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• W1999802541 abstract "The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A-kinase anchoringproteins (AKAPs). AKAPs contain an amphipathic helix domain that binds to the type II regulatory subunit of PKA (RII). Synthetic peptides containing this amphipathic helix domain bind to RII with high affinity and competitively inhibit the binding of PKA with AKAPs. Addition of these anchoring inhibitor peptides to spermatozoa inhibits motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P., and Carr, D. W. (1997) J. Biol. Chem. 272, 4747–4752). However, inhibition of the PKA catalytic activity does not mimic these peptides, suggesting that the peptides are disrupting the interaction of AKAP(s) with proteins other than PKA. Using the yeast two-hybrid system, we have now identified two sperm-specific human proteins that interact with the amphipathic helix region of AKAP110. These proteins, ropporin (a protein previously shown to interact with the Rho signaling pathway) and AKAP-associated sperm protein, are 39% identical to each other and share a strong sequence similarity with the conserved domain on the N terminus of RII that is involved in dimerization and AKAP binding. Mutation of conserved residues in ropporin or RII prevents binding to AKAP110. These data suggest that sperm contains several proteins that bind to AKAPs in a manner similar to RII and imply that AKAPs may have additional and perhaps unique functions in spermatozoa.AF231410 AF239723 The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A-kinase anchoringproteins (AKAPs). AKAPs contain an amphipathic helix domain that binds to the type II regulatory subunit of PKA (RII). Synthetic peptides containing this amphipathic helix domain bind to RII with high affinity and competitively inhibit the binding of PKA with AKAPs. Addition of these anchoring inhibitor peptides to spermatozoa inhibits motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P., and Carr, D. W. (1997) J. Biol. Chem. 272, 4747–4752). However, inhibition of the PKA catalytic activity does not mimic these peptides, suggesting that the peptides are disrupting the interaction of AKAP(s) with proteins other than PKA. Using the yeast two-hybrid system, we have now identified two sperm-specific human proteins that interact with the amphipathic helix region of AKAP110. These proteins, ropporin (a protein previously shown to interact with the Rho signaling pathway) and AKAP-associated sperm protein, are 39% identical to each other and share a strong sequence similarity with the conserved domain on the N terminus of RII that is involved in dimerization and AKAP binding. Mutation of conserved residues in ropporin or RII prevents binding to AKAP110. These data suggest that sperm contains several proteins that bind to AKAPs in a manner similar to RII and imply that AKAPs may have additional and perhaps unique functions in spermatozoa. AF231410 AF239723 cAMP-dependent protein kinase A-kinase anchoringproteins type II regulatory subunit of PKA anchoring inhibitor peptides AKAP-associated sperm protein fibrosheathin II sperm protein 17 PKA inhibitor peptide glutathione S-transferase rapid amplification of cDNA ends kilobase(s) polymerase chain reaction PKA1 is a ubiquitous, multifunctional kinase involved in the regulation of a diverse array of cellular events. The PKA holoenzyme consists of four subunits, two catalytic and two regulatory. The regulatory subunits form dimers through an interaction at the N terminus whereas the C terminus contains two tandem repeat sequences, which form the cAMP binding sites. Binding of cAMP to the regulatory subunits promotes the dissociation and activation of the catalytic subunits. A second function of the regulatory subunits is to target or anchor PKA to specific subcellular locations within the cell. A major advance in signal transduction research in recent years is the understanding that the actions of many signaling molecules are spatially restricted and coordinated through cell- and function-specific targeting of these enzymes and their substrates (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1904) Google Scholar). PKA is anchored to specific cellular compartments through the interaction of the regulatory subunit with a family of proteins referred to as A-kinase anchoringproteins (AKAPs) (reviewed in Refs. 2Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 3Rubin C.S. Biochim. Biophys. Acta. 1994; 1224: 467-479PubMed Google Scholar, 4Scott J.D. Carr D.W. News Physiol. Sci. 1992; 7: 143-148Google Scholar). Numerous AKAPs have been cloned and biochemically characterized. Several AKAPs have been shown to simultaneously bind to PKA and other signal transduction molecules such as calmodulin, protein phosphatase 1 (PP1), calcineurin (PP2B), and protein kinase C (5Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (529) Google Scholar, 6Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (483) Google Scholar, 7Nauert J.B. Klauck T.M. Langeberg L.K. Scott J.D. Curr. Biol. 1997; 7: 52-62Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 8Sarkar D. Erlichman J. Rubin C.S. J. Biol. Chem. 1984; 259: 9840-9846Abstract Full Text PDF PubMed Google Scholar, 9Schillace R.V. Scott J.D. Curr. Biol. 1999; 9: 321-324Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). This has led to a model in which AKAPs act as scaffolding molecules that coordinate the actions of several kinases and phosphatases all located within one cellular compartment. The structural feature of AKAPs that promotes interaction with PKA has been known for some time (10Carr D.W. Stofko-Hahn R.E. Fraser I.D. 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). AKAPs contain an amphipathic helix region and bind to the type II regulatory subunit of PKA via the hydrophobic face. The identification of this binding domain has facilitated the design of reagents that have been used to determine experimentally the physiological consequences of the interaction of PKA with AKAPs. Synthetic peptides encompassing the amphipathic helix binding domain are potent competitive inhibitors of PKA·AKAP interaction (11Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar) and therefore are referred to as anchoring inhibitor peptides (AIPs). Addition of AIPs to a variety of somatic cells inhibits PKA modulation of cellular events. For example, microinjection of AIPs into hippocampal neurons causes a time-dependent decrease in AMPA/kainate-responsive currents whereas control peptides had no effect on channel activity, suggesting that PKA anchoring is required for PKA modulation of the AMPA/kainate channels (12Rosenmund C. Carr D.W. Bergeson S.E. Nilaver G. Scott J.D. Westbrook G.L. Nature. 1994; 368: 853-856Crossref PubMed Scopus (324) Google Scholar). Since this initial finding, several laboratories have used AIPs to demonstrate that anchoring is required for PKA modulation of l-type Ca2+ channels (13Johnson B.D. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11492-11496Crossref PubMed Scopus (157) Google Scholar), calcium-activated potassium channels (14Wang Z.W. Kotlikoff M.I. Am. J. Physiol. 1996; 271: L100-L105PubMed Google Scholar), insulin secretion from pancreatic beta cells (15Lester L.B. Langeberg L.K. Scott J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14942-14947Crossref PubMed Scopus (167) Google Scholar), and phosphatidylinositide turnover in myometrial cells (16Dodge K.L. Carr D.W. Yue C. Sanborn B.M. Mol. Endocrinol. 1999; 13: 1977-1987PubMed Google Scholar, 17Dodge K.L. Carr D.W. Sanborn B.M. Endocrinology. 1999; 140: 5165-5170Crossref PubMed Scopus (37) Google Scholar). In all of the above examples, the action of AIPs is mimicked by the addition of reagents that inhibit the catalytic activity of PKA. These data support a model where AKAPs interact with the PKA regulatory subunit to anchor or target the catalytic subunit to relevant physiological substrates. Several sperm AKAPs have been identified and characterized (18Carrera A. Gerton G.L. Moss S.B. Dev. Biol. 1994; 165: 272-284Crossref PubMed Scopus (189) Google Scholar, 19Erlichman J. Gutierrez-Juarez R. Zucker S. Mei X. Orr G.A. Eur. J. Biochem. 1999; 263: 797-805Crossref PubMed Scopus (29) Google Scholar, 20Horowitz J.A. Wasco W. Leiser M. Orr G.A. J. Biol. Chem. 1988; 263: 2098-2104Abstract Full Text PDF PubMed Google Scholar, 21Lin R.Y. Moss S.B. Rubin C.S. J. Biol. Chem. 1995; 270: 27804Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 22Mei X. Singh I.S. Erlichman J. Orr G.A. Eur. J. Biochem. 1997; 246: 425-432Crossref PubMed Scopus (57) Google Scholar, 23Miki K. Eddy E.M. J. Biol. Chem. 1999; 274: 29057-29062Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 24Pariset C. Weinman S. Mol. Reprod. Dev. 1994; 39: 415-422Crossref PubMed Scopus (36) Google Scholar, 25Reinton N. Collas P. Haugen T.B. Skalhegg B.S. Hansson V. Jahnsen T. Tasken K. Dev. Biol. 2000; 223: 194-204Crossref PubMed Scopus (93) Google Scholar, 26Vijayaraghavan S. Goueli S.A. Davey M.P. Carr D.W. J. Biol. Chem. 1997; 272: 4747-4752Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 27Vijayaraghavan S. Liberty G.A. Mohan J. Winfrey V.P. Olson G.E. Carr D.W. Mol. Endocrinol. 1999; 13: 705-717Crossref PubMed Scopus (0) Google Scholar). The most prominent AKAP detected by RII overlay assay of bovine, human, mouse, and monkey spermatozoa is AKAP110 (20Horowitz J.A. Wasco W. Leiser M. Orr G.A. J. Biol. Chem. 1988; 263: 2098-2104Abstract Full Text PDF PubMed Google Scholar, 21Lin R.Y. Moss S.B. Rubin C.S. J. Biol. Chem. 1995; 270: 27804Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 27Vijayaraghavan S. Liberty G.A. Mohan J. Winfrey V.P. Olson G.E. Carr D.W. Mol. Endocrinol. 1999; 13: 705-717Crossref PubMed Scopus (0) Google Scholar). Northern analysis suggests that AKAP110 is expressed only in spermatozoa, and immunofluorescence studies detecting AKAP110 in the flagella suggest that this protein may be involved in regulating motility. Addition of S-Ht31 (stearated-Ht31 is a cell permeable AIP) to bovine caudal epididymal spermatozoa inhibits motility in a time- and concentration-dependent manner (26Vijayaraghavan S. Goueli S.A. Davey M.P. Carr D.W. J. Biol. Chem. 1997; 272: 4747-4752Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). A control peptide, S-Ht31-P, identical to S-Ht31 except for an isoleucine to proline substitution that prevents amphipathic helix formation, had no effect on motility. Surprisingly, inhibition of PKA catalytic activity by addition of H-89 or S-PKI had little effect on basal motility or motility stimulated by agents previously thought to work via PKA activation (26Vijayaraghavan S. Goueli S.A. Davey M.P. Carr D.W. J. Biol. Chem. 1997; 272: 4747-4752Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). These data suggest that proteins interacting with sperm AKAPs regulate motility in a manner that is independent of PKA catalytic activity. These results have been confirmed by a different approach. McKnight and colleagues (28Burton K.A. Treash-Osio B. Muller C.H. Dunphy E.L. McKnight G.S. J. Biol. Chem. 1999; 274: 24131-24136Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), using mutant mice lacking RIIα, have shown that the catalytic subunit is no longer located along the flagellum but instead is concentrated in the cytoplasmic droplet, yet the spermatozoa are motile and the mice are fertile. These data suggest that neither RIIα nor RIIα-dependent localization of PKA catalytic subunit is necessary to support motility. Thus, it appears that AIPs may be exerting an effect on spermatozoa that is independent of AKAP·RIIα interaction. One hypothesis consistent with the above data is that sperm AKAPs are interacting with proteins other than RII via the amphipathic helix domain. This would explain how AIPs could be regulating motility in a manner independent of both the regulatory and catalytic subunits of PKA. Using the yeast two-hybrid system to screen a human testis library, we have now identified several sperm-specific proteins that bind to fragments of AKAP110 containing the amphipathic helix domain. Binding studies demonstrate that these proteins bind to AKAP110 in a manner homologous with RII. Based on these data, we propose a model for the function of AKAPs in spermatozoa that is very different than the model for AKAPs in somatic cells. The yeast strain EGY48(p2op-lacZ) containing constructs of pLexA human AKAP110, truncated hAKAP110-(1–350), and human ropporin was transformed with a pB42AD-fused human testis cDNA library following protocols for the Matchmaker LexA Two-Hybrid System (CLONTECH). Approximately 2 × 108 transformants were screened with each construct on 150-mm plates containing 5 × 104 clones with SD/Gal/Raf/-His/-Trp/-Ura/-Leu/+Xgal + BU salts. Blue colonies, positive for β-galactosidase activity, were re-streaked on SD/-His/-Trp/-Ura plates, then replated on SD/Gal/Raf/-His/-Trp/-Ura/-Leu/+X-gal + BU salts to re-test for activation of both the lacZ and LEU2 reporter genes. Double positives were then plated on master plates containing SD/-His/-Trp/-Ura and used for yeast PCR and sequence analysis. Ropporin and mutant ropporin (L18A) were expressed as pET30a N-terminal His6-tagged fusion proteins in Escherichia coli(BL21(DE3)) and were purified by fast protein liquid chromatography using Hi-Trap chelating Sepharose columns (Amersham Pharmacia Biotech). One liter of LB broth + 30 μg/ml kanamycin was inoculated and grown to mid-logarithm phase before adding 1 mmisopropyl-1-thio-β-d-galactopyranoside to induce protein expression at 37 °C for 2 h. The cells were then pelleted by centrifugation at 2000 × g for 20 min, sonicated in buffer A on ice (20 mm HEPES, 500 mm NaCl, 20 mm imidazole, 1 mm4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, pH 7.4), and clarified by centrifugation at 15,000 × g for 20 min at 4 °C, and the resultant supernatant was passed through a 0.45-μm filter. The supernatant was then applied to the Ni2+-charged Hi-Trap chelating column. Bound proteins were eluted with a stepwise gradient of imidazole (0–0.5 m) in buffer A. Fractions containing purified protein were identified by Coomassie Blue staining of 10% SDS-polyacrylamide gel electrophoresis gels. pET11d-RIIα and mutant RIIα (L14A) were expressed in E. coli (BL21(DE3)) and purified by cAMP-agarose affinity column as previously described (10Carr D.W. Stofko-Hahn R.E. Fraser I.D. 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). Plasmids for expression of recombinant hAKAP110, hAKAP110-(1–350), mRIIα, h-ropporin, hASP, hSP17, and hFSII in a yeast two-hybrid system, as histidine-tagged or GST fusion proteins inE. coli were prepared as follows. The pET30a hAKAP110 full-length construct and its truncated form hAKAP110-(1–350) (27Vijayaraghavan S. Liberty G.A. Mohan J. Winfrey V.P. Olson G.E. Carr D.W. Mol. Endocrinol. 1999; 13: 705-717Crossref PubMed Scopus (0) Google Scholar) were digested with EcoRI and XhoI, gel-purified, and ligated into pLexA, pB42AD (CLONTECH, Palo Alto, CA), and pGEX5x-1 (Amersham Pharmacia Biotech, Piscataway, NJ) vectors cut with the same restriction enzymes. PCR was performed on pET11d RIIα with forward primer 5′-CCGGAATTCATGAGCCACATCCAGATCCCG-3′ and reverse primer 5′-CCGCTCGAGCACACTGAGAAGGCTCCAAGATTC-3′, respectively, containing anEcoRI and XhoI restriction site. The PCR product was digested with EcoRI/XhoI, gel-purified, and ligated into pLexA and pB42AD. Full-length PCR products of ropporin, SP17, and FSII were obtained by 5′-RACE of a Marathon Ready Human Testis cDNA library (CLONTECH). Ropporin forward primer 5′-GGATTCATGGCTCAGACAGATAAGCCAACATG-3′ and reverse primer 5′-CCCTCGAGAATTGTGCTGTTACTCCAGCCAAACC-3′, SP17 forward primer 5′-AGATCCATGTCGATCCATTCTCCAACACCCA-3′ and reverse primer 5′-ATTTGCGGCCGCTGGAGGTAAAACCAGTGTCCTCACTTG-3′, FSII forward primer 5′-AGATCCATGTCGATCCATTCTCCAACACCCAC-3′ and reverse primer 5′-ATTTGCGGCCGCTGGAGGTAAAACCAGTCTCCTCACTTG-3′. Ropporin hadEcoRI/XhoI restriction sites added to the primers, SP17 and FSII primers had BamHI/NotI restriction sites added. Ropporin was digested and ligated into pET30a, pLexA, and pB42AD; SP17 and FSII were subcloned into pLexA. ASP was obtained using the yeast two-hybrid system and Human Testis Matchmaker LexA cDNA (CLONTECH),EcoRI/XhoI-digested, and ligated into pLexA and pET30a. A Mouse Multiple Tissue Northern blot (CLONTECH), containing 2 μg of poly(A)+ RNA per lane, was screened using the full-length ASP fragment generated in the plasmids section. The probe was32P-labeled using the High Prime DNA labeling kit (Roche Molecular Biochemicals). Hybridization of the probe was carried out at 42 °C for 18 h in Ullrich's buffer/2% SDS. The blot was washed at room temperature 2× in SSC, 2× in SSC/2% SDS at 65 °C and finally in 0.1% SSC at room temperature. An exposure of the blot was then made on x-ray film for 72 h. E. coli transformed with pGEX-5X-1 plasmid encoding human AKAP110-(1–350) or AKAP110-P (containing a proline substitution for leucine 131) were grown to mid-logarithm phase at 37 °C in 1 liter of LB medium. They were cultured for an additional 2 h at 37 °C in the presence of 0.2 mmisopropyl-β-d-thiogalactopyranoside to induce synthesis of the fusion protein. Crude extracts were prepared by sonicating the bacteria in 20 mm Tris-HCl, pH 8.0, 100 mmNaCl, and 1 mm 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride. 2% Tween 20 (v/v) was added to the extract and rotated at room temperature for 1 h. The extract was centrifuged at 15,000 × g for 20 min before passing the supernatant through a 0.45-μm filter. The supernatant was incubated for 30 min at room temperature with 3 ml of glutathione-Sepharose before extensive washing in phosphate-buffered saline to remove nonspecifically bound proteins. Prior to the addition of purified proteins, 200 μl of the GST-hAKAP110-(1–350) was incubated with 750 μl of Blotto and 10 mm dithiothreitol for 30 min at room temperature. 1 μg of RIIα, RIIα (L14A), 3 μg of ropporin and 4 μg of ropporin (L18A) were added to separate tubes containing the Blotto/AKAP110 mix and rotated at room temperature for 2 h. After washing extensively with phosphate-buffered saline and 10 mm dithiothreitol, proteins were eluted by boiling in SDS gel-loading buffer and separated by 10% SDS-polyacrylamide gel electrophoresis. Regulatory subunit RIIα was detected by Western blotting using rabbit antisera against RIIα (6825) and secondary anti-rabbit horseradish peroxidase conjugate (Sigma Chemical Co., St. Louis, MO). Ropporin was detected by conjugated horseradish peroxidase-Anti-S-Protein (Novagen, Madison, WI). PCR was done directly on diethyl pyrocarbonate-H2O suspensions of yeast cell positives using vector-specific primers flanking the multiple cloning site of the pB42AD vector (29Ling M. Merante F. Robinson B.H. Nucleic Acids Res. 1995; 23: 4924-4925Crossref PubMed Scopus (72) Google Scholar, 30Sathe G.M. O'Brien S. McLaughlin M.M. Watson F. Livi G.P. Nucleic Acids Res. 1991; 19: 4775Crossref PubMed Scopus (55) Google Scholar). The resulting PCR products were gel-purified, and sequence analysis was performed using the Big-Dye Terminator Sequencing Kit (Applied Biosystems, Foster City, CA) and the same vector specific primers. The Y-Der yeast DNA extraction kit (Pierce, Rockford, IL) was used to recover plasmid DNA from positives that wouldn't amplify using the above method. Analysis of sequence data, sequence comparison, and alignments was performed using the MacVector ClustalW program (Oxford Molecular Group) and the BLAST program (31Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) provided by the NCBI server at the National Library of Medicine/National Institutes of Health. The 5′-RACE was performed using a Marathon cDNA amplification kit and Human Testis Marathon-Ready cDNA (CLONTECH) as described with the accompanying procedures. The primers used to obtain full-length PCR products were previously stated in the plasmids section. Plasmid constructs pET30a mouse ropporin, pET11d mouse RIIα, and pGEX5x-1 containing hAKAP110-(1–350) were used as PCR templates for the QuikChange 1-day site-directed mutagenesis method (Stratagene, La Jolla, CA). Aligned leucines (see Fig. 4 A below) at positions 18 and 14 of ropporin and RIIα, respectively, were mutated to alanines using the following primers: Ropporin (L18A), forward primer 5′-TGCCGGAATTGGCAAAGCAGTTTAC-3′, reverse primer 5′-GTAAACTGCTTTGCCAATTCCGGCA-3′; RIIα (L14A), forward primer 5′-TCACGGAGCTGGCACAGGGCTACA-3′, reverse primer 5′-TGTAGCCCTGTGCCAGCTCCGTGA-3′. Leucine 131 within the amphipathic helix region of hAKAP110 was mutated to proline using the following primers: forward primer 5′-ATGCTAACCGCCCAACGAATCTAG-3′, reverse primer 5′-CTAGATTCGTTGGGCGGTTAGCAT-3′. The resulting mutated DNA was transformed into E. coli Super Competent JM109 cells (Promega, Madison, WI) and grown on antibiotic-resistant LB Agar plates. The mutations were verified by sequence analysis using vector-specific primers. Adult male mice were sacrificed by CO2 asphyxiation, and a sperm suspension was obtained by mincing the cauda epididymides in buffered saline (145 mmNaCl and 5 mm Hepes, pH 7.4). Sperm were then fixed 30 min in 4% formaldehyde in 0.1 m sodium phosphate buffer, pH 7.4), attached to coverslips, permeabilized in absolute acetone for 10 min at −20 °C and air-dried. For immunostaining, cells were first incubated 1 h in blocking buffer of Tris-saline (TN = 150 mm NaCl, 25 mm Tris-HCl, pH 8.0, and 0.05% Tween 20) containing 2.5% bovine serum albumin and 5% goat serum and then successively incubated in primary and secondary antibodies also diluted in blocking solution. Between all incubation steps, coverslips were washed three times in TN containing 1% goat serum. Primary antibodies, prepared in rabbits, included affinity-purified IgG to AKAP110 and ropporin. Control samples substituted identical levels of affinity-purified non-immune rabbit IgG for immune IgG. Cy3-conjugated affinity-purified secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). AKAP110 is a sperm-specific AKAP that binds to the type II regulatory subunit of PKA via an amphipathic helix-binding motif located at amino acid position 124–143 (27Vijayaraghavan S. Liberty G.A. Mohan J. Winfrey V.P. Olson G.E. Carr D.W. Mol. Endocrinol. 1999; 13: 705-717Crossref PubMed Scopus (0) Google Scholar). To determine if other proteins also bind to AKAP110 via the amphipathic helix binding domain, a human testis cDNA library was screened using an N-terminal fragment of AKAP110-(1–350) as bait in a yeast two-hybrid procedure. Positives were then co-transformed in the two-hybrid system with another fragment of AKAP110-(349–660) that does not contain an amphipathic helix domain. Three positives were identified that bound to the amphipathic helix-containing fragment (1) but not to the other fragment (349) (Fig. 1). The pB42AD plasmids were recovered from these clones, and the cDNA inserts (1.4, 1.3, and 1.1 kb in size) were sequenced. Sequence analysis demonstrated that the 1.4-kb insert encodes for the human type II regulatory subunit of the cAMP-dependent protein kinase, RIIα (GenBank™ accession number NM004147). The first base of the insert lines up with base 142 of the deposited sequence, which lists the coding region as 190–1404. Thus, the 1.4-kb insert contains the full-length coding region for PKA RIIα protein plus an additional 16 amino acids from the 5′-untranslated region. Although this additional region would normally not be translated, there are no stop codons within this segment, and thus these additional amino acids functioned as a bridge between the pB42AD fusion partner and RIIα. The sequence obtained from the 1.3-kb insert is 94% identical to the amino acid sequence of the murine protein ropporin (GenBank™ accession number AF178531), which we previously isolated as a binding partner of a Rho effector, rhophilin (32Fujita A. Nakamura K. Kato T. Watanabe N. Ishizaki T. Kimura K. Mizoguchi A. Narumiya S. J. Cell Sci. 2000; 113: 103-112Crossref PubMed Google Scholar). The human ropporin (h-ropporin) sequence has been submitted to the GenBank™ data base (accession number AF231410). Ropporin is a sperm-specific protein localized in the principal piece and the end piece of sperm flagella (32Fujita A. Nakamura K. Kato T. Watanabe N. Ishizaki T. Kimura K. Mizoguchi A. Narumiya S. J. Cell Sci. 2000; 113: 103-112Crossref PubMed Google Scholar). Ropporin forms homodimers and binds to rhophilin, and both proteins have been shown to co-precipitate in vitro with Rho (32Fujita A. Nakamura K. Kato T. Watanabe N. Ishizaki T. Kimura K. Mizoguchi A. Narumiya S. J. Cell Sci. 2000; 113: 103-112Crossref PubMed Google Scholar). Once again, in addition to sequence that was homologous to the coding region for m-ropporin, the pB42AD insert contained sequence upstream (165 bases) of the ropporin start site, adding 55 amino acids of 5′-untranslated repeat as a bridge between the pB42AD fusion protein and the h-ropporin. The sequence obtained from the 1.1-kb insert is a novel protein. It is 39% identical to h-ropporin but does not match any other protein in the GenBank™ data base and will hereafter be referred to asAKAP-associated sperm protein or ASP. ASP also contains a 5′-untranslated repeat bridge (57 bases, 19 amino acids) between the vector and the start site. This sequence has been submitted to the GenBank™ data base (accession number AF239723). The optimal alignment (MacVector ClustalW Alignment Program) of m-ropporin, h-ropporin, and ASP is shown in Fig.2. ASP is highly homologous with the N-terminal 80 residues of ropporin and only moderately homologous with the rest of the molecule, suggesting the N terminus contains a conserved domain that may have an important function in spermatozoa. We have previously shown that m-ropporin is detected only in the testis and then only in the most inner part of the seminiferous tubules region, suggesting this protein is expressed in developing spermatozoa (32Fujita A. Nakamura K. Kato T. Watanabe N. Ishizaki T. Kimura K. Mizoguchi A. Narumiya S. J. Cell Sci. 2000; 113: 103-112Crossref PubMed Google Scholar). To determine the tissue distribution of ASP, Northern blots containing 2 μg of poly(A)+ RNA from eight different adult mouse tissues were probed with 32P-labeled ASP cDNA. A single message was detected only in the testis (Fig. 3), suggesting this protein is testis-specific and possibly sperm-specific. Using a linear regression analysis of a plot of the log10 versus the RF of the molecular weight markers, the ASP mRNA was calculated to be ∼1.05 kb. This was bigger than expected based on the insert size (850 bases) from the yeast vector. To determine if the insert represented full-length cDNA for ASP, 5′-RACE was performed using Human Testis Marathon-Ready cDNA (CLONTECH) as template. An additional 144 bases were identified using this technique, bringing the total number of bases to 994 or approximately the same size as the calculated mRNA. Although this new region contains an open reading frame continuous with the rest of the protein, it does not contain an alternate start site. At the time of submission of this manuscript, a BLAST search of the human genome data base does not detect either human ropporin or ASP. As mentioned above, the N-terminal regions of ropporin and ASP are the most conserved. The sequence in this region is also similar to the N terminus of the type II regulatory subunit of PKA and two other sperm-specific proteins, SP-17 (GenBankTM accession number Q15506) (33Kong M. Richardson R.T. Widgren E.E. O'Rand M.G. Biol. Reprod. 1995; 53: 579-590Crossref PubMed Scopus (72) Google Scholar,34Lea I.A. Richardson R.T. Widgren E.E. O'Rand M.G. Biochim. Biophys. Acta. 1996; 1307: 263-266Crossref PubMed Scopus (57) Google Scholar) and fibrousheathin II (FSII) (GenBankTM accession number NM_012189). 2A. Mandal, M. J. Wolkowicz, S. Naaby-Hansen, and J. C. Herr, unpublished data. Optimal alignment (MacVector ClustalW alignment program) of RIIα and RIIβ (residues 1–46 or 45, respectively, of both human and mouse) with the corresponding regions of ropporin, ASP, SP17, and FSII is shown in Fig.4 A. The dark gray shading indicates sequence identity, and the light gray shading indicates sequence similarity. Although these sperm proteins share high sequence similarity with the N-terminal region of RII, they have little or no homology with other regions of RII such as the nucleotide binding domains. The only other protein identifiable by sequence homology, sharing the characteristics of having an AKAP binding domain but not a cyclic-nucleotide binding domain, is a hypothetical protein from Caenorhabditis elegans F39H12.3. The location of this protein in C. elegans is still undetermined. The N-terminal 44 amino acids of RII contain the domains responsible for homodimerization and binding to AKAPs (Fig. 4 B). The sequence identity of h-RIIβ, h-ropporin, hASP, hSP17, hFSII, and hRIα with hRIIα (1–44) is 70, 30, 32, 45, 34, and 18%, respectively. It is interesting to note that a" @default.
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• W1999802541 title "Identification of Sperm-specific Proteins That Interact with A-kinase Anchoring Proteins in a Manner Similar to the Type II Regulatory Subunit of PKA" @default.
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