Identification of Tethering Domains for Protein Kinase A Type Iα Regulatory Subunits on Sperm Fibrous Sheath Protein FSC1
1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês
10.1074/jbc.273.51.34384
ISSN1083-351X
Autores Tópico(s)Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities
ResumoThe fibrous sheath is a unique cytoskeletal structure in the sperm flagellum believed to modulate sperm motility. FSC1 is the major structural protein of the fibrous sheath. The yeast two-hybrid system was used to identify other proteins that contribute to the structure of the fibrous sheath or participate in sperm motility. When FSC1 was used as the bait to screen a mouse testis cDNA library, two clones were isolated encoding the type Iα regulatory subunit (RIα) of cAMP-dependent protein kinase. Deletion analysis using the yeast two-hybrid system andin vitro binding assays with glutathioneS-transferase-FSC1 fusion proteins identified two RIα tethering domains on FSC1. A domain located at residues 219–232 (termed domain A) corresponds to the reported tethering domain for a type II regulatory subunit (RII) of cAMP-dependent protein kinase, indicating that this binding domain has dual specificity to RI and RII. Another RIα tethering site (termed domain B) at residues 335–344 shows specific binding of RIα and had no significant sequence homology with known RII tethering domains. However, helical wheel projection analysis indicates that domain B is likely to form an amphipathic helix, the secondary structure of RII tethering domains of protein kinase A anchoring proteins. This was supported by the finding that site-directed mutagenesis to disrupt the amphipathic helix eliminated RIα binding. This is apparently the first report of an RIα-specific protein kinase A anchoring protein tethering domain. The fibrous sheath is a unique cytoskeletal structure in the sperm flagellum believed to modulate sperm motility. FSC1 is the major structural protein of the fibrous sheath. The yeast two-hybrid system was used to identify other proteins that contribute to the structure of the fibrous sheath or participate in sperm motility. When FSC1 was used as the bait to screen a mouse testis cDNA library, two clones were isolated encoding the type Iα regulatory subunit (RIα) of cAMP-dependent protein kinase. Deletion analysis using the yeast two-hybrid system andin vitro binding assays with glutathioneS-transferase-FSC1 fusion proteins identified two RIα tethering domains on FSC1. A domain located at residues 219–232 (termed domain A) corresponds to the reported tethering domain for a type II regulatory subunit (RII) of cAMP-dependent protein kinase, indicating that this binding domain has dual specificity to RI and RII. Another RIα tethering site (termed domain B) at residues 335–344 shows specific binding of RIα and had no significant sequence homology with known RII tethering domains. However, helical wheel projection analysis indicates that domain B is likely to form an amphipathic helix, the secondary structure of RII tethering domains of protein kinase A anchoring proteins. This was supported by the finding that site-directed mutagenesis to disrupt the amphipathic helix eliminated RIα binding. This is apparently the first report of an RIα-specific protein kinase A anchoring protein tethering domain. cAMP-dependent protein kinase protein kinase A anchoring protein glutathione S-transferase polyacrylamide gel electrophoresis phosphate buffered saline regulatory subunit type I regulatory subunit type II synthetic dropout polymerase chain reaction. The mammalian sperm flagellum consists mainly of cytoskeletal structures, including the axoneme, the outer dense fibers, and the fibrous sheath. The axoneme is formed of microtubles organized in a "nine plus two" arrangement, extends the full length of the flagellum, and functions as the motor of the sperm. It is surrounded by the outer dense fibers throughout most of its length. The outer dense fibers are enclosed by the mitochondrial sheath in the middle piece and by the fibrous sheath in the principal piece of the flagellum (1Eddy E.M. O'Brien D.A. Kobil E. Neill J. The Physiology of Reproduction. Raven Press, New York1994: 29-77Google Scholar). The outer dense fibers and the fibrous sheath are believed to stiffen the flagellum and to modulate its bending (2Lindermann C.B. Orlando A. Kanous K.S. J. Cell Sci. 1992; 102: 249-260PubMed Google Scholar, 3Si Y. Okuno M. Exp. Cell Res. 1993; 208: 170-174Crossref PubMed Scopus (43) Google Scholar). In addition, several spermatogenic cell-specific glycolytic enzymes are associated with the fibrous sheath (4Bunch D.O. Welch J.E. Magyar P.L. Eddy E.M. O'Brien D.A. Biol. Reprod. 1998; 58: 834-841Crossref PubMed Scopus (130) Google Scholar, 5Mori C. Nakamura N. Welch J.E. Gotoh H. Goulding E.H. Fujioka M. Eddy E.M. Mol. Reprod. Dev. 1998; 49: 374-385Crossref PubMed Scopus (104) Google Scholar, 6Bradley M.P. Geelan A. Leitch V. Goldberg E. Mol. Reprod. Dev. 1996; 44: 452-459Crossref PubMed Scopus (29) Google Scholar). This structure may be a scaffold for enzymes that produce energy required for hyperactivated motility of sperm at the time of fertilization. Furthermore, cAMP-stimulated protein phosphorylation is essential for initiation or maintenance of sperm motility (7Garbers D.L. Kopf G.S. Greengard P. Robison G.A. Advances in Cyclic Mucleotide Research. Raven Press, New York1980: 251-306Google Scholar, 8Brandt H. Hoskins D.D. J. Biol. Chem. 1980; 255: 982-987Abstract Full Text PDF PubMed Google Scholar, 9Tash J.S. Kakar S.S. Means A.R. Cell. 1984; 38: 551-559Abstract Full Text PDF PubMed Scopus (93) Google Scholar) and proteins in the fibrous sheath are subject to phosphorylation. The coordinated regulation of many cellular mechanisms is mediated through cAMP-dependent protein kinase (PKA).1 There has been much interest in how cAMP can act as a second messenger to regulate different mechanisms at specific sites distributed throughout the cytoplasm and nucleus. One way this is achieved appears to be through diversity of PKA components. PKA is a tetramer of two catalytic (C) and two regulatory (R) subunits, and genes encoding three C subunits and four R subunits have been identified (10Takio K. Smith S.B. Krebs E.G. Walsh K.A. Titani K. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2544-2548Crossref PubMed Scopus (83) Google Scholar, 11Lee D.C. Carmichael D.F. Krebs E.G. McKnight G.S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3608-3612Crossref PubMed Scopus (132) Google Scholar, 12Jahnsen T. Hedin L. Kidd V.J. Beatie W.G. Lohmann S.M. Walter U. Durica J. Schulz T.Z. Schiltz E. Browner M. Lawrence C.B. Goldman D. Ratoosh S.L. Richards J.S. J. Biol. Chem. 1986; 261: 12352-12361Abstract Full Text PDF PubMed Google Scholar, 13Clegg C.H. Cadd G.G. McKnight G.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3703-3707Crossref PubMed Scopus (167) Google Scholar). RIα and RIIα subunits are expressed ubiquitously, while RIβ and RIIβ subunits are expressed mainly in brain and testis. In addition, protein kinase A anchoring proteins (AKAPs) have been identified that are present in specific subcellular sites. AKAPs have tethering domains for regulatory subunits that place PKA in close proximity to specific organelles or cytoskeletal components to localize phosphorylation events that occur in response to activation signals (14Glantz S.B. Li Y. Rubin C.S. J. Biol. Chem. 1993; 268: 12796-12804Abstract Full Text PDF PubMed Google Scholar, 15Glantz S.B. Amat J.A. Rubin C.S. Mol. Biol. Cell. 1992; 3: 1215-1228Crossref PubMed Scopus (108) Google Scholar, 16Hirsch A.H. Glantz S.B. Li Y. You Y. Rubin C.S. J. Biol. Chem. 1992; 267: 2131-2134Abstract Full Text PDF PubMed Google Scholar). Ligand overlay assays have indicated that only RII subunits of PKA are tethered to AKAPs. The observation that RII subunits bind to structural proteins in the sperm flagellum (17Horowitz J.A. Toeg H. Orr G.A. J. Biol. Chem. 1984; 259: 832-838Abstract Full Text PDF PubMed Google Scholar, 18Horowitz J.A. Wasco W. Leiser M. Orr G.A. J. Biol. Chem. 1988; 263: 2098-2104Abstract Full Text PDF PubMed Google Scholar, 19Macleod J. Mei X. Erlichman J. Orr G.A. Eur. J. Biochem. 1994; 225: 107-114Crossref PubMed Scopus (16) Google Scholar) was consistent with RII subunits being found mainly in the particulate fraction and RI subunits in the cytosolic fraction of cell homogenates. However, yeast two-hybrid screens recently isolated D-AKAP1 and D-AKAP2 proteins that interact with both RIα and RIIα (20Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. J. Biol. Chem. 1997; 272: 8057-8064Crossref PubMed Scopus (257) Google Scholar, 21Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11184-11189Crossref PubMed Scopus (202) Google Scholar). Although D-AKAP1 has a 25-fold lower affinity for RIα than for RIIα, this suggests that RI subunits also may determine the subcellular localization of PKA. Deletion and point mutagenesis approaches have been used with ligand overlay assays to map RII tethering domains on AKAPs. These domains have limited consensus sequences but are predicted to form secondary structures of amphipathic helices that containing acidic amino acids in the hydrophilic region and amino acids with a long aliphatic side chain in the hydrophobic region (14Glantz S.B. Li Y. Rubin C.S. J. Biol. Chem. 1993; 268: 12796-12804Abstract Full Text PDF PubMed Google Scholar, 22Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D. J. Biol. Chem. 1991; 266: 14188-14192Abstract Full Text PDF PubMed Google Scholar, 23Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar). The location of the RI tethering domain was not determined for D-AKAP1 or D-AKAP2, but the binding region for RI partially or completely overlaps that for RII in D-AKAP1 (20Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. J. Biol. Chem. 1997; 272: 8057-8064Crossref PubMed Scopus (257) Google Scholar, 21Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11184-11189Crossref PubMed Scopus (202) Google Scholar). To better understand the function of the fibrous sheath of mouse sperm, we have used immunological, biochemical, and molecular biology approaches to identify some of its components. These include three enzymes, glutathione S-transferase (24Fulcher K.D. Welch J.E. Klapper D.G. O'Brien D.A. Eddy E.M. Mol. Reprod. Dev. 1995; 42: 415-424Crossref PubMed Scopus (56) Google Scholar), glyceraldehyde-3-phosphate dehydrogenase (4Bunch D.O. Welch J.E. Magyar P.L. Eddy E.M. O'Brien D.A. Biol. Reprod. 1998; 58: 834-841Crossref PubMed Scopus (130) Google Scholar), and type I hexokinase (5Mori C. Nakamura N. Welch J.E. Gotoh H. Goulding E.H. Fujioka M. Eddy E.M. Mol. Reprod. Dev. 1998; 49: 374-385Crossref PubMed Scopus (104) Google Scholar), and the major structural protein of the fibrous sheath, referred to as fibrous sheath component 1 (25Eddy E.M. O'Brien D.A. Fenderson B.A. Welch J.E. Ann. N. Y. Acad. Sci. 1991; 637: 224-239Crossref PubMed Scopus (31) Google Scholar, 26Fulcher K.D. Mori C. Welch J.E. O'Brien D.A. Klapper D.G. Eddy E.M. Biol. Reprod. 1995; 52: 41-49Crossref PubMed Scopus (83) Google Scholar). It is remarkable that three of these are products of genes expressed only in spermatogenic cells (Gstm5, Gapds, and Fsc1), while the other is the product of spermatogenic cell-specific transcripts (Hk1-s). Northern analysis and in situhybridization demonstrated that Fsc1 transcription first occurs in the postmeiotic phase of spermatogenesis when the fibrous sheath is assembled. Other investigators isolated a cDNA encoding a fibrous sheath protein referred to as AKAP82 that is identical to FSC1, except for lacking 9 amino acids at the N terminus (27Carrera A. Gerton G.L. Moss S.B. Dev. Biol. 1994; 165: 272-284Crossref PubMed Scopus (189) Google Scholar). An important finding was that RII subunits bind to a 14-amino acid region of AKAP82 (28Visconti P.E. Johnson L.R. Oyaski M. Fornés M. Moss S.B. Gerton G.L. Kopf G.S. Dev. Biol. 1997; 192: 351-363Crossref PubMed Scopus (175) Google Scholar). This region was predicted to form an amphipathic helix, and the binding in the ligand overlay assay was inhibited by a synthetic peptide of the corresponding sequence (28Visconti P.E. Johnson L.R. Oyaski M. Fornés M. Moss S.B. Gerton G.L. Kopf G.S. Dev. Biol. 1997; 192: 351-363Crossref PubMed Scopus (175) Google Scholar). The studies reported here used the yeast two-hybrid system, deletion mutagenesis, and an in vitro binding assay to demonstrate that two RIα and one RIIα tethering domains are present on FSC1. While RIα and RIIα bound to domain A, only RIα bound to domain B. This suggests that the amphipathic helix formed by domain B has a secondary structure distinct from that required for RII binding. Taken together with the immunocytochemical evidence that RI associates with the fibrous sheath and outer dense fibers (29Moos J. Peknicová J. Geussová G. Philimonenko V. Hozák P. Mol. Reprod. Dev. 1998; 50: 79-85Crossref PubMed Scopus (18) Google Scholar), these results suggest that tethering of RIα to FSC1 may provide a mechanism for the subcellular localization of PKA to a region of the flagellum important for sperm motility. The Saccharomyces cerevisiaestrain Y190 (MATa, ura3–52,his3-Δ200, ade2–101, trp1–901,leu2–3, 112, gal4Δgal80Δ,URA::GAL-lacZ,cyh r 2,LYS2::GAL-HIS3) and yeast culture media used in these yeast two-hybrid studies were obtained fromCLONTECH (Palo Alto, CA). Synthetic dropout (SD) and dropout supplements were used to prepare tryptophan and/or leucine amino acid-deficient media (SD/Trp− Leu−, SD/Trp−, or SD/Leu−). Transactivation ofHis3 was assayed using medium deficient in tryptophan, leucine, and histidine (SD/Trp− Leu−His−) and supplemented with 25 mm3-amino-1,2,4-triazole (Sigma). PCR was used to produce the full-length Fsc1 protein coding sequence (GenBankTM accession number U10341) or various deletion mutants. Forward primers containing NdeI sites and reverse primers containing SalI sites were used to generate PCR products for ligation into the pAS2-1 (CLONTECH) yeast two-hybrid system expression plasmid (TableI). Numbering of the amino acid sequence of FSC1 was that of Fulcher et al. (26Fulcher K.D. Mori C. Welch J.E. O'Brien D.A. Klapper D.G. Eddy E.M. Biol. Reprod. 1995; 52: 41-49Crossref PubMed Scopus (83) Google Scholar). The PCR reaction contained 20 ng of mouse Fsc1 cDNA in λ gt11 (26Fulcher K.D. Mori C. Welch J.E. O'Brien D.A. Klapper D.G. Eddy E.M. Biol. Reprod. 1995; 52: 41-49Crossref PubMed Scopus (83) Google Scholar), 10 pmol of each primer, a 0.1 mm concentration of each of the four deoxynucleotide triphosphates, and Pfu DNA polymerase and reaction buffer (Stratagene, La Jolla, CA) in a 50-μl reaction volume. Amplification was performed for 30 cycles with a temperature profile of 30 s at 94 °C, 1 min at 55 °C, and 4.5 min at 72 °C. The PCR products were digested with NdeI andSalI restriction enzymes, ligated into plasmid pAS2-1 digested with NdeI and SalI, and then transformed in Escherichia coli DH5α competent cells (Life Technologies, Inc.). The resulting constructs allowed FSC1 to be expressed in yeast as a fusion protein with GAL4.Table IPCR primer sequences used for construction of expression plasmid for FSC1PositionDirectionPrimer sequence1Forward5′-CCGCATATGATTGCCTACTGTGGTACTACAACG-3′125Forward5′-CCGCATATGCAACATGCATTAAGCCCCTCAGCC-3′202Forward5′-CCGCATATGCAGAGGTCAGTTGCCACTCCTGAG-3′237Forward5′-CCGCATATGAAGGACAAATTGGAAGGTGGAAGC-3′311Forward5′-CCGCATATGGGCGAAAAGCAGCAGATGTGCCCA-3′326Forward5′-CCGCATATGGATTCCATCAGCAAGGGGCTTATGG-3′335Forward5′-CCGCATATGTATGCAAATCAAGTAGCATCTGAC-3′343Forward5′-CCGCATATGATGGTCTCTGTTATGAAAACTTTG-3′359Forward5′-CCGCATATGCCAATTCCGGCTTGTGTGGTCCTG-3′397Forward5′-CCGCATATGACAGACTCAGACTTCGTTTCTGC-3′218Reverse5′-CCGGTCGACGGAAAGGTCGTCCATAGAACATTC-3′247Reverse5′-CCGGTCGACATGGAGACATTTGCTTCCACCTTC-3′311Reverse5′-CCGGTCGACCGCCACACTTGTTCTTGTTACACA-3′325Reverse5′-CCGGTCGACCGCAAATTCTTTGCTGTCTTTTGG-3′334Reverse5′-CCGGTCGACAACCATAAGCCCCTTGCTGATGGA-3′344Reverse5′-CCGGTCGACCATCATGTCAGATGCTACTTGATT-3′361Reverse5′-CCGGTCGACCCGGAATTGGCTTCCCACAGCTGT-3′849Reverse5′-CCGGTCGACAGGGGAGTCAAAGGAATTCTCAGC-3′ Open table in a new tab To produce constructs that expressed FSC1 protein in E. colias a fusion protein with glutathione S-transferase, PCR forward primers were used that contained the BamHI sequence to allow ligation into the corresponding site in pGEX-4T-1 (Amersham Pharmacia Biotech). Other conditions for construction of these plasmids were as described above. The coding sequence of Fsc1 was subcloned into pBluescript II KS+ (Stratagene), and then used as a template to introduce the point mutation by using the QuikChangeTMsite-directed mutagenesis kit (Stratagene). Sequencing of the targeted site was carried out using T7 Sequenase version 2.0 (Amersham Pharmacia Biotech). PCRs using the appropriate primer set were used to amplify DNA sequences corresponding to mutant FSC1 proteins covering residues 237–361. The products were ligated into pGEX-4T-1 to produce constructs that expressed mutant FSC1 proteins in E. coli as described above. Yeast Y190 competent cells were prepared with the Yeastmaker yeast transformation system (CLONTECH), based on the lithium acetate method described by Gietz et al. (30Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2898) Google Scholar), transformed with plasmid pAS/Fsc/1–849 containing the full-length Fsc1cDNA, and cultured on SD/Trp− agar plates for 4 days at 30 °C. Selected yeast cells containing pAS/Fsc/1–849 were cultured in SD/Trp− medium and used as the carrier of the bait vector for sequential transformation. A mouse testis cDNA library constructed in the pGAD10 vector (CLONTECH) was screened using the yeast two-hybrid procedure. The pAS/Fsc/1–849 and pGAD10 co-transformants were cultured on SD/Trp−Leu− His− agar plates for 7 days at 30 °C. Selected transformants were tested for β-galactosidase activity by the colony lift assay using 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside as the substrate on ProtranTM nitrocellulose membranes (Schleicher & Schuell). To assay binding activity of candidate cDNA clones andFsc1 deletion mutants, lifts of transformants were cultured for 2 days at 30 °C on YPD or SD/Trp− Leu−agar plate, respectively, and then permeabilized and assayed for β-galactosidase activity. Membranes were frozen for 10 min at −70 °C and thawed at room temperature to permeabilize yeast cells and then soaked in Z-buffer (0.3 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, 40 mm β-mercaptoethanol, 10 mm KCl, 1 mm MgSO4, and 0.1 m phosphate buffer, pH 7.0) for 8 h at 37 °C. Yeast cells were transformed with pAS2–1 deletion mutants of Fsc1 and cultured overnight in 1 ml of SD/Trp− Leu−selection medium at 30 °C. Nine ml of YPD medium then was added, and growth was allowed to proceed to the midlogarithmic phase. Yeast proteins were extracted by the urea/SDS method as described (31Printen J.A. Sprague Jr., G.F. Genetics. 