Phosphorylation of rat spermatidal protein TP2 by sperm-specific protein kinase A and modulation of its transport into the haploid nucleus.
Transition protein 2 (TP2), which is expressed during stages 12-15 of mammalian spermiogenesis, has been shown to undergo phosphorylation immediately after its synthesis. We reported earlier that TP2 is phosphorylated in vitro at threonine 101 and serine 109 by the salt extract of sonication-resistant (elongating and elongated) spermatid nuclei and the protein kinase phosphorylating TP2 was identified to be protein kinase A (PKA). We now report that the cytosol from haploid spermatids but not from premeiotic germ cells is able to phosphorylate recombinant TP2 in vitro at threonine 101 and serine 109. The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA). Reverse transcription-PCR analysis indicated that Cs-PKA was present in the haploid spermatids and absent from premeiotic germ cells. The rat Cs-PKA transcript was amplified and sequenced using the isoform-specific primers. The sequence of rat Cs-PKA at the N terminus differs from mouse and human by one amino acid. Western blot analysis using specific anti-Calpha1 antibodies revealed that Calpha1-PKA is absent in haploid spermatid cytosol. We have also established an in vitro nuclear transport assay for the haploid round spermatids. Using this assay, we have found that the cytoplasmic factors and ATP are absolutely essential for translocation of TP2 into the nucleus. Phosphorylation was found to positively modulate the NLS dependent import of TP2 into the nucleus.
D021581:Active Transport, Cell Nucleus; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D002868:Chromosomal Proteins, Non-Histone; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006238:Haploidy; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry; D012756:Sheep; D013094:Spermatozoa / Q000201:enzymology; D013912:Threonine / Q000737:chemistry; D014162:Transfection
D002868:Chromosomal Proteins, Non-Histone; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C489505:Tnp2 protein, mouse; C489504:Tnp2 protein, rat; C053886:spermatid transition proteins; D049452:Green Fluorescent Proteins; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases