both the precursor and mature forms of AKAP4 are tightly anchored to the fibrous sheath [8]. These observations mean that AKAP4 probably binds to proteins in addition to AKAP3 during its incorporation into the fibrous sheath.
AKAP4 Binding Regions
AKAP4 has RI-specific and RI/RII-dual binding domains [9–11]. RI and RII subunits of PKA form homodimers between their N-terminal 40–45 aa and the dimerized region in turn binds to the AKAP anchoring domain [39–41]. Although RII is reported to bind to AKAPs with 500-fold greater affinity than RI [42], only RI clones were isolated in screens with the precursor and the mature forms of AKAP4. In addition, the 50 aa portion of ropporin containing the RII-homologous domain bound strongly to AKAP4, but binding by full-length ropporin was negligible. It will be of interest to determine whether RI, RII and ropporin bind to AKAP4 with different affinities than to other AKAPs.
The shortest AKAP4 construct to which FSIP1 bound contained aa 351–731. However, FSIP1 bound to an AKAP4 construct beginning at aa 351, but not to one beginning at aa 359, and to an AKAP4 construct ending at aa 721, but not to one ending at aa 692. This strongly suggests that aa 351–359 and aa 692–721 of AKAP4 contain the minimum binding domains for FSIP1. The AKAP3 and AKAP4 sequences are 35% identical, and several segments of 20 or more amino acids are highly similar [14]. Three of these segments are contained within the shortest AKAP4 construct to which FSIP1 bound, but do not coincide with either minimum binding domains.
The shortest AKAP4 construct to which FSIP2 was able to bind contained aa 202–247. However, FSIP2 did bind to an AKAP4 construct beginning at aa 202, but not to one beginning at aa 237, and to an AKAP4 construct ending at aa 247, but not to one ending at aa 218. This strongly suggests that aa 202–218 and aa 237–247 of AKAP4 contain the minimum binding domains for FSIP2. The regions of AKAP4 to which FSIP2 binds encompass the RI/RII-dual binding domain (aa 220–229). Although FSIP2 lacks a region of homology with RII and binds to residues flanking this domain on AKAP4, it might influence PKA binding by masking the RI/RII-dual binding domain or by inducing a conformational change in AKAP4 that alters the binding affinity for RI or RII [10, 11].
The regions of AKAP4 to which AKAP3, FSIP1, and FSIP2 bind are not homologous with known protein-protein binding motifs. However, surface probability (Emini) and hydrophilicity (Kyte-Doolittle) determinations using the GCG PeptideStructure program (Pharmacopia) predict that the AKAP3, FSIP1, and FSIP2 binding regions correspond to one or more peptide loops exposed on the surface of AKAP4.
Recent studies have demonstrated that AKAPs anchor other proteins in addition to PKA. For example, protein phosphatase 2B, protein kinase C, and other kinases are associated with AKAP79 in postsynaptic densities of neurons [43, 44]. A current concept is that AKAPs often are scaffolds for multiunit complexes containing upstream activators or downstream targets of signal transduction processes [4, 5]. It is now known that PKA, FSIP1, FSIP2, and AKAP3 bind to AKAP4 and that ropporin and ASP bind to this complex through AKAP3 [15]. In addition, ropporin binds rhophilin [16, 17]. Ropporin was not extracted from sperm with Triton X-100 treatment, but was soluble in 4 M urea, whereas rhophilin remained bound to the fibrous sheath in the presence of 6 M urea [17]. This suggests that rhophilin is bound more strongly to another protein than to ropporin and might provide an additional link between the AKAP4 complex and the fibrous sheath.
Additional proteins reported to be bound to the fibrous sheath include GAPDS [18, 19], GSTm5 [24, 45], HK1S [20–23], TAKAP-80 [12], CABYR [25], and FS39 [26]. With the exception of CABYR and HK1S, these proteins are either highly insoluble (FS39) or resistant to being solubilized from the fibrous sheath in 4 M urea and 2 mM DTT. Although further studies might find that one or more of these proteins binds directly to AKAP4, the studies might also reveal that the proteins bind to the fibrous sheath through linker proteins, possibly including FSIP1 and FSIP2. In addition, it is unknown when during spermiogenesis most of the fibrous sheath proteins are synthesized or when they become associated with the fibrous sheath. If they bind to the ribs and longitudinal columns before AKAP4, this would suggest that they are anchored to AKAP3 or another component of the nascent fibrous sheath.
Most of the proteins associated with the fibrous sheath are products of genes transcribed only during the postmeiotic period of spermatogenesis. These include homologues of genes expressed in somatic cells (i.e., Gapds, Gstm5), genes for proteins that share characteristic functional domains with proteins in somatic cells (i.e., Akap4, Akap3, Takap-80), and genes for proteins unique to spermatogenic cells (rhophilin, Fs39, CABYR, Fsip1, Fsip2). Although the fibrous sheath is a unique cytoskeletal structure, it is not obvious why so many of its components are products of novel genes. Fibrous sheath-like structures are present in the sperm of some birds [46, 47] and lizards [48] that appear to be the predecessors of the fibrous sheath in mammalian sperm. The characterization of genes responsible for these structures in birds and reptiles may provide insights into why so many novel genes are involved in the formation and function of the fibrous sheath.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 26 November 2002.
First decision: 6 December 2002.
Accepted: 21 January 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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M. Bajpai, S. E. Fiedler, Z. Huang, S. Vijayaraghavan, G. E. Olson, G. Livera, M. Conti, and D. W. Carr AKAP3 Selectively Binds PDE4A Isoforms in Bovine Spermatozoa Biol Reprod, January 1, 2006; 74(1): 109 - 118. [Abstract] [Full Text] [PDF]
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 B. Baccetti, G. Collodel, M. Estenoz, D. Manca, E. Moretti, and P. Piomboni Gene deletions in an infertile man with sperm fibrous sheath dysplasia Hum. Reprod., October 1, 2005; 20(10): 2790 - 2794. [Abstract] [Full Text] [PDF]
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 E. Horowitz, Z. Zhang, B. H. Jones, S. B. Moss, C. Ho, J. R. Wood, X. Wang, M. D. Sammel, and J. F. Strauss III Patterns of expression of sperm flagellar genes: early expression of genes encoding axonemal proteins during the spermatogenic cycle and shared features of promoters of genes encoding central apparatus proteins Mol. Hum. Reprod., April 1, 2005; 11(4): 307 - 317. [Abstract] [Full Text] [PDF]
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Z. Huang, P. R. Somanath, R. Chakrabarti, E. M. Eddy, and S. Vijayaraghavan Changes in Intracellular Distribution and Activity of Protein Phosphatase PP1{gamma}2 and Its Regulating Proteins in Spermatozoa Lacking AKAP4 Biol Reprod, February 1, 2005; 72(2): 384 - 392. [Abstract] [Full Text] [PDF]
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 M. Luconi, V. Carloni, F. Marra, P. Ferruzzi, G. Forti, and E. Baldi Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A J. Cell Sci., March 1, 2004; 117(7): 1235 - 1246. [Abstract] [Full Text] [PDF]
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 R. M. Turner Tales From the Tail: What Do We Really Know About Sperm Motility? J Androl, November 1, 2003; 24(6): 790 - 803. [Full Text] [PDF]
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