1994; 138: 609-619Crossref PubMed Google Scholar) using 250 μl of cracking buffer. Proteins from 5 μl of extract were separated by SDS-PAGE using 10% (w/v) acrylamide gel and transferred onto ImobilonTM nylon membranes (Millipore Corp., Bedford, MA). The membranes were soaked in TBST (150 mm NaCl, 0.1% (v/v) Tween 20, 50 mm Tris-HCl, pH 7.4) solution containing 2% (w/v) gelatin for 1 h and then incubated for 1 h with monoclonal antibody (0.5 μg/ml) to the GAL4 DNA-binding domain (CLONTECH) in TBST. After extensive washing with TBST, membranes were incubated for 30 min with 10% (v/v) goat serum in TBST. After washing briefly, membranes were incubated for 30 min with horseradish peroxidase-conjugated goat antiserum to mouse IgG (1:30,000 dilution in TBST) (Sigma) preabsorbed for 30 min with 0.5 mg/ml yeast proteins to eliminate nonspecific antibodies against common yeast proteins. Yeast proteins were prepared from Y190 strain cells sonicated in PBS for 3 min followed by incubation for 30 min with 1% (v/v) Triton X-100 to solubilize membrane proteins. After extensive washing of the membrane with TBST solution, FSC1-GAL4 fusion protein was detected using ECLTM reagents (Amersham Pharmacia Biotech) according to procedures recommended by the supplier. E. coli transformed with pGEX-4T-1 plasmid encoding Fsc1were grown to the midlogarithmic phase at 37 °C in LB medium. They were cultured for an additional 90 min at 30 °C in the presence of 0.1 mm isopropyl-β-d-thiogalactopyranoside to induce synthesis of the fusion protein. Crude extracts were prepared by sonicating bacteria in PBS for 30 s followed by incubation for 30 min with 1% (v/v) Triton X-100. Crude extract (15–150 μl) was incubated for 2 h at 4 °C with 30 μl of glutathione-Sepharose resin (Amersham Pharmacia Biotech) in 0.5 ml of PBS, followed by extensive washing with PBS to remove nonspecifically bound proteins. Extracts from testes of 3–5-month-old CD-1 mice were prepared by homogenization in extraction buffer (140 mm NaCl, 0.1% (v/v) Triton X-100, 0.1 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 mm Tris-HCl, pH 7.5). After centrifugation at 10,000 × g for 10 min, supernatant corresponding to 150 μg or 1.5 mg of protein was added to the tube containing GST-FSC1 fusion protein immobilized on resin. The volume was adjusted to 0.5 ml with extraction buffer, and the mixture was incubated for 2 h at 4 °C. After washing extensively with PBS, proteins were eluted by boiling in SDS gel-loading buffer, the eluates were divided into three parts, and proteins were separated by SDS-PAGE using 10% (w/v) acrylamide gels prepared according to Laemmli (32Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207461) Google Scholar). Each lane contained that portion of 50 or 500 μg of protein from testis extracts that bound to GST-FSC1, while the positive control lane contained 10 μg (Fig. 3, lane T) or 5 μg (Fig. 5,lane 1) of total testis extract. Regulatory subunits of PKA were detected by Western blotting using rabbit antisera against mouse RIα, RIIα (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:1000 dilution each) or RIβ (1:500 dilution; a gift of Dr. Daniel W. Carr, Oregon Health Sciences University, Portland, OR). Secondary antibody was horseradish peroxidase-conjugated goat antiserum to rabbit IgG (1:10,000 dilution; Santa Cruz Biotechnology). Western blotting was performed as described above except that second antibody was not preabsorbed. Other gels were stained with Coomassie Brilliant Blue to detect proteins. Prestained standard proteins (Bio-Rad) were used to estimate protein mass.Figure 5Disruption of RIα binding activity in FSC1 domain B. Point mutations were introduced into the Fsc1cDNA to disturb the putative amphipathic helix in domain B, and then RIα binding activity was determined by in vitrobinding assays, as in Fig. 3. The proteins that bound truncated FSC1 were analyzed by Western blotting using antiserum to RIα (a). Eluted proteins were visualized by Coomassie staining (b). Lane 1 received testis extract not subjected to the binding assay. Lanes 2 and3–7 received GST fusion proteins containing truncated FSC1 proteins with the intervening region (N) or domain B (B) as in Fig. 3 a, respectively. Lane 3 received fusion protein with wild type domain B. The other lanes received fusion proteins that contained domain B with the following mutations: replacement of serine 327 with proline (S327P) (lane 4); replacement of alanine 340 with proline (A340P) (lane 5); replacement of valine 339 with serine (V339S) (lane 6); exchange of valine 339 and serine 341 (V339S/S341V) (lane 7). Thearrow to the left of a indicates the position of RIα. The dots to the left ofa and an open triangle in bidentify the same proteins as in Fig. 3.View Large Image Figure ViewerDownload (PPT) The yeast two-hybrid procedure (33Brent R. Ptashne M. Cell. 1985; 43: 729-736Abstract Full Text PDF PubMed Scopus (435) Google Scholar) was used to screen a mouse testis cDNA library to identify proteins that interact directly with FSC1. Yeast Y190 cells were transfected with a bait plasmid containing the mouse Fsc1 cDNA protein coding region followed by sequential transformation with library plasmid DNA. Secondary transformants corresponding to 5 × 106 clones were screened for interaction with FSC1 based on growth on medium lacking histidine. Positive clones were tested for transactivation ofLacZ as another marker gene. Of the eight clones positive in both assays, two (FA7 and FC9) were subjected to further analysis in this study. To confirm association with FSC1, plasmids for the FA7 and FC9 clones were rescued from yeast cells, cloned and amplified in E. coli, and then used to retransform yeast cells. Transformants were selected on medium lacking tryptophan and/or leucine, according to the type and combination of plasmid vectors used. Only transformants that received pAS/Fsc/1–849 in combination with plasmids containing pFA7 or pFC9 grew on SD/Trp− Leu− His−, while transformants that received pAS/Fsc/1–849, pFA7, or pFC9 alone did not grow (data not shown). Transformants that received plasmid vector(s) were also cultured on YPD medium and used for colony lift assays to detect β-galactosidase expression (data not shown). This assay gave the same results as the growth selection assay, confirming that transactivation was due to association between FSC1 and proteins encoded by pFA7 and pFC9. These results also suggested that the interaction is direct and that no additional factors are required for association. Sequence analysis demonstrated that both the pFA7 and pFC9 cDNAs encode PKA RIα. The pFC9 cDNA encoded the full-length protein of 381 amino acids, while the pFA7 cDNA encoded 190 residues in the N-terminal half (data not shown). Differences from the previously reported nucleotide sequence (13Clegg C.H. Cadd G.G. McKnight G.