AAT-1, a novel testis-specific AMY-1-binding protein, forms a quaternary complex with AMY-1, A-kinase anchor protein 84, and a regulatory subunit of cAMP-dependent protein kinase and is phosphorylated by its kinase.
AMY-1 has been identified by us as a c-Myc-binding protein and was found to stimulate c-Myc transcription activity. AMY-1 was also found to be associated with protein kinase A anchor protein 84/149 (S-AKAP84/AKAP149) in the mitochondria in somatic cells and sperm, suggesting that it plays a role in spermatogenesis. To determine the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human testis cDNA library, and a clone encoding a novel protein, AAT-1, was obtained. Three isoforms of AAT-1, AAT-1alpha, -beta, and -gamma, were found to be derived from an alternative splicing of the transcripts of the aat-1 gene, which was mapped at human chromosome 3q13-3q21. AAT-1 was found to be specifically expressed in the testis during the course of spermatogenesis and also to be present in the spermatid and mature sperm, as was AMY-1. AAT-1alpha was found to bind to and be colocalized in mitochondria with AMY-1 in human HeLa and mouse GC-1 cells. Furthermore, AAT-1alpha was found to bind to the N-terminal half of S-AKAP84/AKAP149 in a quaternary complex with AMY-1 and a regulatory subunit (RII) of cAMP-dependent kinase (PKA), in which AAT-1alpha was associated with RII via S-AKAP84/AKAP149, in rat testis and HeLa cells. It was then found that AAT-1alpha weakly stimulated a phosphorylation activity of PKA and also that AAT-1 itself was phosphorylated by PKA in vivo and in vitro. These results suggest that both AAT-1 and AMY-1 play roles in spermatogenesis.
Yukitake
Hiroshi
H
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan.
Furusawa
Makoto
M
Taira
Takahiro
T
Iguchi-Ariga
Sanae M M
SM
Ariga
Hiroyoshi
H
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002893:Chromosomes, Human, Pair 3; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016259:Genes, myc; D006801:Humans; D046911:Macromolecular Substances; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D055291:Salivary alpha-Amylases; D013091:Spermatogenesis / Q000502:physiology; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D013737:Testis / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D014018:Tissue Distribution; D014157:Transcription Factors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; C486333:Akap1 protein, mouse; C486332:Akap1 protein, rat; C470011:CFAP91 protein, human; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C116115:MYCBP protein, human; D046911:Macromolecular Substances; D008565:Membrane Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D017868:Cyclic AMP-Dependent Protein Kinases; C524799:Amy1 protein, mouse; D055291:Salivary alpha-Amylases