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3703-3707Crossref PubMed Scopus (167) Google Scholar) were found at the third position in five codons, but this did not alter the deduced amino acid sequence (data not shown). Only the pFC9 RIα cDNA was used for further experiments. The region of the FSC1 protein responsible for RIα binding was identified by deletional mutagenesis in the yeast two-hybrid system. The amino acid numbering was based on the FSC1 sequence (26Fulcher K.D. Mori C. Welch J.E. O'Brien D.A. Klapper D.G. Eddy E.M. Biol. Reprod. 1995; 52: 41-49Crossref PubMed Scopus (83) Google Scholar). A series of deletion mutants were constructed in the pAS2-1 plasmid to allow expression of truncated FSC1 polypeptides in yeast cells. These plasmids were used to retransform primary transformants expressing the full-length RIα protein. Transformants containing plasmids both for deletion mutants ofFsc1 and full-length RIα were selected on SD/Trp− Leu− and examined for β-galactosidase activity by the colony lift assay. The first series examined were sequential deletions from the N terminus of FSC1. The β-galactosidase activity was lost after the deletions reached residue 343 (343–849), while a longer construct (335–849) gave β-galactosidase activity (Fig.1 b). This strongly suggests that the peptide sequence within the 335–342 residue region is required for RIα binding. We next examined another series of deletion mutations that lacked domain A (see below) in the N-terminal region of FSC1. The polypeptide that included residues 237–334 did not result in β-galactosidase activity, while activity was seen with the polypeptide that included residues 237–344 (Fig. 1 b). These results confirm that RIα binding is associated with residues 335–344. We term this region domain B (Fig. 1 c). The second series of mutants involved plasmids with deletions producing truncations from the C terminus of FSC1. It was found that β-galactosidase activity occurred until the deletion reached residue 218 (125–218) (Fig. 1 b). Taken together with other results showing that β-galactosidase activity was present with polypeptide 125–247 and absent with polypeptide 237–334, this indicated that an RIα binding domain lies between residue 219 and 236 in the FSC1 polypeptide (Fig. 1 b). Visconti et al. (28Visconti P.E. Johnson L.R. Oyaski M. Fornés M. Moss S.B. Gerton G.L. Kopf G.S. Dev. Biol. 1997; 192: 351-363Crossref PubMed Scopus (175) Google Scholar) recently mapped an RII tethering domain of AKAP82 to a 57-amino acid region that includes a 14-amino acid sequence similar to an RII tethering domain in other AKAPs. This region of AKAP82 corresponds to residues 219–232 of FSC1, and our results strongly suggest that this region is also capable of binding RIα. We term this region domain A (Fig. 1 c). Transactivation of the His3 gene was also examined for all of the deletion mutants (Fig. 1, a and c). Transformants with plasmids encoding deletion mutants of FSC1 and full-length RIα were selected on SD/Trp−Leu− medium and then transferred onto SD/Trp−Leu− His− medium to test for growth. Representative results (Fig. 1 a) show that mutants with deletions up to domain A or B grew on medium lacking histidine (335–849, 237–344, and 125–247), while further deletions resulted in the loss of growth activity (343–849, 237–334, and 125–218). These results were consistent with the β-galactosidase activity assays (Fig. 1, b and c). Yeast homogenates were examined for the presence of fusion proteins to determine if failure to transactivate LacZ andHis3 was due to failure of the vectors to express. However, Western blotting with an antibody to the GAL4 DNA binding domain demonstrated that the truncated FSC1 proteins were expressed in yeast in which transactivation did not occur (Fig.2). Fusion protein was detected in homogenates of transformants in which neither LacZ norHis3 genes were expressed (343–8
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